RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 15/776,725, filed May 16, 2018, which is a national stage application, filed under 35 U.S.C. § 371, of International Application No. PCT/US2016/050297 filed Sep. 2, 2016, which claims benefit of priority to U.S. Provisional Application No. 62/257,796, filed Nov. 20, 2015, and U.S. Provisional Application No. 62/257,784, filed Nov. 20, 2015, the entire contents of each of which are incorporated herein by reference.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named “35327-516001WO_Sequence_Listing.txt”, which was created on Sep. 2, 2016 and is 587 KB in size, is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for detecting and determining the concentration of glucose and/or galactose.

BACKGROUND

Biosensors are analytical tools that can be used to measure the presence of a single molecular species in a complex mixture by combining the exquisite molecular recognition properties of biological macromolecules with signal transduction mechanisms that couple ligand binding to readily detectable physical changes (Hall, Biosensors, Prentice-Hall, Englewood Cliffs, N.J.; Scheller et al., Curr. Op. Biotech. 12:35-40, 2001). Ideally, a biosensor is reagentless and, in contrast to enzyme-based assays or competitive immunoassays, does not change composition as a consequence of making the measurement (Hellinga & Marvin, Trends Biotech. 16:183-189, 1998). Most biosensors combine a naturally occurring macromolecule such as an enzyme or an antibody, with the identification of a suitable physical signal particular to the molecule in question, and the construction of a detector specific to that system (Meadows, Adv. Drug Deliv. Rev. 21:177-189, 1996). Recently, molecular engineering techniques have been explored to develop macromolecules that combine a wide range of binding specificities and affinities with a common signal transduction mechanism, to construct a generic detection system for many different analytes (Hellinga & Marvin, Trends Biotech. 16:183-189, 1998).

Escherichia coli periplasmic binding proteins are members of a protein superfamily (bacterial periplasmic binding proteins, bPBPs) (Tam & Saier, Microbiol. Rev. 57:320-346, 1993). These proteins comprise two domains linked by a hinge region (Quiocho & Ledvina, Molec. Microbiol. 20:17-25, 1996). The ligand-binding site is located at the interface between the two domains. The proteins typically adopt two conformations: a ligand-free open form, and a ligand-bound closed form, which interconvert via a hinge-bending mechanism upon ligand binding. This global, ligand-mediated conformational change has been exploited to couple ligand binding to changes in fluorescence intensity by positioning single, environmentally sensitive fluorophores in locations that undergo local conformational changes in concert with the global change (Brune et al., Biochemistry 33:8262-8271, 1994; Gilardi et al., Prot. Eng. 10:479-486, 1997; Gilardi et al., Anal. Chem. 66:3840-3847, 1994; Marvin et al., Proc. Natl. Acad. Sci. USA 94:4366-4371, 1997, Marvin and Hellinga, J. Am. Chem. Soc. 120:7-11, 1998; Tolosa et al., Anal. Biochem. 267:114-120, 1999; Dattelbaum & Lakowicz, Anal. Biochem. 291:89-95, 2001; Marvin & Hellinga, Proc. Natl. Acad. Sci. USA 98:4955-4960, 2001; Salins et al., Anal. Biochem. 294:19-26, 2001).

SUMMARY OF THE INVENTION

The invention provides improved biosensors that rapidly, reliably, and accurately detect and quantify glucose and/or galactose with significant advantages over previous systems. The present disclosure provides a biosensor for ligand, comprising a ligand-binding protein that is attached to a reporter group. The ligand may be glucose and/or galactose, and the ligand-binding protein includes a domain that binds the glucose and/or the galactose. The binding of a ligand to the ligand-binding domain of the ligand-binding protein causes a change in signaling by the reporter group. In various implementations, the biosensor may produce a signal when a ligand is bound to the ligand binding domain that is not produced (and/or that is different from a signal that is produced) when the ligand is absent from the ligand binding domain. These biosensors have widespread utility including in clinical, industrial, and environmental settings.

The glucose-binding proteins (biosensors) described herein are characterized by one conformational shape when bound to glucose and a different conformational shape when unbound to glucose, this change in shape affects the signal of a detectable label such as a fluorophore. The proteins are engineered to include a single cysteine to which the detectable label, e.g., a fluorophore is covalently attached. The biosensors are reagentless in that their monitoring mechanism requires neither additional substrates for a signal to develop, nor measurement of substrate consumption or product generation rates to determine glucose concentrations.

Among the advantages of these fluorophone-containing protein constructs is their high durability. The constructs retain their ability to bind glucose, change shape and thus detect the analyte, glucose, (a) even when immobilized (directly or indirectly)onto a solid surface such as a bead, plate, or sheet; (b) even after dessication (and subsequent reconstitution in a physiological buffer solution); (c) even when subjected to ambient conditions, e.g., conditions that can be encountered in storage and/or transportation; and (d) even when aged/stored for extended periods of time, e.g., weeks, months, or even years. Thus, the biosensors do not require refrigeration or a cold chain for distribution, permitting a wider range of applicability such as in-the-field use and reducing the cost of the sensor product.

For clinical applications, microliter volumes, e.g., 10 μl or less of a bodily fluid such as blood may be used. Moreover compared to conventional enzyme-based or antibody based assay systems, the results are achieved virtually instantaneously, e.g., within 30-60 seconds. A further advantage is that the sensors consistently and reliably bind to and detect the analyte (glucose) in complex fluids such as whole blood. Thus in a clinical setting, whole blood need not be processed, thereby reducing time and cost of the diagnostic procedure.

In non-clinical situations, e.g., industrial of commercial settings such as analysis of waste water, food or beverage production, or bioreactor/fermentation monitoring, the samples to be analyzed can be used directly upon sampling without further purification or processing, similarly reducing time and expense of the test. Moreover, the immobilized sensors need not be washed to remove unbound material following contacting the test sample with the sensors, because the unbound material (“contaminants”) do not materially affect the production of a precise, reliable detectable assay signal.

The glucose biosensors produce a dichromatic, ratiometric signal, i.e., the signal is defined as the quotient of the intensities at two independent wavelengths. The advantage of such a signal is that it provides an internally consistent reference. The self-calibrating nature of a ratiometric measurement removes the necessity for carrying out on-board calibration tests prior to each measurement.

Thus, reagentless, fluorescently responsive glucose sensors present a number of advantages over enzyme-based biosensors, including elimination of chemical transformations, elimination of substrate requirements, and self-calibration, which together lead to rapid response times, continuous monitoring capabilities, simple sample-handling, and lower cost due to simplified manufacturing and distribution processes.

Ligand-Binding Proteins

Aspects of the present subject matter provide biosensors comprising a ligand-binding protein that binds glucose and/or galactose. Ligand binding proteins that bind both glucose and galactose may be referred to herein as “glucose-galactose binding proteins” (GGBPs). Typically, a natural GGBP has a glucose dissociation constant (KD) of about 10 μM or less at room temperature. However, GGBPs may be selected, designed, or engineered to detect different (e.g., higher or lower) levels of glucose and/or galactose. The ligand-binding protein may comprise a naturally occurring protein or a protein that is modified compared to a naturally occurring protein. For example, the ligand-binding protein may comprise one or more mutations compared to a naturally occurring protein. In some embodiments, the naturally occurring protein is a naturally occurring counterpart of the ligand-binding protein (e.g., the ligand-binding protein is a mutant of the naturally occurring counterpart).

A “naturally occurring counterpart” of a mutant polypeptide is a polypeptide produced in nature from which the mutant polypeptide has been or may be derived (e.g., by one or more mutations). For example, the naturally occurring counterpart is an endogenous polypeptide produced by an organism in nature, wherein the endogenous polypeptide typically does not have one or more of the mutations present in the mutant polypeptide. For convenience and depending on context, a naturally occurring counterpart may be referred to herein for the purpose of comparison and to illustrate the location and/or presence of one or more mutations, binding activities, and/or structural features.

As used herein, a “mutation” is a difference between the amino acid sequence of a modified polypeptide/protein and a naturally occurring counterpart. A polypeptide having a mutation may be referred to as a “mutant.” Non-limiting examples of mutations include insertions, deletions, and substitutions. However, the term “mutation” excludes (i) the addition of amino acids to the N-terminus or C-terminus of a polypeptide, and (ii) the omission/deletion/replacement of a polypeptide's signal peptide (e.g., replacement with another signal peptide or with a methionine).

The addition of amino acids to the N-terminus or C-terminus of a protein via a peptide bond may be referred to herein as a “fusion” of the amino acids to the protein. Similarly, an exogenous protein fused to amino acids (e.g., another protein, a fragment, a tag, or a polypeptide moiety) at its N-terminus or C-terminus may be referred to as a “fusion protein.” The added amino acids may comprise a heterologous polypeptide, e.g., a polypeptide reporter group such as a fluorescent protein, a moiety that facilitates the isolation or modification of a polypeptide, or a moiety that facilitates the attachment of a polypeptide to a substrate or surface. As used herein, “heterologous” when referring to the added amino acids (e.g., a “polypeptide”) of a fusion protein indicates that the polypeptide is not naturally part of the protein to which it is fused in the fusion protein. For example, the sequence of a heterologous polypeptide (“added amino acids”) that is fused to a protein is encoded by an organism other than the organism from which the protein is derived, is not known to be naturally encoded by any organism, or is encoded by a gene other than the wild-type gene that encodes an endogenous version of the protein.

As used herein the term “signal peptide” refers to a short (e.g., 5-30 or 10-60 amino acids long) stretch of amino acids at the N-terminus of a protein that directs the transport of the protein. In various embodiments, the signal peptide is cleaved off during the post-translational modification of a protein by a cell. Signal peptides may also be referred to as “targeting signals,” “leader sequences,” “signal sequences,” “transit peptides,” or “localization signals.” In instances where a signal peptide is not defined for a GGBP discussed herein, the signal peptide may optionally be considered to be, e.g., the first 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 5-15, 5-20, 5-25, 5-100, 10-15, 10-20, 10-25, 10-50, 10-100, 25-50, 25-75, or 25-100 amino acids from the N-terminus of the translated protein (compared to a protein that has not had the signal peptide removed, e.g., compared to a naturally occurring protein).

In some embodiments, the ligand-binding protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 1-10, 1-15, 1-20, 5-15, 5-20, 10-25, 10-50, 20-50, 25-75, 25-100 or more mutations compared to a naturally occurring protein while retaining at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5%, or about 100% of the activity of the naturally occurring protein. Mutations include but are not limited to substitutions, insertions, and deletions. Non-limiting examples of ligand-binding proteins may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 1-10, 1-15, 1-20, 5-15, 5-20, 10-25, 10-50, 20-50, 25-75, 25-100, or more substitution mutations compared to a naturally occurring protein while retaining at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5%, or about 100% of the activity of the naturally occurring protein. In embodiments, at least one amino acid of the ligand-binding protein has been substituted with a cysteine. Alternatively or in addition, a ligand-binding protein may include one or more mutations that remove a cysteine, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more substitutions or deletions of a cysteine compared to a naturally occurring protein.

Alternatively, the ligand-binding protein is not a mutant. For example, a reporter group is fused to the N-terminus or the C-terminus of the ligand-binding protein.

In various embodiments, a ligand-binding protein may comprise a stretch of amino acids (e.g., the entire length of the ligand-binding protein or a portion comprising at least about 50, 100, 200, 250, 300, or 350 amino acids) in a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% identical to an amino acid sequence of a naturally occurring protein.

In some embodiments, the mutations are conservative, and the present subject matter includes many ligand-binding proteins in which the only mutations are substitution mutations. In non-limiting examples, a ligand-binding protein has no deletions or insertions compared to a naturally occurring protein (e.g., a naturally occurring counterpart). Alternatively, a ligand-binding protein may have (i) less than about 5, 4, 3, 2, or 1 inserted amino acids, and/or (ii) less than about 5, 4, 3, 2, or 1 deleted amino acids compared to a naturally occurring protein.

In various embodiments, a naturally occurring protein to which a ligand-binding protein is compared or has been derived (e.g., by mutation, fusion, or other modification) from a prokaryotic ligand-binding protein such as a bacterial ligand-binding protein. For example, the prokaryotic ligand-binding protein is a mutant, fragment, or variant of a natural (i.e., wild-type) bacterial protein. In various embodiments, the bacterial ligand-binding protein is from a thermophilic, mesophilic, or cryophilic prokaryotic microorganism (e.g., a thermophilic, mesophilic, or cryophilic bacterium).

A microorganism is “thermophilic” if it is capable of surviving, growing, and reproducing at temperatures between 41 and 140° C. (106 and 284° F.), inclusive. In various embodiments, a thermophilic organism has an optimal growth temperature between 41 and 140° C., or that is at least about 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, or 140° C. Many thermophiles are archaea. Thermophilic eubacteria are suggested to have been among the earliest bacteria. Thermophiles are found in various geothermally heated regions of the Earth, such as hot springs and deep sea hydrothermal vents, as well as decaying plant matter, such as peat bogs and compost. Unlike other types of microorganisms, thermophiles can survive at much hotter temperatures, whereas other bacteria would be damaged and sometimes killed if exposed to the same temperatures. Thermophiles may be classified into three groups: (1) obligate thermophiles; (2) facultative thermophiles; and (3) hyperthermophiles. Obligate thermophiles (also called extreme thermophiles) require such high temperatures for growth, whereas facultative thermophiles (also called moderate thermophiles) can thrive at high temperatures, but also at lower temperatures (e.g. below 50° C.). Hyperthermophiles are particularly extreme thermophiles for which the optimal temperatures are above 80° C. Some microorganisms can live at temperatures higher than 100° C. at large depths in the ocean where water does not boil because of high pressure. Many hyperthermophiles are also able to withstand other environmental extremes such as high acidity or radiation levels. A compound (e.g., a protein or biosensor) is “thermotolerant” if it is capable of surviving exposure to temperatures above 41° C. For example, in some embodiments a thermotolerant biosensor retains its function and does not become denatured when exposed to a temperature of about 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, or 140° C. for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or more minutes. In some embodiments, the thermotolerant compound survives exposure to 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, or 140° C. under pressure.

A microorganism is “mesophilic” if it is capable of surviving, growing, and reproducing at temperatures between 20 and 40° C. (68 and 104° F.), inclusive. “Psychrophiles” or “cryophiles” are microorganisms that are capable of growth and reproduction in cold temperatures. In various embodiments, a psychrophile is capable of growth and reproduction at a temperature of 10° C. or less, e.g., between −20° C. and +10° C.

In some embodiments, the microbial protein is produced by a bacterial microorganism, an archaean microorganism, an algal microorganism, a protozoan microorganism, or a fungal microorganism. In non-limiting examples, the microbial protein is produced by a Gram-positive bacterium or a Gram-negative bacterium. In various embodiments, a biosensor comprises a modified (e.g., mutated, fused, and/or conjugated) periplasmic binding protein or a cytoplasmic binding protein.

In non-limiting examples in which the ligand-binding protein is (1) an Escherichia coli (E. coli) glucose-galactose binding protein (GGBP) (e.g., has been derived from an E. coli GGBP via mutation) or (2) has an amino acid sequence that is at least 95%, 96, 97%, 98%, or 99% identical to the amino acid sequence of E. coli GGBP, the ligand-binding protein comprises a mutation other than a mutation of amino acid Y10, D14, N15, F16, N91, K92, E93, S112, S115, E149, H152, D154, A155, R158, M182, W183, N211, D236, L255, N256, D257, P294, or V296, wherein each amino acid position is numbered as in (SEQ ID NO: 17).

In certain embodiments, the ligand-binding protein is not derived from (e.g., by mutation, fusion, or other modification) an E. coli protein (such as ecGGBP) and does not comprise an amino acid sequence that is identical to the amino acid sequence of ecGGBP. For example, the naturally occurring counterpart of the ligand-binding protein is not an E. coli GGBP (e.g., the ligand-binding protein is not a mutant of, fusion protein comprising, or other variant of an E. coli GGBP). In some embodiments, the amino acid sequence of the ligand-binding protein is less than about 100%, 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, or 15% identical to an E. coli GGBP protein having amino acids in the sequence set forth as SEQ ID NO: 1, 16, or 17.

Aspects of the present subject matter provide a ligand-binding protein with a mutation that alters the interaction of the ligand-binding protein with a ligand (e.g., glucose and/or galactose). For example, the ligand-binding protein comprises a mutation that alters the interaction of the ligand-binding protein with the ligand compared to a naturally occurring counterpart. In some embodiments, the ligand-binding protein comprises a mutation that alters the interaction of an amino acid of the ligand-binding protein with a water molecule compared to a naturally occurring counterpart.

In some embodiments, the ligand-binding protein does not comprise a signal peptide. For example, the signal peptide (e.g., that is present in a naturally occurring counterpart) may be replaced with a methionine.

Exemplary implementations relate to a ligand such as glucose or galactose, wherein the ligand-binding protein comprises a GGBP. For example, the GGBP may comprise a mutant of, a fragment of, or a fusion protein comprising a microbial GGBP. In embodiments, the GGBP is not a mutant or fragment to which a heterologous polypeptide has been attached or added. In some embodiments, the ligand-binding protein has an affinity (KD) for glucose and/or galactose within the concentration range of glucose and/or galactose in a subject. In certain embodiments, the ligand-binding protein has an affinity (KD) for glucose in the range of about 0.2 mM to about 100 mM, about 0.1 mmol/L to about 120 mmol/L, or about 4 mmol/L to about 33 mmol/L. In various embodiments, the ligand-binding protein has an affinity (KD) for galactose in the range of about 0.8 mM to about 100 mM or about 0.2 mM to about 400 mM. The biosensor is capable of detecting glucose in, e.g. the hypoglycemic, euglycemic, hyperglycemic, or hyperglycemic-hyperosmotic range. Thus, unlike previous glucose sensors, the ratiometric reagentless glucose biosensors produce precise measurements over an extended glucose concentration range from hypoglycemic, euglycemic, hyperglycemic, as well as the hyperglycemic-hyperosmotic range in sample volumes of less than 10 μl. In some embodiments, the ligand-binding protein comprises a mutation that alters (e.g., increases or decreases) the interaction of the mutant with bound glucose compared to a naturally occurring protein (e.g., a microbial GGBP), wherein the interaction is with a portion of the glucose selected from the group consisting of 1-hydroxyl, 2-hydroxyl, 3-hydroxyl, 4-hydoxyl, 6-hydroxyl, pyranose ring, or any combination thereof. In non-limiting examples, the ligand-binding protein comprises a mutation that alters (e.g., increases or decreases) the mutant's affinity and/or specificity for glucose and/or galactose compared to a the unmutated ligand-binding protein (e.g., a microbial GGBP). In certain embodiments, the ligand-binding protein comprises a mutation that alters the interaction between the protein and bound glucose and/or galactose, a mutation that alters the equilibrium between the open and closed states of the ligand-binding protein, a mutation that alters the interaction between the ligand-binding protein and a reporter group (such as a fluorescent conjugate, e.g., the interaction with a carbonyl group or a naphthalene ring of a prodan-derived fluorophore such as Acrylodan or Badan), and/or a mutation that impacts indirect interactions that alter the geometry of the ligand binding site. In various embodiments, the mutation does not reduce, or negligibly impacts, the thermostability of the ligand-binding protein. In some embodiments, the mutation alters the thermostability of the ligand-binding protein by less than about 1, 2, 3, 4, 5, or 10° C.

The present subject matter provides a GGBP that is or is a mutant of: an Escherichia sp. (e.g., E. albertii, E. coli, E. fergusonii, E. hermannii, or E. vulneris) GGBP; a Thermoanaerobacter sp. (e.g., T. acetoethylicus, T. brockii, T. ethanolicus, T. italicus, T. kivui, T. mathranii, T. pseudethanolicus, T. siderophilus, T. sulfurigignens, T. sulfurophilus, T. thermocopriae, T. thermohydrosulfuricus, T. thermosaccharolyticum, T. uzonensis, or T. wiegelii) GGBP; a Clostridium sp. (e.g., C. absonum, C. aceticum, C. acetireducens, C. acetobutylicum, C. acidisoli, C. aciditolerans, C. acidurici, C. aerotolerans, C. aestuarii, C. akagii, C. aldenense, C. aldrichii, C. algidicarni, C. algidixylanolyticum, C. algifaecis, C. algoriphilum, C. alkalicellulosi, C. aminophilum, C. aminovalericum, C. amygdalinum, C. amylolyticum, C. arbusti, C. arcticum, C. argentinense, C. asparagiforme, C. aurantibutyricum, C. autoethanogenum, C. baratii, C. barkeri, C. bartlettii, C. beijerinckii, C. bifermentans, C. bolteae, C. bornimense, C. botulinum, C. bowmanii, C. bryantii, C. butyricum, C. cadaveris, C. caenicola, C. caminithermale, C. carboxidivorans, C. carnis, C. cavendishii, C. celatum, C. celerecrescens, C. cellobioparum, C. cellulofermentans, C. cellulolyticum, C. cellulosi, C. cellulovorans, C. chartatabidum, C. chauvoei, C. chromiireducens, C. citroniae, C. clariflavum, C. clostridioforme, C. coccoides, C. cochlearium, C. colletant, C. colicanis, C. colinum, C. collagenovorans, C. cylindrosporum, C. difficile, C. diolis, C. disporicum, C. drakei, C. durum, C. estertheticum, C. estertheticum estertheticum, C. estertheticum laramiense, C. fallax, C. felsineum, C. fervidum, C. fimetarium, C. formicaceticum, C. frigidicarnis, C. frigoris, C. ganghwense, C. gasigenes, C. ghonii, C. glycolicum, C. glycyrrhizinilyticum, C. grantii, C. haemolyticum, C. halophilum, C. hastiforme, C. hathewayi, C. herbivorans, C. hiranonis, C. histolyticum, C. homopropionicum, C. huakuii, C. hungatei, C. hydrogeniformans, C. hydroxybenzoicum, C. hylemonae, C. jejuense, C. indolis, C. innocuum, C. intestinale, C. irregulare, C. isatidis, C. josui, C. kluyveri, C. lactatifermentans, C. lacusfryxellense, C. laramiense, C. lavalense, C. lentocellum, C. lentoputrescens, C. leptum, C. limosum, C. litorale, C. lituseburense, C. ljungdahlii, C. lortetii, C. lundense, C. magnum, C. malenominatum, C. mangenotii, C. mayombei, C. methoxybenzovorans, C. methylpentosum, C. neopropionicum, C. nexile, C. nitrophenolicum, C. novyi, C. oceanicum, C. orbiscindens, C. oroticum, C. oxalicum, C. papyrosolvens, C. paradoxum, C. paraperfringens, C. paraputrificum, C. pascui, C. pasteurianum, C. peptidivorans, C. perenne, C. perfringens, C. pfennigii, C. phytofermentans, C. piliforme, C. polysaccharolyticum, C. populeti, C. propionicum, C. proteoclasticum, C. proteolyticum, C. psychrophilum, C. puniceum, C. purinilyticum, C. putrefaciens, C. putrificum, C. quercicolum, C. quinii, C. ramosum, C. rectum, C. roseum, C. saccharobutylicum, C. saccharogumia, C. saccharolyticum, C. saccharoperbutylacetonicum, C. sardiniense, C. sartagoforme, C. scatologenes, C. schirmacherense, C. scindens, C. septicum, C. sordellii, C. sphenoides, C. spiroforme, C. sporogenes, C. sporosphaeroides, C. stercorarium, C. stercorarium leptospartum, C. stercorarium stercorarium, C. stercorarium thermolacticum, C. sticklandii, C. straminisolvens, C. subterminale, C. sufflavum, C. sulfidigenes, C. symbiosum, C. tagluense, C. tepidiprofundi, C. termitidis, C. tertium, C. tetani, Clostridium tetanomorphum, C. thermaceticum, C. thermautotrophicum, C. thermoalcaliphilum, C. thermobutyricum, C. thermocellum, C. thermocopriae, C. thermohydrosulfuricum, C. thermolacticum, C. thermopalmarium, C. thermopapyrolyticum, C. thermosaccharolyticum, C. thermosuccinogenes, C. thermosulfurigenes, C. thiosulfatireducens, C. tyrobutyricum, C. uliginosum, C. ultunense, C. villosum, C. vincentii, C. viride, C. xylanolyticum, or C. xylanovorans) GGBP; a Salmonella sp. [e.g., S. bongori, S. enterica, S. enterica subspecies enterica, S. enterica subspecies salamae, S. enterica subspecies arizonae, S. enterica subspecies diarizonae, S. enterica subspecies houtenae, S. enterica subspecies indica, or S. enterica subspecies enterica serovar Typhimurium (S. typhimurium)] GGBP; a Caldicellulosiruptor sp. (e.g., C. saccharolyticus, C. acetigenus, C. bescii, C. changbaiensis, C. hydrothermalis, Caldicellulosiruptor hydrother, C. kristjanssonii, C. kronotskyensis, C. lactoaceticus, C. owensensis, or C. obsidiansis) GGBP; a Paenibacillus sp. (e.g., P. agarexedens, P. agaridevorans, P. alginolyticus, P. alkaliterrae, P. alvei, P. amylolyticus, P. anaericanus, P. antarcticus, P. assamensis, P. azoreducens, P. azotofixans, P. barcinonensis, P. borealis, P. brasilensis, P. brassicae, P. campinasensis, P. chinjuensis, P. chitinolyticus, P. chondroitinus, P. cineris, P. cookii, P. curdlanolyticus, P. daejeonensis, P. dendritiformis, P. durum, P. ehimensis, P. elgii, P. favisporus, P. glucanolyticus, P. glycanilyticus, P. gordonae, P. graminis, P. granivorans, P. hodogayensis, P. illinoisensis, P. jamilae, P. kobensis, P. koleovorans, P. koreensis, P. kribbensis, P. lactis, P. larvae, P. lautus, P. lentimorbus, P. macerans, P. macquariensis, P. massiliensis, P. mendelii, P. motobuensis, P. naphthalenovorans, P. nematophilus, P. odorifer, P. pabuli, P. peoriae, P. phoenicis, P. phyllosphaerae, P. polymyxa, P. popilliae, P. pulvifaciens, P. rhizosphaerae, P. sanguinis, P. stellifer, P. terrae, P. thiaminolyticus, P. timonensis, P. tylopili, P. turicensis, P. validus, P. vortex, P. vulneris, P. wynnii, P. xylanilyticus) GGBP; a Butyrivibrio sp. (e.g., B. proteoclasticus, B. crossotus, B. fibrisolvens, or B. hungatei) GGBP; a Roseburia sp. (e.g., R. intestinalis, R. faecis, R. hominis, or R. inulinivorans) GGBP; a Faecalibacterium sp. (e.g., F. prausnitzii) GGBP; an Erysipelothrix sp. (e.g., E. rhusiopathiae, E. inopinata, or E. tonsillarum) GGBP; or an Eubacterium sp. (e.g., E. rectale, E. acidaminophilum, E. nodatum, E. oxidoreducens, or E. foedans) GGBP.

In various embodiments, a biosensor comprises a GGBP that is or is a mutant of: an Escherichia coli GGBP (ecGGBP; SEQ ID NO: 1), a Thermoanaerobacter thermosaccharolyticum GGBP (ttGGBP; SEQ ID NO: 2), a Salmonella typhimurium GGBP (stGGBP; SEQ ID NO: 3), a Caldicellulosiruptor hydrothermalis GGBP (chyGGBP; SEQ ID NO: 4), a Caldicellulosiruptor obsidiansis GGBP (cobGGBP; SEQ ID NO: 5), a Paenibacillus sp. GGBP (pspGGBP; SEQ ID NO: 6); a Clostridium saccharolyticum GGBP (csaGGBP; SEQ ID NO: 7); a Clostridium autoethanogenum GGBP (cauGGBP; SEQ ID NO: 12); a Clostridium ljungdahlii GGBP (cljGGBP; SEQ ID NO: 11); a Butyrivibrio proteoclasticus GGBP (bprGGBP; SEQ ID NO: 8); a Roseburia intestinalis GGBP (rinGGBP_A; SEQ ID NO: 9 or rinGGBP_B; SEQ ID NO: 13); a Faecalibacterium prausnitzii GGBP (fprGGBP; SEQ ID NO: 10); a Erysipelothrix rhusiopathiae GGBP (erhGGBP; SEQ ID NO: 14); or a Eubacterium rectale GGBP (ereGGBP; SEQ ID NO: 15). In some embodiments, the GGBP comprises an amino acid sequence that is between 75% and 10% identical (e.g., between 25% and 50% identical) to the amino acid sequence of ecGGBP (SEQ ID NO: 1 or 17), ttGGBP (SEQ ID NO: 2 or 18), stGGBP (SEQ ID NO: 3 or 19), chyGGBP (SEQ ID NO: 4 or 20), pspGGBP (SEQ ID NO: 6 or 22); csaGGBP (SEQ ID NO: 7 or 23); bprGGBP (SEQ ID NO: 8 or 24); rinGGBP_A (SEQ ID NO: 9 or 25); rinGGBP_B (SEQ ID NO: 13 or 29); fprGGBP (SEQ ID NO: 10 or 26); cljGGBP (SEQ ID NO: 11 or 27); cauGGBP (SEQ ID NO: 12 or 28); erhGGBP (SEQ ID NO: 14 or 30); and/or ereGGBP (SEQ ID NO: 15 or 31).

Aspects of the present subject matter include a GGBP that is or is a mutant of a protein listed in Table 1, e.g., the protein numbered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138 in Table 1.

With regard to a defined polypeptide, % identity figures higher or lower than those provided herein will encompass various embodiments. Thus, where applicable, in light of a minimum % identity figure, a polypeptide may comprises an amino acid sequence which is at least 60%, 65%, 70%, 75%, 76%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the reference SEQ ID NO or to each of the reference SEQ ID NOs. Where applicable, in light of a maximum % identity to a reference sequence, a polypeptide may comprise an amino acid sequence which is less than 75%, 70%, 65%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, or 15% identical to the reference SEQ ID NO or to each of the reference SEQ ID NOs. In certain embodiments, a polypeptide comprises amino acids in a sequence that is preferably at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% and less than about 75%, 70%, 65%, 60%, 55%, 50%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, or 30% identical to the reference SEQ ID NO or to each of the reference SEQ ID NOs. Non-limiting examples of reference proteins and amino acid sequences disclosed herein include:

- (i) a glucose-galactose binding protein from Escherichia coli (ecGGBP; genome, NC_002695; protein, WP_032329053, SEQ ID NO: 1);
- (ii) a glucose-galactose binding protein from Thermoanaerobacterium thermosaccharolyticum (ttGGBP; genome, NC_014410; protein, YP_003852930.1, SEQ ID NO: 2);
- (iii) a glucose-galactose binding protein from Salmonella typhimurium (stGGBP; genome, NC_003197; protein, WP_001036943, SEQ ID NO: 3);
- (iv) a glucose-galactose binding protein from Caldicellulosiruptor hydrothermalis (chyGGBP; genome, NC_014652; protein identifier, YP_003991244.1, SEQ ID NO: 4);
- (v) a glucose-galactose binding protein from Caldicellulosiruptor obsidiansis (cobGGBP; genome, NC_014392; protein, YP_003839461.1, SEQ ID NO: 5);
- (vi) a glucose-galactose binding protein from Paenibacillus sp. (pspGGBP; genome, NC_013406; protein, YP_003243743.1, SEQ ID NO: 6);
- (vii) a glucose-galactose binding protein from Clostridium saccharolyticum (csaGGBP; genome, NC_014376; protein, YP_003822565.1, SEQ ID NO: 7);
- (viii) a glucose-galactose binding protein from Butyrivibrio proteoclasticus (bprGGBP; genome, NC_014387; protein, YP_003830205.1, SEQ ID NO: 8);
- (ix) a glucose-galactose binding protein from Roseburia intestinalis (rinGGBP_A; genome, NC_021012; protein, YP_007778116.1, SEQ ID NO: 9);
- (x) a glucose-galactose binding protein from Faecalibacterium prausnitzii (fprGGBP; genome, NC_021020; protein, YP_007799070.1, SEQ ID NO: 10);
- (xi) a glucose-galactose binding protein from Clostridium ljungdahlii (cljGGBP; genome, NC_014328; protein, CLJU_c08950, SEQ ID NO: 11);
- (xii) a glucose-galactose binding protein from Clostridium autoethanogenum (cauGGBP; genome, NC_022592; protein, CAETHG_2989, SEQ ID NO: 12);
- (xiii) a glucose-galactose binding protein from Roseburia intestinalis (rinGGBP_B; genome, NC_021012; protein, YP_007778124.1, SEQ ID NO: 13);
- (xiv) a glucose-galactose binding protein from Erysipelothrix rhusiopathiae (erhGGBP; genome, NC_015601; protein, YP_004561181.1, SEQ ID NO: 14); and
- (xv) a glucose-galactose binding protein from Eubacterium rectale (ereGGBP; genome, NC_012781; protein, YP_002936409.1, SEQ ID NO: 15).

The GGBPs disclosed herein may optionally be fused (e.g., at their N-terminal and/or C-terminal ends) to a motif comprising a stretch of amino acids that facilitates the isolation or other manipulation such as conjugation to a moiety or immobilization on a substrate such as a plastic, a cellulose product such as paper, polymer, metal, noble metal, semi-conductor, or quantum dot (e.g., a fluorescent quantum dot). A non-limiting example of such a stretch of amino acids has the sequence: GGSHHHHHH (SEQ ID NO: 104). This motif is not required for and is not believed to influence or affect ligand-binding activity or signal transduction. For example, each of SEQ ID NOs: 32-103 (and the non-limiting examples of otherproteins used in the experiments disclosed herein) comprises this motif (SEQ ID NO: 104). Alternatively or in addition, a ligand-binding protein may be fused to a heterologous polypeptide or “added amino acids” that facilitates the attachment therof to a surface, such as the surface of a device.

In some embodiments, a polypeptide comprises 1, 2, 3, 4, 5, or more substitutions or deletions of a cysteine compared to the naturally occurring counterpart of the polypeptide (i.e., 1, 2, 3, 4, 5, or more native cysteines have been removed), e.g., 1, 2, 3, 4, 5, or more cysteine to alanine substitutions compared to the naturally occurring counterpart of the polypeptide. In some embodiments, all of cysteines of a polypeptide have been deleted and/or substituted compared to its natural counterpart.

In embodiments, the amino acid sequence of a protein comprises no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mutations compared to its naturally occurring counterpart. In some embodiments, less than 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 of the mutations is a deletion or insertion of 1, 2, 3, 4, or 5 or no more than 1, 2, 3, 4, or 5 amino acids. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or more of the mutations is a substitution mutation. In certain embodiments, every mutation to a protein compared to its naturally occurring counterpart is a substitution mutation. In various embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more or all of the mutations to a protein compared to its naturally occurring counterpart is a conservative substitution mutation.

In various embodiments, a polypeptide does not have any insertion or deletion compared to its natural counterpart, other than (optionally) the removal of the signal peptide and/or the fusion of compounds such as another polypeptide at the N-terminus or C-terminus thereof.

Ligand-Binding Proteins Comprising a Primary Complementary Surface (PCS)

The following BLAST parameters are used to identify sequence homologues of GGBP: (1) Expect threshold is 10.0; (2) Gap cost is Existence:11 and Extension:1; (3) The Matrix employed is BLOSUM62; (4) The filter for low complexity regions is “on.” Such an alignment may be generated using the ProteinHunter program. The ProteinHunter package always executes BLAST searches, with the following command

“blastall -p blastp -m 8 -b 50000 -d %s -i <INPUT FILE> -o <OUTPUT FILE>”

where <INPUT FILE> and <OUTPUT FILE> specify the input and output files, respectively for a given calculation. This command executes the BLAST alignment program for protein sequences with default parameters, intrinsically set by the program. The BLAST program version is 2.2.24.

Sequence homologues of GGBP identified using BLAST may be aligned with ecGGBP using ClustalW to identify homologues that share a PCS with ecGGBP as discussed below.

Aspects of the present subject matter provide ligand-binding proteins that share a PCS with a GGBP disclosed herein. In embodiments, the PCS comprises at least about 5, 6, 7, 8, 9, or 10 amino acid positions used to identify a glucose-galactose binding protein. For example, the PCS of ecGGBP may comprise positions 14, 16, 91, 152, 154, 158, 183, 211, 236, and 256, wherein each position is counted as in SEQ ID NO: 17. In various embodiments, a protein shares a PCS with ecGGBP if the amino acid sequence of the protein has

(i) D or N at the position that aligns with position 14 of ecGGBP;

(ii) F, Y, or W at the position that aligns with position 16 of ecGGBP;

(iii) N or D at the position that aligns with position 91 of ecGGBP;

(iv) H, N, or Q at the position that aligns with position 152 of ecGGBP;

(v) D or N at the position that aligns with position 154 of ecGGBP;

(vi) R at the position that aligns with position 158 of ecGGBP;

(vii) W, F, or Y at the position that aligns with position 183 of ecGGBP;

(viii) N or D at the position that aligns with position 211 of ecGGBP;

(ix) D or N at the position that aligns with position 236 of ecGGBP; and

(x) N or D at the position that aligns with position 256 of ecGGBP,

wherein the alignment between ecGGBP (SEQ ID NO: 17) and the protein is constructed using the ClustalW alignment program.

The ProteinHunter package always executes multiple sequence alignments with the following command

“clustalw -infile=<INPUT FILE> -outfile=<OUTPUTFILE> -align -quiet”

This command executes the CLUSTALW multi-sequence alignment program for protein sequences. There are no user-specified parameter settings that alter the alignment behavior of the program. The CLUSTALW program version is 2.1.

For convenience and depending on context, a position that aligns with a stated position of ecGGBP may be referred to herein as “equivalent” to the stated position.

Exemplary Ligand-Binding Proteins

Various biosensors provided herein comprise glucose-galactose binding proteins, such as glucose-galactose binding proteins that have altered amino acid sequences compared to their naturally occurring counterparts. In embodiments, such proteins are conjugated to reporter groups. ecGGBP is a non-limiting reference protein respect to glucose-galactose binding proteins. An alignment of ecGGBP with other polypeptides is provided in FIG. 3.

In various embodiments, a polypeptide of the present disclosure comprises

- (a) an amino acid that is preferably (i) at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%, and (ii) less than about 75%, 70%, 65%, 60%, 55%, 50%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, or 35% identical to ecGGBP;
- (b) a cysteine substitution (compared to its naturally occurring counterpart) within a stretch of at least 5, 10, or 20 amino acids having at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 183 of ecGGBP (e.g., a tryptophan to cysteine substitution);
- (c) a stretch of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids having at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 14 of ecGGBP;
- (d) a stretch of amino acids in the sequence
  - a. IYKX₁DDX₂FM (where X₁is any amino acid, or where X₁is Y, F, Q, or K; and where X₂is any amino acid, or where X₂is N or T) (SEQ ID NO: 182),
  - b. YKX₁DDX₂FM (where X₁is any amino acid, or where X₁is Y, F, Q, or K; and where X₂is any amino acid, or where X₂is N or T) (SEQ ID NO: 183),
  - c. YKX₁DDX₂F (where X₁is any amino acid, or where X₁is Y, F, Q, or K; and where X₂is any amino acid, or where X₂is N or T) (SEQ ID NO: 184),
  - d. KX₁DDX₂F (where X₁is any amino acid, or where X₁is Y, F, Q, or K; and where X₂is any amino acid, or where X₂is N or T) (SEQ ID NO: 185),
  - e. YKXDD (where X is any amino acid, or where X is Y, F, Q, or K) (SEQ ID NO: 186),
  - f. KXDD (where X is any amino acid, or where X is Y, F, Q, or K) (SEQ ID NO: 187),
  - g. DDXF (where X is any amino acid, or where X is N or T) (SEQ ID NO: 188),
  - h. DX₁FMX₂X₃VR (where X₁is any amino acid, or where X₁is N or T; where X₂is any amino acid, or where X₂is S, T, G; and where X₃is any amino acid, or where X₃is V, G, E, or L) (SEQ ID NO: 189),
  - i. DX₁FMX₂X₃V (where X₁is any amino acid, or where X₁is N or T; where X₂is any amino acid, or where X₂is S, T, G; and where X₃is any amino acid, or where X₃is V, G, E, or L) (SEQ ID NO: 190),
  - j. DXFM (where X is any amino acid, or where X is N or T) (SEQ ID NO: 191),
  - k. IYKX₁DNX₂FM (where X₁is any amino acid, or where X₁is Y, F, Q, or K; and where X₂is any amino acid, or where X₂is N or T) (SEQ ID NO: 192),
  - l. YKX₁DNX₂FM (where X₁is any amino acid, or where X₁is Y, F, Q, or K; and where X₂is any amino acid, or where X₂is N or T) (SEQ ID NO: 193),
  - m. YKX₁DNX₂F (where X₁is any amino acid, or where X₁is Y, F, Q, or K; and where X₂is any amino acid, or where X₂is N or T) (SEQ ID NO: 194),
  - n. KX₁DNX₂F (where X₁is any amino acid, or where X₁is Y, F, Q, or K; and where X₂is any amino acid, or where X₂is N or T) (SEQ ID NO: 195),
  - o. YKXDN (where X is any amino acid, or where X is Y, F, Q, or K) (SEQ ID NO: 196),
  - p. KXDN (where X is any amino acid, or where X is Y, F, Q, or K) (SEQ ID NO: 197),
  - q. DNXF (where X is any amino acid, or where X is N or T) (SEQ ID NO: 198),
  - r. NX₁FMX₂X₃VR (where X₁is any amino acid, or where X₁is N or T; where X₂is any amino acid, or where X₂is S, T, G; and where X₃is any amino acid, or where X₃is V, G, E, or L) (SEQ ID NO: 199),
  - s. NX₁FMX₂X₃V (where X₁is any amino acid, or where X₁is N or T; where X₂is any amino acid, or where X₂is S, T, G; and where X₃is any amino acid, or where X₃is V, G, E, or L) (SEQ ID NO: 200),
  - t. NXFM (where X is any amino acid, or where X is N or T) (SEQ ID NO: 201);
- (e) a stretch of amino acids in the sequence
  - a. IYKX₁DDX₂FM (where X₁is any amino acid, or where X₁is Y, F, Q, or K; and where X₂is any amino acid, or where X₂is N or T) (SEQ ID NO: 182),
  - b. YKX₁DDX₂FM (where X₁is any amino acid, or where X₁is Y, F, Q, or K; and where X₂is any amino acid, or where X₂is N or T) (SEQ ID NO: 183),
  - c. YKX₁DDX₂F (where X₁is any amino acid, or where X₁is Y, F, Q, or K; and where X₂is any amino acid, or where X₂is N or T) (SEQ ID NO: 184),
  - d. KX₁DDX₂F (where X₁is any amino acid, or where X₁is Y, F, Q, or K; and where X₂is any amino acid, or where X₂is N or T) (SEQ ID NO: 185),
  - e. YKXDD (where X is any amino acid, or where X is Y, F, Q, or K) (SEQ ID NO: 186),
  - f. KXDD (where X is any amino acid, or where X is Y, F, Q, or K) (SEQ ID NO: 187),
  - g. DDXF (where X is any amino acid, or where X is N or T) (SEQ ID NO: 188),
  - h. DX₁FMX₂X₃VR (where X₁is any amino acid, or where X₁is N or T; where X₂is any amino acid, or where X₂is S, T, G; and where X₃is any amino acid, or where X₃is V, G, E, or L) (SEQ ID NO: 189),
  - i. DX₁FMX₂X₃V (where X₁is any amino acid, or where X₁is N or T; where X₂is any amino acid, or where X₂is S, T, G; and where X₃is any amino acid, or where X₃is V, G, E, or L) (SEQ ID NO: 190),
  - j. DXFM (where X is any amino acid, or where X is N or T) (SEQ ID NO: 191),
  - k. IYKX₁DNX₂FM (where X₁is any amino acid, or where X₁is Y, F, Q, or K; and where X₂is any amino acid, or where X₂is N or T) (SEQ ID NO: 192),
  - l. YKX₁DNX₂FM (where X₁is any amino acid, or where X₁is Y, F, Q, or K; and where X₂is any amino acid, or where X₂is N or T) (SEQ ID NO: 193),
  - m. YKX₁DNX₂F (where X₁is any amino acid, or where X₁is Y, F, Q, or K; and where X₂is any amino acid, or where X₂is N or T) (SEQ ID NO: 194),
  - n. KX₁DNX₂F (where X₁is any amino acid, or where X₁is Y, F, Q, or K; and where X₂is any amino acid, or where X₂is N or T) (SEQ ID NO: 195),
  - o. YKXDN (where X is any amino acid, or where X is Y, F, Q, or K) (SEQ ID NO: 196),
  - p. KXDN (where X is any amino acid, or where X is Y, F, Q, or K) (SEQ ID NO: 197),
  - q. DNXF (where X is any amino acid, or where X is N or T) (SEQ ID NO: 198),
  - r. NX₁FMX₂X₃VR (where X₁is any amino acid, or where X₁is N or T; where X₂is any amino acid, or where X₂is S, T, G; and where X₃is any amino acid, or where X₃is V, G, E, or L) (SEQ ID NO: 199),
  - s. NX₁FMX₂X₃V (where X₁is any amino acid, or where X₁is N or T; where X₂is any amino acid, or where X₂is S, T, G; and where X₃is any amino acid, or where X₃is V, G, E, or L) (SEQ ID NO: 200), or
  - t. NXFM (where X is any amino acid, or where X is N or T) (SEQ ID NO: 201),
- within about 50, 45, 40, 35, 20, or 15 amino acids of the N-terminus of the polypeptide, including or not including the amino acids of the signal peptide (also referred to herein as the leader peptide) of its natural counterpart;
- (f) a stretch of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids having at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 91 of ecGGBP
- (g) a stretch of amino acids having the sequence

(SEQ ID NO: 202)

a. PVVFFNKEP,

- - b. PVVFXNKEP (where X is any amino acid, or where X is F, L, V, or I) (SEQ ID NO: 203),
  - c. PVVFFNXEP (where X is any amino acid, or where X is K, R, E, or G) (SEQ ID NO: 204),
  - d. PVVFX₁NX₂EP (where X₁is any amino acid, or where X₁is F, L, V, or I; and where X₂is any amino acid, or where X₂is K, R, E, or G) (SEQ ID NO: 205),
  - e. VVFX₁NX₂EP (where X₁is any amino acid, or where X₁is F, L, V, or I; and where X₂is any amino acid, or where X₂is K, R, E, or G) (SEQ ID NO: 206),
  - f. VFX₁NX₂EP (where X₁is any amino acid, or where X₁is F, L, V, or I; and where X₂is any amino acid, or where X₂is K, R, E, or G) (SEQ ID NO: 207),
  - g. PVVFX₁NX₂E (where X₁is any amino acid, or where X₁is F, L, V, or I; and where X₂is any amino acid, or where X₂is K, R, E, or G) (SEQ ID NO: 208),
  - h. PVVFXN (where X is any amino acid, or where X is F, L, V, or I) (SEQ ID NO: 209),
  - i. PX₁VFX₂N (where X₁is any amino acid, or where X₁is V, I, L, or T; and where X₂is any amino acid, or where X₂is F, L, V, or I) (SEQ ID NO: 210),
  - j. PVX₁FX₂N (where X₁is any amino acid, or where X₁is V, I, L, or T; and where X₂is any amino acid, or where X₂is F, L, V, or I) (SEQ ID NO: 211),
  - k. FX₁NX₂EP (where X₁is any amino acid, or where X₁is F, L, V, or I; and where X₂is any amino acid, or where X₂is K, R, E, or G) (SEQ ID NO: 212), or
  - l. FX₁NX₂E (where X₁is any amino acid, or where X₁is F, L, V, or I; and where X₂is any amino acid, or where X₂is K, R, E, or G) (SEQ ID NO: 213);
- (h) a stretch of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids having at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 150, 155, or 160 of ecGGBP;
- (i) a stretch of amino acids having the sequence

(SEQ ID NO: 214)

a. PGHPDAEART,

- - b. PGHX₁DAX₂X₃RT (where X₁is any amino acid, or where X₁is P, Q, V, I, or E; where X₂is any amino acid, or where X₂is E, I, K, or Q; and where X₃is any amino acid, or where X₃is A, L, V, K, Q, or Y) (SEQ ID NO: 215),
  - c. GHX₁DAX₂X₃RT (where X₁is any amino acid, or where X₁is P, Q, V, I, or E; where X₂is any amino acid, or where X₂is E, I, K, or Q; and where X₃is any amino acid, or where X₃is A, L, V, K, Q, or Y) (SEQ ID NO: 216),
  - d. HX₁DAX₂X₃RT (where X₁is any amino acid, or where X₁is P, Q, V, I, or E; where X₂is any amino acid, or where X₂is E, I, K, or Q; and where X₃is any amino acid, or where X₃is A, L, V, K, Q, or Y) (SEQ ID NO: 217),
  - e. DAX₁X₂RT (where X₁is any amino acid, or where X₁is E, I, K, or Q;

and where X₂is any amino acid, or where X₂is A, L, V, K, Q, or Y) (SEQ ID NO: 218),

- - f. PGHX₁DAX₂X₃R (where X₁is any amino acid, or where X₁is P, Q, V, I, or E; where X₂is any amino acid, or where X₂is E, I, K, or Q; and where X₃is any amino acid, or where X₃is A, L, V, K, Q, or Y) (SEQ ID NO: 219),
  - g. PGHXDA (where X is any amino acid, or where X is P, Q, V, I, or E) (SEQ ID NO: 220),
  - h. PGHXD (where X is any amino acid, or where X is P, Q, V, I, or E) (SEQ ID NO: 221),
  - i. PGNPDAEART (SEQ ID NO: 222),
  - j. PGNX₁DAX₂X₃RT (where X₁is any amino acid, or where X₁is P, Q, V, I, or E; where X₂is any amino acid, or where X₂is E, I, K, or Q; and where X₃is any amino acid, or where X₃is A, L, V, K, Q, or Y) (SEQ ID NO: 223),
  - k. GNX₁DAX₂X₃RT (where X₁is any amino acid, or where X₁is P, Q, V, I, or E; where X₂is any amino acid, or where X₂is E, I, K, or Q; and where X₃is any amino acid, or where X₃is A, L, V, K, Q, or Y) (SEQ ID NO: 224),
  - l. NX₁DAX₂X₃RT (where X₁is any amino acid, or where X₁is P, Q, V, I, or E; where X₂is any amino acid, or where X₂is E, I, K, or Q; and where X₃is any amino acid, or where X₃is A, L, V, K, Q, or Y) (SEQ ID NO: 225),
  - m. PGNX₁DAX₂X₃R (where X₁is any amino acid, or where X₁is P, Q, V, I, or E; where X₂is any amino acid, or where X₂is E, I, K, or Q; and where X₃is any amino acid, or where X₃is A, L, V, K, Q, or Y) (SEQ ID NO: 226),
  - n. PGNXDA (where X is any amino acid, or where X is P, Q, V, I, or E) (SEQ ID NO: 227), or
  - o. PGNXD (where X is any amino acid, or where X is P, Q, V, I, or E) (SEQ ID NO: 228);
- (j) a stretch of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids having at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 183 of ecGGBP;
- (k) a stretch of amino acids having the sequence

(SEQ ID NO: 229)

a. DTAMWD,

(SEQ ID NO: 230)

b. DTAMCD,

(SEQ ID NO: 231)

c. DTAMW,

(SEQ ID NO: 232)

d. DTAMC,

(SEQ ID NO: 233)

e. TAMWD,

(SEQ ID NO: 234)

f. TAMCD,

- - g. AX₁WX₂X₃(where X₁is any amino acid, or where X₁is M or N; where X₂is any amino acid, or where X₂is D or N; and where X₃is any amino acid, or where X₃is T or R) (SEQ ID NO: 235), or
  - h. AX₁CX₂X₃(where X₁is any amino acid, or where X₁is M or N; where X₂is any amino acid, or where X₂is D or N; and where X₃is any amino acid, or where X₃is T or R) (SEQ ID NO: 236);
- (l) a stretch of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids having at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 210 of ecGGBP;
- (m) a stretch of amino acids having the sequence

(SEQ ID NO: 237)

a. IEVVIANND,

(SEQ ID NO: 238)

b. EVVIANND,

(SEQ ID NO: 239)

c. IEVVIANN,

(SEQ ID NO: 240)

d. EVVIANN,

- - e. IEX₁VX₂X₃NND (where X₁is any amino acid, or where X₁is V, A, L, or C; where X₂is any amino acid, or where X₂is I, F, or L; and where X₃is any amino acid, or where X₃is A or C) (SEQ ID NO: 241),
  - f. IEX₁VX₂X₃NN (where X₁is any amino acid, or where X₁is V, A, L, or C; where X₂is any amino acid, or where X₂is I, F, or L; and where X₃is any amino acid, or where X₃is A or C) (SEQ ID NO: 242),
  - g. EX₁VX₂X₃NND (where X₁is any amino acid, or where X₁is V, A, L, or C; where X₂is any amino acid, or where X₂is I, F, or L; and where X₃is any amino acid, or where X₃is A or C) (SEQ ID NO: 243), or
  - h. EXiVX₂X₃NN (where X₁is any amino acid, or where X₁is V, A, L, or C; where X₂is any amino acid, or where X₂is I, F, or L; and where X₃is any amino acid, or where X₃is A or C) (SEQ ID NO: 244);
- (n) a stretch of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids having at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 235 or 240 of ecGGBP;
- (o) a stretch of amino acids having the sequence

(SEQ ID NO: 245)

a. PVFGVDA,

(SEQ ID NO: 246)

b. VFGVDA,

(SEQ ID NO: 247)

c. PVFGVD,

(SEQ ID NO: 248)

d. FGVDA,

(SEQ ID NO: 249)

e. PVXGVDA,

(where X is any amino acid or where X is F, V,

or I)

- - f. VXGVDA (where X is any amino acid or where X is F, V, or I) (SEQ ID NO: 250),
  - g. PVXGVD (where X is any amino acid or where X is F, V, or I) (SEQ ID NO: 251), or
  - h. VXGVD (where X is any amino acid or where X is F, V, or I) (SEQ ID NO: 252);
- (p) a stretch of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids having at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 255 or 260 of ecGGBP
- (q) a stretch of amino acids having the sequence GTVLNDA (SEQ ID NO: 253), GTVLND (SEQ ID NO: 254), GTVLN (SEQ ID NO: 255), or TVLND (SEQ ID NO: 256);
- (r) no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 15 deleted or inserted amino acids compared to ecGGBP, not including added amino acids added to the N-terminus or C-terminus of the polypeptide compared to its natural counterpart, and including or not including the signal peptide of the natural counterpart of the polypeptide;
- (s) at least 8, 9, 10, or 11, or exactly 8, 9, 10, or 11 α-helices; and/or
- (t) at least 9, 10, 11, or 12 β-sheets or exactly 9, 10, 11, or 12 β-sheets.

In embodiments, two or more or each of features (c)-(q) above occurs in the polypeptide in the order listed above as the amino acid sequence of the polypeptide is viewed or read from the N-terminus to the C-terminus (with additional features and/or amino acid sequences therebetween). For example, the polypeptide may have an N-terminus, followed by feature (c), (d), or (e), followed by feature (f) or (g), followed by feature (h) or (i), followed by feature (j) or (k), followed by feature (l) or (m), followed by feature (n) or (o), followed by feature (p) or (q), followed by the C-terminus.

As used herein when referring to the order of features in an amino acid read from the N terminus to the C-terminus, a first feature is “followed by” a second feature when the second feature occurs after the first feature in the amino acid sequence. The words “followed by” do not require that the second feature immediately follow or be close to the first feature. For example, the N-terminus is followed by the C-terminus.

The features listed above are not limiting and may be combined with any other relevant features disclosed herein, including those listed below.

In some embodiments the polypeptide has the following sequence:

ZZZZ!GVXIY K%DDXFMXXV RXAXXXXXXX XXXX#XXZZD

XQNXQXXQN# X!DXXXXKXX XX$X1NXVDX XAAGXXI#KA

XXXNXPVVFX NXEPXXXX$X XXDKXYYVGX XXX#SGXXXG

#XXXXXWXXX XXXDXNX#GX x#%V$xxG#P GHXDAxxRTX

%X!XXXXXXG IXXXXLXXDX AXWDXXXXXX KMXXXLXXZX

X#XIEXVXXN NDXMA$GA!E ALKXZZZZXX XXXXPVXGVD

AXXXXXXXXX XGX$XGTVLN DAXXQXKAXX XXAXXLXXGZ

XXXX#XXXXX IZ#XKX!X!X YXX!XKDNXX #ZZZZZZZZ

wherein each

Z is, individually, any amino acid or is absent,

X is, individually, any amino acid,

! is, individually, I or V,

S is, individually, L or M,

% is, individually, F or Y, and

# is, individually, N, D, Q, or E.

In a non-limiting example, the glucose-galactose binding polypeptide comprises an N-terminal domain and a C-terminal domain connected by a flexible hinge, with the ligand-binding site (the ligand binding domain) located in the cleft between the N-terminal and the C-terminal domain.

In some embodiments, the glucose-galactose binding polypeptide comprises, from the N-terminus to the C-terminus, a first β-sheet (β1), followed by a first α-helix (α1), followed by a second β-sheet (β2), followed by a second α-helix (α2), followed by a third β-sheet (β3), followed by a third α-helix (α3), followed by a fourth β-sheet (β4), followed by a fourth α-helix (α4), followed by a fifth β-sheet (β5), followed by a first inter-domain hinge segment (h1), followed by a fifth α-helix (α5), followed by a sixth β-sheet (β6), followed by a sixth α-helix (α6), followed by a seventh β-sheet (β7), followed by a seventh α-helix (α7), followed by an eighth β-sheet (β8), followed by an eighth α-helix (α8), followed by a ninth β-sheet (β9), followed by a ninth α-helix (α9), followed by a tenth β-sheet (α10), followed by a second inter-domain hinge segment (h2), followed by a tenth α-helix (α10), followed by a third inter-domain hinge segment (h3), followed by an eleventh β-sheet (β11). In some embodiments, the polypeptide comprises (i) 1, 2, or 3 amino acid substitutions between β1 and α1; (ii) 1, 2, or 3 amino acid substitutions between β2 and α2; (iii) 1, 2, or 3 amino acid substitutions between the β3 and α3; (iv) 1, 2, or 3 amino acid substitutions between the β4 and α4; (v) 1, 2, or 3 amino acid substitutions in β6; (vi) 1, 2, or 3 amino acid substitutions in α6; (vii) 1, 2, or 3 amino acid substitutions between the β7 and α7; (viii) 1, 2, or 3, amino acid substitutions in α8; and/or (ix) 1, 2, or 3 amino acid substitutions between the β9 and α9. In some embodiments, the substitutions are conservative substitutions. In various embodiments, the polypeptide comprises a cysteine substitution at β7, α7, or between β7 and α7.

The glucose-galactose binding polypeptide may further comprise 1, 2, or more Ca²⁺ binding sites.

In various embodiments, the C_α root-mean-square deviation (RMSD) between the backbone of the glucose-galactose binding polypeptide and ecGGBP, ttGGBP, stGGBP, chyGGBP, cobGGBP, pspGGBP, csaGGBP, bprGGBP, rinGGBP_A, rinGGBP_B, fprGGBP, cljGGBP, cauGGBP, erhGGBP, ereGGBP, and/or chyGGBP is, e.g., between about 0.1-3 Å, 0.5-1 Å, 0.5-1.5 Å, or 0.5-2 Å, or less than about 0.1 Å, 0.2 Å, 0.3 Å, 0.4 Å, 0.5 Å, 0.6 Å, 0.7 Å, 0.8 Å, 0.9 Å, 1.0 Å, 1.5 Å, 1.6 Å, 1.7 Å, 1.8 Å, 1.9 Å, 2.0 Å, 2.5 Å, or 3 Å. In some embodiments, the C_α RMSD between the N-terminal domain (i.e., the portion of the protein at the N-terminal side of the binding domain hinge) backbone of the glucose-galactose binding polypeptide and the corresponding domain of ecGGBP, ttGGBP, stGGBP, chyGGBP, cobGGBP, and/or pspGGBP is, e.g., between about 0.1-3 Å, 0.5-1 Å, 0.5-1.5 Å, or 0.5-2 Å, or less than about 0.1 Å, 0.2 Å, 0.3 Å, 0.4 Å, 0.5 Å, 0.6 Å, 0.7 Å, 0.8 Å, 0.9 Å, 1.0 Å, 1.5 Å, 1.6 Å, 1.7 Å, 1.8 Å, 1.9 Å, 2.0 Å, 2.5 Å, or 3 Å. In certain embodiments, the C_α RMSD between the C-terminal domain (i.e., the portion of the protein at the C-terminal side of the binding domain hinge) backbone of the glucose-galactose binding polypeptide and the corresponding domain of ecGGBP, ttGGBP, stGGBP, chyGGBP, cobGGBP, pspGGBP, csaGGBP, bprGGBP, rinGGBP_A, rinGGBP_B, fprGGBP, cljGGBP, cauGGBP, erhGGBP, ereGGBP, and/or chyGGBP is, e.g., between about 0.1-3 Å, 0.5-1 Å, 0.5-1.5 Å, or 0.5-2 Å, or less than about 0.1 Å, 0.2 Å, 0.3 Å, 0.4 Å, 0.5 Å, 0.6 Å, 0.7 Å, 0.8 Å, 0.9 Å, 1.0 Å, 1.5 Å, 1.6 Å, 1.7 Å, 1.8 Å, 1.9 Å, 2.0 Å, 2.5 Å, or 3 Å. Non-limiting considerations relating to the sequence and structural differences between homologous proteins are discussed in Chothia and Lesk (1986) The EMBO Journal, 5(4):823-826, the entire content of which is incorporated herein by reference.

Non-limiting examples of glucose-galactose binding polypeptides that are useful in biosensors provided herein include ecGGBP, ttGGBP, stGGBP, chyGGBP, cobGGBP, pspGGBP, csaGGBP, bprGGBP, rinGGBP_A, rinGGBP_B, fprGGBP, cljGGBP, cauGGBP, erhGGBP, ereGGBP, and chyGGBP. In embodiments, a biosensor comprises a modified ecGGBP, ttGGBP, stGGBP, chyGGBP, cobGGBP, pspGGBP, csaGGBP, bprGGBP, rinGGBP_A, rinGGBP_B, fprGGBP, cljGGBP, cauGGBP, erhGGBP, ereGGBP, or chyGGBP polypeptide having an amino acid substitution compared to its naturally occurring counterpart, such that the polypeptide has a cysteine at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, or 383 or any combination of 1, 2, 3, 4, or 5 thereof, wherein the position corresponds a SEQ ID NO disclosed herein for ecGGBP, ttGGBP, stGGBP, chyGGBP, cobGGBP, pspGGBP, csaGGBP, bprGGBP, rinGGBP_A, rinGGBP_B, fprGGBP, cljGGBP, cauGGBP, erhGGBP, ereGGBP, or chyGGBP. In embodiments, the cysteine is conjugated to a reporter group.

In embodiments, a biosensor comprises a modified ecGGBP. In non-limiting examples, the modified ecGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: Y10X, D14X, N15X, F16X, P70X, N91X, K92X, S112X, S115X, E149X, H152X, P153X, D154X, A155X, R158X, M182X, W183X, N211X, D212X, D236X, L238X, D257X, P294X, and V296X, where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in ecGGBP without including the signal peptide (SEQ ID NO: 17). In some embodiments, the modified ecGGBP comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the following substitutions: Y10A, Y10C, D14C, D14A, D14Q, D14N, D14S, D14T, D14E, D14H, D14L, D14Y, D14F, F16L, F16A, F16C, N91C, N91A, K92A, K92C, E149C, E149K, E149Q, E1495, H152C, H152A, H152F, H152Q, H152N, D154C, D154A, D154N, A155C, A1555, A155H, A155L, A155F, A155Y, A155N, A155K, A155M, A155W, A155Q, R158C, R158A, R158K, M182C, M182W, W183C, N211C, N211F, N211W, N211K, N211Q, N2115, N211H, N211M, N211C, D212C, L238C, D236C, D236A, D236N, N256A, N256D, D257C, P294C, and V293C.

In various embodiments, a biosensor comprises a modified ttGGBP. In non-limiting examples, the modified ttGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: Y11X, D15X, T16X, F17X, G20X, N42X, V67X, R69X, R91X, E92X, A111X, Q148X, H151X, Q152X, A154X, N181X, W182X, D183X, D211X, T237X, T240X, L257X, N258X, D259X, A260X, and K300X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in ttGGBP with the signal peptide replaced with a methionine (SEQ ID NO: 18). In some embodiments, the modified ttGGBP comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the following substitutions: Y11C, D15A, D15E, D15N, D15C, T16S, T16N, T16C, F17C, G20A, G20C, N42C, V67C, R69P, R69C, R91K, E92C, A111C, Q148S, Q148K, Q148E, Q148C, H151Q, H151N, H151F, H151C, Q152P, Q152C, A154S, A154N, A154M, A154F, A154C, N181C, W182C, D183C, D211A, D211C, T237C, T240A, L257C, N258D, N258S, N258A, N258C, D259C, A260N, A260Q, A260R, A260K, A260W, A260F, A260Y, A260S, A260C, and K300C.

In embodiments, a biosensor comprises a modified stGGBP. In non-limiting examples, the modified stGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: Y11X, Y13X, D15X, N16X, F17X, P71X, N92X, K93X, P113X, S116X, E150X, H153X, P154X, D155X, A156X, R159X, M183X, W184X, N211X, N212X, D213X, A214X, D237X, L239X, D258X, P295X, and V297X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in stGGBP with the signal peptide replaced with a methionine (SEQ ID NO: 19). In some embodiments, the modified stGGBP comprises 1, 2 or 3 of the following mutations: Y13C, F17C, and W184C.

In embodiments, a biosensor comprises a modified chyGGBP. In non-limiting examples, the modified chyGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: F12X, D14X, T15X, F16X, R68X, N89X, R90X, A110X, S113X, E147X, H150X, Q151X, D152X, A153X, R156X, M180X, W181X, N207X, N208X, D209X, D210X, D237X, T239X, D258X, V296X, and Y298X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in chyGGBP with the signal peptide replaced with a methionine (SEQ ID NO: 20). In some embodiments, the modified chyGGBP comprises 1, 2, or 3 of the following mutations: F12C, F16C, C39A, W181C, and C206A.

In embodiments, a biosensor comprises a modified cobGGBP. In non-limiting examples, the modified cobGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: F12X, D14X, T15X, F16X, C39X, R68X, N89X, R90X, A110X, S113X, E147X, H150X, Q151X, D152X, A153X, R156X, C173X, M180X, W181X, C206X, N207X, N208X, D209X, D210X, D237X, T239X, D258X, P297X, and Q299X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in cobGGBP with the signal peptide replaced with a methionine (SEQ ID NO: 21). In some embodiments, the modified cobGGBP comprises 1, 2, or 3 of the following mutations: F12C, F16C, C39A, C173A, W181C, and C206A.

In embodiments, a biosensor comprises a modified pspGGBP. In non-limiting examples, the modified pspGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: F9X, D11X, T12X, F13X, R65X, N86X, R87X, A107X, S110X, E144X, H147X, Q148X, D149X, A150X, R153X, M177X, W178X, N204X, N205X, D206X, D207X, D234X, T236X, 255X, A294X, and K296X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in pspGGBP with the signal peptide replaced with a methionine (SEQ ID NO: 22). In some embodiments, the modified pspGGBP comprises 1, 2, or 3 of the following mutations: F9C, F13C, and W178C.

In embodiments, a biosensor comprises a modified csaGGBP. In non-limiting examples, the modified csaGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: Y14X, D16X, F18X, C62X, I72X, C82X, N93X, R94X, C113A, S118X, A121X, E152X, N155X, E156X, D157X, S158X, R161X, N185X, W186X, C211X, D241X, L243X, D262X, D290X, I292X, I297X, F299X, Q301X, and T302X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in csaGGBP with the signal peptide replaced with a methionine (SEQ ID NO: 23). In some embodiments, the modified csaGGBP comprises 1, 2, 3, 4, 5, 6, 7, or 8 of the following mutations: Y14C, F18C, C62A, C82A, C113A, W186C, and C211A.

In embodiments, a biosensor comprises a modified bprGGBP. In non-limiting examples, the modified bprGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: C8X, K12X, D14X, N15X, F16X, S72X, N93X, R94X, C112X, C116X, A118X, S121X, A153X, N156X, I157X, D158X, A159X, C179X, N186X, W187X, C211X, N212X, N213X, D214X, A215X, D241X, D243X, K251X, C289X, D290X, and V292X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in bprGGBP with the signal peptide replaced with a methionine (SEQ ID NO: 24). In some embodiments, the modified bprGGBP comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the following mutations: CBA, K12C, F16C, C112A, C116A, C179A, W187C, C211A, and C289A.

In embodiments, a biosensor comprises a modified rinGGBP_A. In non-limiting examples, the modified rinGGBP_A may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: C6X, F10X, D12X, N13X, F14X, S70X, N91X, R92X, C114X, A116X, Q118X, D151X, N154X, V155X, D156X, A157X, R160X, C177X, N184X, W185X, C210X, N211X, N212X, D213X, A214X, D240X, L242X, L250X, C288X, D289X, and V291X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in rinGGBP_A with the signal peptide replaced with a methionine (SEQ ID NO: 25). In some embodiments, the modified rinGGBP_A comprises 1, 2, 3, 4, 5, 6, 7, or 8 of the following mutations: C6A, F10C, F14C, C114A, C177A, W185C, C210A, and C288A.

In embodiments, a biosensor comprises a modified rinGGBP_B. In non-limiting examples, the modified rinGGBP_B may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: Q13X, D15X, T16X, F17X, C66X, C70A, R76X, N97X, R98X, A118X, S121X, E155X, H158X, Q159X, D160X, A161X, R164X, N188X, W189X, N215X, N216X, D217X, D218X, D244X, T246X, D265X, P301X, A303X, and C306X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in rinGGBP_B with the signal peptide replaced with a methionine (SEQ ID NO: 29). In some embodiments, the modified rinGGBP_B comprises 1, 2, 3, 4, 5, or 6 of the following mutations: Q13C, F17C, C66A, C70A, W189C, and C306A.

In embodiments, a biosensor comprises a modified fprGGBP. In non-limiting examples, the modified fprGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: C8A, F12X, D14X, N15X, F16X, T69X, N90X, R91X, C105X, C106X, A113X, S116X, C143X, D146X, N149X, I150X, D151X, A152X, R155X, N179X, W180X, C205A, N206X, N207X, D208X, A209X, D235X, L237X, N243X, D284X, and V286X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in fprGGBP with the signal peptide replaced with a methionine (SEQ ID NO: 26). In some embodiments, the modified fprGGBP comprises 1, 2, 3, 4, 5, 6, or 7 of the following mutations: C8A, F12C, F16C, C105A, C106A, C143A, W180C, and C205A.

In embodiments, a biosensor comprises a modified cljGGBP. In non-limiting examples, the modified cljGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: F11x, N13X, T14X, W15X, V67X, C77X, N88X, R89X, A109X, S112X, E142X, N145X, Q146X, D147X, A148X, R151X, M175X, W176X, C198X, N201X, N202X, D203X, D204X, D231X, T233X, D252X, D291X, and K294X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in cljGGBP with the signal peptide replaced with a methionine (SEQ ID NO: 27). In some embodiments, the modified cljGGBP comprises 1, 2, 3, 4, or 5 of the following mutations: F11C, W15C, C77A, W176C, and C198A.

In embodiments, a biosensor comprises a modified cauGGBP. In non-limiting examples, the modified cauGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: F12X, N14X, T15X, W16X, V68X, C78X, N89X, R90X, A110X, S113X, E143X, N146X, Q147X, D148X, A149X, R152X, M176X, W177X, C199X, N203X, N204X, D205X, D206X, D233X, T235X, D254X, D293X, and K295X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in cauGGBP with the signal peptide replaced with a methionine (SEQ ID NO: 28). In some embodiments, the modified cauGGBP comprises 1, 2, 3, 4, or 5 of the following mutations: F12C, W16C, C78A, W177C, and C199A.

In embodiments, a biosensor comprises a modified erhGGBP. In non-limiting examples, the modified erhGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: F13X, D15X, N16X, F17X, P76X, N97X, R98X, A119X, S122X, D153X, N156X, V157X, D158X, A159X, R162X, M187X, W188X, N214X, N215X, D216X, G217X, D243X, I245X, D264X, E312X, and V314X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in erhGGBP with the signal peptide replaced with a methionine (SEQ ID NO: 30). In some embodiments, the modified erhGGBP comprises 1, 2, or 3 of the following mutations: F13C, F17C, and W188C.

In embodiments, a biosensor comprises a modified ereGGBP. In non-limiting examples, the modified ereGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: Q13X, D15X, T16X, F17X, C29X, C65X, C69X, R75X, N96X, R97, A117X, S120X, E154X, H157X, Q158X, D159X, A160X, R163X, C183X, N187X, W188X, N214X, N215X, D216X, A217X, D243X, T245X, D264X, P301X, and E303X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in ereGGBP with the signal peptide replaced with a methionine (SEQ ID NO: 31). In some embodiments, the modified ereGGBP comprises 1, 2, 3, 4, 5, 6, 7, or 8 of the following mutations: C10A, Q13C, F17C, C29A, C65A, C69A, C183A, and W188C.

In various embodiments, the mutant glucose-galactose binding polypeptide's glucose and/or galactose disassociation constant differs by at least about 1 μM, 5 μM, 10 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 75 μM, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM (increase or decrease) compared to its naturally occurring counterpart.

The biosensors and ligand-binding proteins provided herein are robust and useful at a wide range of physical conditions, e.g., pressure, temperature, salinity, osmolality, and pH conditions. For example, biosensors and ligand-binding proteins provided herein may survive substantial periods of time after being dried or exposed to high temperatures. In some embodiments, the biosensor maintains at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more of its signal transduction activity after exposure to a temperature of about 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, or 125, or 40-125° C. for about 1, 2, 3, 4, 5, 6, 15, 30, 60, 120, 180, 240, or 360 minutes. In certain embodiments, the biosensor maintains at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more of its signal transduction activity after 1, 2, 3, 4, or 5 freeze-thaw cycles in an aqueous solution. In various embodiments, the biosensor maintains at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more of its signal transduction activity after storage at a temperature of between 20-37° C. for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, or 1-24 months in dry form. In some embodiments, the optimal functional temperature of the biosensor is between 41 and 122° C., between 20 and 40° C., or less than about 10° C. (e.g., between −20 and +10° C.). Devices, compositions, and biosensors provided herein may be stored, e.g., with or without protection from exposure to light. In some embodiments, the devices, compositions, and biosensors are stored in the dark, e.g., with protection from light.

Reporter Group Attachment

Aspects of the present subject matter provide a biosensor that comprises a one or more reporter groups attached to a ligand-binding protein, wherein binding of a ligand to a ligand-binding domain of the ligand-binding protein causes a change in signaling by the reporter group. In various embodiments, the reporter group is attached to an endosteric site, an allosteric site, or a peristeric site of the ligand-binding protein. In embodiments, the reporter group is covalently or noncovalently attached to the ligand-binding protein.

For convenience and depending on context, a reporter group may be referred to by a name of an unattached form of the reporter group regardless of whether the reporter group is attached to a ligand-binding protein. For example, a compound known as “Compound A” when in an unconjugated form may be referred to herein as “Compound A” when in a form that is attached to a ligand-binding protein. In a specific example, the term “Acrylodan” is used to refer to unreacted/unconjugated Acrylodan, as well as Acrylodan that is conjugated to a ligand-binding protein.

In certain embodiments, a biosensor comprises a reporter group that is conjugated to a ligand-binding protein, and the reporter group is conjugated to an amino acid of the protein that is at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 4, 6, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 angstroms (Å) from the ligand when the ligand is bound to the protein. In some embodiments, the reporter group is conjugated to an amino acid of the protein that is within an α-helix or a β-sheet. In some embodiments, the reporter group is conjugated to an amino acid that (i) is not within an α-helix or a β-sheet, but is within about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of an amino acid of the protein's amino acid sequence that is within α-helix or β-sheet. In some embodiments, the reporter group is conjugated to an amino acid that is in an inter-domain hinge amino acid region between two domains of a protein. In some embodiments, the reporter group is conjugated to an amino acid that is in an inter-domain hinge amino acid region between (i) α-helix and a β-sheet; (ii) two α-helixes; or (iii) two β-sheets of a protein. In some embodiments, the reporter group is conjugated to an amino acid (e.g., a cysteine such as a cysteine added by substitution compared to a naturally corresponding polypeptide) between positions 1-25, 25-50, 50-75, 75-100, 100-125, 125-150, 150-175, 175-200, 200-225, 225-250, 250-275, 275-350, 275-300, 275-325, 300-325, 300-350, 300-383, or 350-383 (inclusive) of a polypeptide (e.g., not including N-terminal fusion proteins compared to the polypeptide's naturally occurring counterpart).

Direct signaling relationships between proteins and reporter groups are readily designed by replacing a residue known to form a ligand contact with a cysteine to which the fluorophore is attached (“endosteric” attachment site). Other, indirect signaling relationships can be established in two ways. The first relies on visual inspection of the ligand complex structure, and identifying residues that are located in the vicinity of the binding site, but do not interact directly with the ligand, and that are likely to be involved in conformational changes. Typically, such “peristeric” sites are located adjacent to the residues that form direct contacts with the bound ligand. In the case of the bPBPs, such residues are located at the perimeter of the inter-domain cleft that forms the ligand binding site. The environment of these peristeric sites changes significantly upon formation of the closed state. These are examples of positions which are proximal to the ligand-binding pocket/domain. The second, most general, approach identifies sites in the protein structure that are located anywhere in the protein, including locations at some distance away from the ligand-binding site (i.e., distal to the ligand-binding pocket/domain), and undergo a local conformational change in concert with ligand binding. If the structures of both the open and closed states are known, then such “allosteric” sites can be identified using a computational method that analyzes the conformational changes that accompany ligand binding (Marvin et al., Proc. Natl. Acad. Sci. USA 94:4366-4371, 1997). Alternatively, once allosteric sites have been identified in one bPBP, modeling and structural homology arguments can be invoked to identify such sites in other bPBPs in which only one state has been characterized (Marvin & Hellinga, J. Am. Chem. Soc. 120:7-11, 1998). This generalized conformational analysis also may identify peristeric and endosteric sites, which were identified and classified by visual inspection.

In non-limiting implementations, the reporter group is attached to said ligand-binding protein via a biotin-avidin interaction. The reporter group may be, e.g., conjugated to biotin and the ligand-binding protein is conjugated to avidin. In an example, the avidin is bound to four biotin molecules wherein each biotin molecule is individually conjugated to a reporter group. Alternatively, the reporter group is conjugated to avidin and the ligand-binding protein is conjugated to biotin. For example, the avidin is bound to four biotin molecules, wherein each biotin molecule is individually conjugated to a ligand-binding protein.

As used herein, “conjugated” means covalently attached. One compound may be directly conjugated to another compound, or indirectly conjugated, e.g., via a linker.

In some embodiments, the reporter group is directly attached to said ligand-binding protein. In various embodiments, the reporter group is attached to an amino acid of the ligand-binding protein that is at least about 2, 4, 6, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 angstroms (Å) from the ligand when the ligand is bound to the ligand-binding protein. In certain embodiments, the reporter group is conjugated to an amino acid having a position within positions 1-25, 25-50, 50-75, 75-100, 100-125, 125-150, 150-175, 175-200, 200-225, 225-250, 250-275, or 275-300 of the ligand-binding protein, wherein position 1 is the N-terminal amino acid of the ligand-binding protein. In non-limiting examples, the reporter group is conjugated to an amino acid of the ligand-binding protein that is (a) within an α-helix or a β-sheet of the ligand-binding protein; (b) not within an α-helix; (c) not within a β-sheet; (d) within about 5 or 10 amino acids of an amino acid that is within an α-helix or β-sheet; (e) within a stretch of consecutive amino acids that links two domains of the ligand-binding protein; (f) within a stretch of consecutive amino acids that links an α-helix and a β-sheet; (g) within a stretch of consecutive amino acids that links two α-helices; or (h) within a stretch of consecutive amino acids that links two β-sheets. In some embodiments, the reporter group is directly attached to the N-terminus or the C-terminus of the ligand-binding protein.

The reporter group may be conjugated to the ligand-binding protein a variety of linkers or bonds, including (but not limited to) a disulfide bond, an ester bond, a thioester bond, an amide bond, or a bond that has been formed by a click reaction. In some embodiments, the click reaction is a reaction between (a) an azide and an alkyne; (b) an azide and an alkyne in the presence of Cu(I); (c) an azide and a strained cyclooctyne; (d) an azide and a dibenzylcyclooctyne, a difluorooctyne, or a biarylazacyclooctynone; (e) a diaryl-strained-cyclooctyne and a 1,3-nitrone; (f) an azide, a tetrazine, or a tetrazole and a strained alkene; (g) an azide, a tetrazine, or a tretrazole and a oxanorbornadiene, a cyclooctene, or a trans-cycloalkene; (h) a tetrazole and an alkene; or (i) a tetrazole with an amino or styryl group that is activated by ultraviolet light and an alkene. These exemplary click chemistry reactions have high specificity, efficient kinetics, and occur in vivo under physiological conditions. See, e.g., Baskin et al. Proc. Natl. Acad. Sci. USA 104(2007):16793; Oneto et al. Acta biomaterilia (2014); Neves et al. Bioconjugate chemistry 24(2013):934; Koo et al. Angewandte Chemie 51(2012):11836; Rossin et al. Angewandte Chemie 49(2010):3375, and U.S. Patent Application Publication No. 20160220686, published Aug. 4, 2016, the entire content of each of which is incorporated herein by reference. For a review of a wide variety of click chemistry reactions and their methodologies, see e.g., Nwe K and Brechbiel M W, 2009 Cancer Biotherapy and Radiopharmaceuticals, 24(3): 289-302; Kolb H C et al., 2001 Angew. Chem. Int. Ed. 40: 2004-2021. The entire contents of each of the foregoing references are incorporated herein by reference.

As used herein, the term “linker” refers to a molecule or sequence (such as an amino acid sequence), that attaches, as in a bridge, one molecule or sequence to another molecule or sequence. “Linked” means attached or bound by covalent bonds, or non-covalent bonds, or other bonds, such as van der Waals forces. In some embodiments, a linker comprises a chemical structure that has resulted from a reaction used to attach one molecule to another.

In various implementations of the present subject matter, the reporter group is conjugated to a cysteine of the ligand-binding protein. The cysteine may be present on a natural counterpart or version of the ligand-binding protein or added to the ligand-binding protein by a substitution mutation. In some embodiments, the cysteine is at the N-terminus or the C-terminus of said ligand-binding protein.

Non-limiting examples relate to the conjugation of a reporter group to a primary amine of the ligand-binding protein. In certain embodiments, the primary amine is present in a lysine of said ligand-binding protein. The lysine may be present on a natural counterpart or version of the ligand-binding protein or added to the ligand-binding protein by a substitution mutation. In various embodiments, the lysine is at the N-terminus or the C-terminus of the ligand-binding protein.

Aspects of the present subject matter provide a biosensor in which the reporter group is attached to the ligand-binding protein via a linker. In some embodiments, the linker comprises an organic compound that is less than about 30, 20, 15, or 10 Å long. Non-limiting examples of spacers include O, S, NH, PH, and alkyl spacers.

“Alkyl,” as used herein, refers to the radical of saturated or unsaturated aliphatic groups, including straight-chain alkyl, alkenyl, or alkynyl groups, branched-chain alkyl, alkenyl, or alkynyl groups, cycloalkyl, cycloalkenyl, or cycloalkynyl (alicyclic) groups, alkyl substituted cycloalkyl, cycloalkenyl, or cycloalkynyl groups, and cycloalkyl substituted alkyl, alkenyl, or alkynyl groups. Unless otherwise indicated, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C₁-C₃₀for straight chain, C₃-C₃₀for branched chain), more preferably 20 or fewer carbon atoms, more preferably 12 or fewer carbon atoms, and most preferably 8 or fewer carbon atoms. Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure. The ranges provided above are inclusive of all values between the minimum value and the maximum value. The term “alkyl” includes both “unsubstituted alkyls” and “substituted alkyls,” the latter of which refers to alkyl moieties having one or more substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents include, but are not limited to, halogen, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, a phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. Unless the number of carbons is otherwise specified, “lower alkyl” as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Likewise, “lower alkenyl” and “lower alkynyl” have similar chain lengths. Preferred alkyl groups are lower alkyls. The alkyl groups may also contain one or more heteroatoms within the carbon backbone. Preferably the heteroatoms incorporated into the carbon backbone are oxygen, nitrogen, sulfur, and combinations thereof. In certain embodiments, the alkyl group contains between one and four heteroatoms.

In some embodiments, the linker comprises a bond formed by a chemical reaction involving a reactive group such as a maleimide group. Alternatively or in addition, the linker comprises a stretch of amino acids. In a non-limiting example, the linker comprises a polyglycine linker. In embodiments, the polyglycine linker comprises 2, 3, 4, 5, or more glycines. Optionally, the polyglycine linker further comprises a serine.

In various implementations, the reporter group is attached to a linker via a covalent bond and the linker is attached to a ligand-binding protein via a covalent bond. In embodiments, the covalent bond between the linker and the reporter group and/or the covalent bond between the linker and the ligand-binding protein is a disulfide bond, an ester bond, a thioester bond, an amide bond, a carbamate bond, or a bond that has been formed by a click reaction. Non-limiting examples of click reactions include reactions between an azide and an alkyne; an azide and an alkyne in the presence of Cu(I); an azide and a strained cyclooctyne; an azide and a dibenzylcyclooctyne, a difluorooctyne, or a biarylazacyclooctynone; a diaryl-strained-cyclooctyne and a 1,3-nitrone; an azide, a tetrazine, or a tetrazole and a strained alkene; an azide, a tetrazine, or a tretrazole and a oxanorbornadiene, a cyclooctene, or a trans-cycloalkene; a tetrazole and an alkene; or a tetrazole with an amino or styryl group that is activated by ultraviolet light and an alkene.

Reporter Groups

Various types of reporter groups may be used in embodiments of the present subject matter. For example, the reporter group may comprise a fluorophore that produces a fluorescent signal. Biosensors comprising a fluorophore may be referred to herein as fluorescently responsive sensors (FRSs).

Preferably, the binding of ligand to an FRS results in a change in ratiometric ΔR in the signal from a reporter group. A ratiometric signal (R_1,2) is defined as the quotient of two intensities, I_λ1and λ_λ2, measured at two independent wavelengths, λ₁and λ₂and may be calculated according to the following equation:

R_1,2=I_λ1/I_λ2

In some embodiments, intensities are integrated over a range of wavelengths in a recorded emission spectrum. In some embodiments, intensities are integrated over 10-nm, 15-nm, 20-nm, 25-nm, 30-nm, 35-nm, 40-nm, 45-nm, 50-nm, 75-nm, 100-nm, 10-40-nm, 10-50-nm, 20-50-nm, or 10-100-nm regions, centered between 400-800nm, e.g. between 420 nm and 520 nm for λ₁, and 400-800 nm, e.g. between 500 nm to 600 nm for λ₂. In some embodiments, intensities are recorded through a bandpass filter. A non-limiting example of a bandpass filter is a 10-nm, 15-nm, 20-nm, 25-nm, 30-nm, 35-nm, 40-nm, 45-nm, 50-nm, 75-nm, 100-nm, 10-40-nm, 10-50-nm, 20-50-nm, or 10-100-nm bandpass filter, centered between 400-800nm, e.g. at 452 nm for λ₁and at 400-800nm, e.g. at 528 nm (λ₂).

Aspects of the present subject matter provide FRSs whose emission spectra change (e.g., the shape of the emission spectra change) in response to ligand binding. In various embodiments, the ratio of intensities at two chosen wavelengths of an FRS's emission spectrum changes upon ligand binding. In some embodiments, the emission wavelength and/or intensity of the fluorophore changes when the position of atoms within the fluorophore changes with respect to each other (e.g., due to the rotation of bound atoms with respect to each other or a change in the angle of a bond). In non-limiting examples, the emission wavelength and/or intensity of the fluorophore changes when (i) one portion of the fluorophore rotates around a bond axis compared to another portion of the fluorophore and/or (ii) when the angle of a bond between two atoms of said fluorophore changes. In a non-limiting example, the fluorophore is a prodan-derived fluorophore (e.g., Acrylodan or Badan) and binding of ligand alters the orientation of a dimethylamino group, a naphthalene ring, and/or a carbonyl with respect to the ligand-binding protein and/or each other. In a non-limiting example, the degree of polarization of a dipole on the fluorophore changes in response to ligand binding. In various embodiments, the emission wavelength and/or intensity of the fluorophore changes when an atom electrostatically interacts with said fluorophore. For example, the emission wavelength and/or intensity of the fluorophore changes when the source of a positive or negative charge changes its distance with respect to the fluorophore within about 1, 2, 3, 4, 5, or 10 Å of the fluorophore. In some embodiments, the fluorophore exhibits hypsochromicity or bathochromicity upon ligand binding to the ligand-binding domain of the ligand-binding protein. In certain embodiments, the fluorophore has an emission wavelength comprising a wavelength of about 550 nanometers (nm), 575 nm, 600 nm, 625 nm, 650 nm, or 550-650 nm.

In some embodiments, the signal comprises the emission intensity of the fluorophore recorded at a single wavelength or range of wavelengths. The change in signal may be a shift in the single wavelength or range of wavelengths. In some embodiments, the shift in the wavelength is at least about 1 nm, at least about 2 nm, at least about 3 nm, at least about 4 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, at least about 11 nm, at least about 12 nm, at least about 13 nm, at least about 14 nm, at least about 15 nm, at least about 16 nm, at least about 17 nm, at least about 18 nm, at least about 19 nm, at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, at least about 45 nm, at least about 50 nm, at least about 55 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nm, at least about 100 nm, at least about 105 nm, at least about 110 nm, at least about 115 nm, at least about 120 nm, at least about 125 nm, or at least about 130 nm. In some embodiments, the shift in the wavelength is about 1 nm to about 20 nm, about 2 nm to about 20 nm, about 3 nm to about 20 nm, about 4 nm to about 20 nm, about 5 nm to about 20 nm, about 1 nm to about 19 nm, about 1 nm to about 18 nm, about 1 nm to about 17 nm, 1 nm to about 16 nm, about 1 nm to about 15 nm, about 1 nm to about 14 nm, about 1 nm to about 13 nm, about 1 nm to about 12 nm, about 1 nm to about 11 nm, or about 1 nm to about 10 nm. In some embodiments, the shift in the wavelength is about 1 nm to about 20 nm. In some embodiments, the shift in the wavelength is about 1 nm to about 130 nm.

In certain embodiments, the signal comprises the ratio or quotient of the emission intensities recorded at two distinct wavelengths or ranges of wavelengths, i. e. , a ratiometric signal. For example, as shown in FIGS. 1A-C, ligand binding may be determined by measuring the ratio of blue to green emission intensities. The change in signal may be decreased emission intensity at one wavelength, and no change in emission intensity at the other wavelength. The change in signal may be increased emission intensity at one wavelength, and no change in emission intensity at the other wavelength. The change in signal may be increased emission intensity at one wavelength, and increased emission intensity at the other wavelength. The change in signal may be decreased emission intensity at one wavelength, and decreased emission intensity at the other wavelength. The change in signal may be increased emission intensity at one wavelength, and decreased emission intensity at the other wavelength. In some embodiments, the change in ratio of the emission intensities recorded at two distinct wavelengths or ranges of wavelengths may be at least about 1.1-fold, at least about 1.2-fold, at least about 1.4-fold, at least about 1.6-fold, at least about 1.8-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 12-fold, at least about 14-fold, at least about 16-fold, at least about 18-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 65-fold, at least about 70-fold, at least about 75-fold, at least about 80-fold, at least about 85-fold, at least about 90-fold, at least about 95-fold, or at least about 100-fold. In some embodiments, the change in ratio of the emission intensities recorded at two distinct wavelengths or ranges of wavelengths may be a decrease of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or of 5-25%, 25-50%, 25-75%, 50-75%, 50-90%, or 75-99% or the reciprocal thereof.

The change in signal may be a change in the ratio of the two distinct wavelengths or ranges of wavelengths. The change in signal may be a shift in the two distinct wavelengths or ranges of wavelengths. In some embodiments, one wavelength shifts. In some embodiments, both wavelengths shift. In some embodiments, the shift in the wavelength is at least about 1 nm, at least about 2 nm, at least about 3 nm, at least about 4 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, at least about 11 nm, at least about 12 nm, at least about 13 nm, at least about 14 nm, at least about 15 nm, at least about 16 nm, at least about 17 nm, at least about 18 nm, at least about 19 nm, at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, at least about 45 nm, at least about 50 nm, at least about 55 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nm, at least about 100 nm, at least about 105 nm, at least about 110 nm, at least about 115 nm, at least about 120 nm, at least about 125 nm, or at least about 130 nm. In some embodiments, the shift in the wavelength is about 1 nm to about 20 nm, about 2 nm to about 20 nm, about 3 nm to about 20 nm, about 4 nm to about 20 nm, about 5 nm to about 20 nm, about 1 nm to about 19 nm, about 1 nm to about 18 nm, about 1 nm to about 17 nm, 1 nm to about 16 nm, about 1 nm to about 15 nm, about 1 nm to about 14 nm, about 1 nm to about 13 nm, about 1 nm to about 12 nm, about 1 nm to about 11 nm, or about 1 nm to about 10 nm. In some embodiments, the shift in the wavelength is about 1 nm to about 20 nm. In some embodiments, the shift in the wavelength is about 1 nm to about 130 nm.

A fluorophore may comprise, e.g., a fluorescent protein or an organic compound having a molecular weight less than about 2000 Daltons (Da). Non-limiting examples of commercially available fluorophores include such as 5-iodoacetamidofluorescein (SIAF) or 6-iodoacetamidofluorescein (6IAF), rhodamine, Oregon Green, eosin, Texas Red, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, Badan, Acrylodan, IAEDANS, comprising 3-cyano-7-hydroxycoumarin, 7-hydroxycoumarin-3-carboxylic acid, 6,8-difluoro-7-hydroxy-4-methylcoumarin, or 7-amino-4-methylcoumarin, pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, DRAQS, DRAQ7, or CyTRAK Orange, cascade blue, Nile red, Nile blue, cresyl violet, oxazine 170, proflavin, acridine orange, acridine yellow, auramine, crystal violet, malachite green, porphin, phthalocyanine, bilirubin, pyrene, N,N′-dimethyl-N-(iodoacetyl)-N′-(7-nitrobenz-2-ox-a-1,3-diazol-4-yl)ethylenediamide (NBD), N-((2-(iodoacetoxy)ethyl)-N-methy-1)amino-7-nitrobenz-2-oxa-1,3-diazole (NBDE), Acrylodan, JPW4039, JPW4042, JPW4045, Oregon Green, Pacific Blue, CPM, N,N′-Dimethyl-N-(Iodoacetyl)-N′-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Ethylenediamine (IANBD), 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin (CPM), BODIPY 499, BODIPY 507/545, BODIPY 499/508, Alexa 432, Alexa488, Alexa532, Alexa546, Cy5, or 1-(2-maleimidylethyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridinium methanesulfonate (PyMPO maleimide) (PyMPO). In various embodiments, the reporter group was thiol-reactive prior to being conjugated to a polypeptide disclosed herein. In embodiments, the reporter group is linked to a polypeptide disclosed herein via a disulfide bond. Additional non-limiting examples of commercially available fluorophores include fluorescent proteins such as Blue Fluorescent Protein (BFP), TagBFP, mTagBFP2, Azurite, Enhanced Blue Florescent Protein 2 (EBFP2), mKalamal, Sirius, Sapphire, T-Sapphire, Cyan Fluorescent Protein (CFP); Enhanced Cyan Fluorescent Protein (ECFP), Cerulean, SCFP3A, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP, mTFP1, AmCyan1, Green Fluorescent Protein (GFP), Enhanced Green Fluorescent Protein (EGFP), Emerald, Superfolder GFP, AcGFP1, ZsGreen1, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, Clover, mNeonGreen, Yellow Fluorescent Protein (YFP), Enhanced Yellow Fluorescent Protein (EYFP), Citrine, Venus, Super Yellow Fluorescent Protein 2 (SYFP2), TagYFP, ZsYellow 1, mBanana, Orange Fluorescetn Protein (OFP), Monomeric Kusabira-Orange (mKO), mKOκ, mKO2, mOrange, mOrange2, Red Fluorescent Protein (RFP), DsRed-Express, DsRed-Express2, DsRed2, AsRed2, mRaspberry, mCherry, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mRuby2, mPlum, HcRed-Tandem, mKate2, mNeptune, HcRed1, E2-Crimson, NirFP, TagRFP657, IFP1.4, or iRFP.

In some embodiments, the fluorophore comprises xanthene, a xanthene derivative, cyanine, a cyanine derivative, squaraine, a squaraine derivative, naphthalene, a naphthalene derivative, coumarin, a coumarin derivative, oxadiazole, an oxadiazole derivative, anthracene, an anthracene derivative, a boradiazaindacine (BODIPY) family fluorophore, pyrene, a pyrene derivative, acridine, an acridine derivative, arylmethine, an arylmethine derivative, tetrapyrrole, or a tetrapyrrole derivative. For example, the fluorophore may comprise a xanthene derivative comprising fluorescein or a fluorescein derivative, rhodamine, Oregon Green, eosin, or Texas Red. Non-limiting examples of fluorescein derivatives include 5-fluorescein, 6-carboxyfluorescein, 3′6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachlorofluorescein, 6-tetrachlorofluorescein, or isothiocyanate. In some embodiments, the fluorophore comprises a cyanine derivative comprising indocarbocyanine, oxacarbocyanine, thiacarbocyanine, or merocyanine. In certain embodiments, the fluorophore comprises a squaraine derivative comprising a ring-substituted squaraine. In various embodiments, the fluorophore comprises a naphthalene derivative comprising a dansyl or prodan naphthalene derivative. In a non-limiting example, the fluorophore comprises prodan or a derivative thereof. In certain embodiments, the fluorophore comprises Badan, Acrylodan, or N-(Iodoacetaminoethyl)-1-naphthylamine-5-sulfonic acid (IAEDANS). In some embodiments, the fluorophore comprises a coumarin derivative such as 3-cyano-7-hydroxycoumarin, 7-hydroxycoumarin-3-carboxylic acid, 6,8-difluoro-7-hydroxy-4-methylcoumarin (DiFMU), or 7-amino-4-methylcoumarin. In various embodiments, the fluorophore comprises an oxadiazole derivative such as pyridyloxazole, nitrobenzoxadiazole, or benzoxadiazole. In certain embodiments, the fluorophore comprises an anthracene derivative comprising an anthraquinone such as DRAQS, DRAQ7, or CyTRAK Orange. In various embodiments, the fluorophore comprises a pyrene derivative comprising cascade blue. In non-limiting examples the fluorophore comprises an oxazine derivative such as Nile red, Nile blue, cresyl violet, or oxazine 170. In some embodiments, the fluorophore comprises an acridine derivative such as proflavin, acridine orange, or acridine yellow. In certain embodiments, the fluorophore comprises an arylmethine derivative such as auramine, crystal violet, or malachite green. In various embodiments, the fluorophore comprises a tetrapyrrole derivative comprising porphin, phthalocyanine, or bilirubin.

Aspects of the present subject matter relate to the use of fluorophores that may readily be attached to a ligand-binding protein disclosed herein, e.g., at a cysteine residue. For example, a fluorophore may comprise a sulfhydryl group prior to attachment to a ligand-binding protein that is reacted with a moiety of the ligand-binding protein to attach the fluorophore to the ligand-binding protein. In some embodiments, the fluorophore comprised a thiol group prior to attachment to the ligand-binding protein. For example, the fluorophore was thiol reactive prior to attachment to said ligand-binding protein. Non-limiting examples of fluorophores that may readily be attached to ligand-binding proteins using thiol reactions include fluorescein, pyrene, NBD, NBDE, Acrylodan (6-acryloy 1-2-dimethylaminonaphthalene), Badan (6-bromo-acetyl-2-dimethylamino-naphthalene), JPW4039, JPW4042, or JPW4045.

In certain embodiments, the fluorophore comprises a derivative of a Prodan-based fluorophore such as Acrylodan or Badan. The excitation and emission properties of the Prodan-based fluorophores Acrylodan and Badan can be altered by manipulating the fluorescent ring system, while preserving the dimethylamino donor group, and the twistable carbonyl acceptor (Klymchenko 2013 Progress in Molecular Biology and Translational Science, 35-58). Replacement of the two-ring naphthalene with a three-ring anthracene (Lu 2006 J. Org. Chem., 71, 9651-9657), fluorene (Kucherak 2010 J. Phys. Chem. Lett., 1, 616-620), pyrene (Niko 2013 Chem. Eur. J., 19, 9760-9765), or styrene (Benedetti 2012 J. Am. Chem. Soc., 134, 12418-12421) cores significantly red-shift the excitation and emission properties, and in the case of the latter two, improve brightness through improvements in their excitation peak extinction coefficients. The entire content of each of the references cited above (as well as all other references referred to herein including the contents of nucleic acid and amino acid sequence accession number references) are incorporated herein by reference. Non-limiting examples of prodan analogues include 2-cyano-6-dihexylaminoanthracene and 2-propionyl-6-dihexylaminoanthracene, as well as fluorophores comprising the following structures:

[Image Omitted]

In some embodiments, the fluorophore comprises a fluorescent protein. Fluorescent proteins that emit blue, cyan, green, yellow, orange, red, far-red, or near infrared radiation when contacted with excitation radiation are known in the art and commercially available as proteins and via the expression of vectors that encode the fluorescent protein. Non-limiting examples of fluorescent proteins include Blue Fluorescent Protein (BFP), TagBFP, mTagBFP2, Azurite, Enhanced Blue Florescent Protein 2 (EBFP2), mKalama1, Sirius, Sapphire, T-Sapphire, Cyan Fluorescent Protein (CFP); Enhanced Cyan Fluorescent Protein (ECFP), Cerulean, SCFP3A, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP, mTFP1, AmCyan1, Green Fluorescent Protein (GFP), Enhanced Green Fluorescent Protein (EGFP), Emerald, Superfolder GFP, AcGFP1, ZsGreenl, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, Clover, mNeonGreen, Yellow Fluorescent Protein (YFP), Enhanced Yellow Fluorescent Protein (EYFP), Citrin, Venus, Super Yellow Fluorescent Protein 2 (SYFP2), TagYFP, ZsYellow1, mBanana, Orange Fluorescetn Protein (OFP), Monomeric Kusabira-Orange (mKO), mKOK, mKO2, mOrange, mOrange2, Red Fluorescent Protein (RFP), DsRed-Express, DsRed-Express2, DsRed2, AsRed2, mRaspberry, mCherry, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mRuby2, mPlum, HcRed-Tandem, mKate2, mNeptune, HcRed1, E2-Crimson, NirFP, TagRFP657, IFP1.4, or iRFP.

In some embodiments, the fluorophore comprises a quantum dot (Medintz et al. 2005) (Sapsford, Berti and Medintz 2006 Angew Chem Int Ed Engl, 45, 4562-89; Resch-Genger et al. 2008 Nat Methods, 5, 763-75). In some embodiments the emission properties of the conjugated protein are enhanced by immobilization on or near metallic nanoparticles (Zeng et al. 2014 Chem Soc Rev, 43, 3426-52; Shen et al. 2015 Nanoscale, 7, 20132-41).

In various embodiments, the peak emission wavelength and/or the emission intensity of the biosensor change when the ligand binds to the ligand-binding protein. In some embodiments, the biosensor exhibits a dichromatic signaling change when the ligand binds to the ligand-binding protein. In various embodiments, the peak emission wavelength of the biosensor shifts by at least about 5, 10, 15, 20, 30, 40, 50, or by about 5-50 nm when the biosensor binds to ligand. In certain embodiments, the emission intensity of the biosensor increases by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or 300% when the biosensor binds to ligand. In various embodiments, the signal produced by said reporter group persists for at least 1 nanoseconds (ns), 5 ns, 10 ns, 25 ns, 50 ns, 75 ns, 100 ns, 200 ns, 300 ns, 400 ns, 500 ns, 600 ns, 700 ns, 800 ns, 900 ns, 0.001 milliseconds (ms), 0.01 ms, 0.1 ms, 1 ms, 5 ms, 10 ms, 20 ms, 25 ms, 50 ms, 100 ms, or 500 ms when the ligand binds to the ligand-binding protein.

Exemplary Methods of Using Biosensors Provided Herein

Aspects of the present subject matter provide a method of assaying for a ligand in a sample. The method may include contacting the sample with a biosensor disclosed herein under conditions such that the ligand-binding protein of the biosensor binds to the ligand if ligand is present in said sample. The method also comprises detecting (i) whether a signal is produced by a reporter group of the biosensor; and/or (ii) the a signal produced by a reporter group of the biosensor. In a non-limiting example, a reporter group of the biosensor is fluorescent, and the method further comprises contacting the reporter group with electromagnetic radiation having a wavelength that comprises a wavelength within the band of excitation wavelengths of the reporter group.

In various embodiments, the method further comprises (i) comparing a signal produced by a reporter group of the biosensor when the biosensor is contacted with the sample with a signal produced by a control sample containing a known quantity of ligand; and (ii) detecting the presence or absence of ligand in said sample based on this comparison. Alternatively or in addition, the method further comprises (i) comparing a signal produced by a reporter group of the biosensor when the biosensor is contacted with the sample with signals produced by a series of control samples containing known quantities of ligand; and (ii) determining the quantity of ligand in the sample based on this comparison. In some embodiments, the series of control samples comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 control samples, and wherein each control sample comprises a different quantity of ligand. Alternatively or in addition, the method further comprises determining the concentration of a ligand in a sample, wherein determining the concentration of the ligand in the sample comprises comparing the signal to a standard hyperbolic ligand binding curve to determine the concentration of the ligand in the test sample, wherein the standard hyperbolic ligand binding curve is prepared by measuring the signal produced by the reporter group of the biosensor when the biosensor is contacted with control samples containing known concentrations of ligand. In various embodiments, the method comprises (i) measuring a ratiometric change (ΔR) and/or an intensity change (ΔI) of a signal produced by the reporter group. In some embodiments, the method includes quantitating the level of ligand present in said sample.

In various embodiments, the ligand comprises glucose and/or galactose and said ligand-binding protein comprises a glucose-galactose binding protein.

Aspects of the present subject matter also provide a method of assaying for multiple ligands in a sample, wherein the multiple ligands comprise a first ligand and a second ligand. Such a method may include contacting the sample with (i) a first biosensor a first ligand provided herein and (ii) a second biosensor for said second ligand, under conditions such that the ligand-binding protein of said first biosensor binds to said first ligand, if said first ligand is present in said sample, and detecting (i) a signal produced by a reporter group of said first biosensor, or (ii) whether a signal is produced by a reporter group of said first biosensor. In some embodiments, the second biosensor is also a biosensor provided herein, and the second biosensor is contacted with the second ligand under conditions such that the ligand-binding protein of the second biosensor binds to the second ligand it is present in the sample. The method may further comprise detecting (i) a signal produced by a reporter group of the second biosensor, or (ii) whether a signal is produced by a reporter group of the second biosensor.

In some embodiments, the signal produced by the reporter group of said first biosensor is different than the signal produced by the reporter group of said second biosensor. In a non-limiting example, the reporter group of said first biosensor and the reporter group of said second biosensor are each fluorescent, and the peak emission wavelength of the reporter group of the first biosensor is at least about 10, 25, 50, 75, or 100 nm greater or lower than the peak emission wavelength of the reporter group of the second biosensor.

Non-limiting examples of biosensors that may be used as the second biosensor include biosensors with ligand-binding proteins comprising a GGBP (e.g., an E. coli GGBP) or a derivative or mutant thereof; (ii) an E. coli arabinose binding protein (e.g., an E. coli arabinose binding protein) or a derivative or mutant thereof; (iii) a dipeptide binding protein (e.g., an E. coli dipeptide binding protein) or a derivative or mutant thereof; (iv) a histidine binding protein (e.g., an E. coli, histidine binding protein) or a derivative or mutant thereof; (v) a ribose binding protein (e.g., an E. coli ribose binding protein) or a derivative or mutant thereof; (vi) a sulfate binding protein (e.g., an E. coli sulfate binding protein) or a derivative or mutant thereof; (vii) a maltose binding protein (e.g., an E. coli maltose binding protein) or a derivative or mutant thereof; (viii) a glutamine binding protein (e.g., an E. coli glutamine binding protein) or a derivative or mutant thereof; (ix) a glutamate/aspartate binding protein (e.g., an E. coli glutamate/aspartate binding protein) or a derivative or mutant thereof; (x) a phosphate binding protein (e.g., an E. coli phosphate binding protein) or a derivative or mutant thereof; or (xi) an iron binding protein [e.g., a Haemophilus influenza (H. influenzae) iron binding protein] or a derivative or mutant thereof. For example, the second biosensor comprises an E. coli GGBP having a Y10C, Y10A, D14A, D14Q, D14N, D14S, D14T, D14E, D14H, D14L, D14Y, D14F, N15C, F16L, F16A, F16Y, K92A, N91A, E93C, S112A, S115A, E149C, E149K, E149Q, E149S, H152A, H152F, H152Q, H152N, H152C, D154A, D154N, A155S, A155H, A155L, A155F, A155Y, A155N, A155K, A155M, A155W, A155Q, R158A, R158K, M182W, W183C, W183A, N211F, N211W, N211K, N211Q, N211S, N211H, N211M, D236A, D236N, L255C, N256A, N256D, D257C, P294C, or V296C mutation (e.g., comprising 1, 2, 3, 4, 5 or more of these mutations), wherein each amino acid position is numbered as in (SEQ ID NO: 17); (ii) an E. coli arabinose binding protein having a D257C, F23C, K301C, L253C, or L298C mutation (e.g., comprising 1, 2, 3, 4, or 5 of these mutations) (see, e.g., U.S. Patent Application Publication No. 2004/0118681, the entire contents of which are incorporated herein by reference) (see, e.g., U.S. Patent Application Publication No. 2004/0118681, the entire contents of which are incorporated herein by reference); (iii) an E. coli dipeptide binding protein having a D450C, K394C, R141C, S111C, T44C, or W315C mutation (e.g., comprising 1, 2, 3, 4, 5 or 6 of these mutations) (see, e.g., U.S. Patent Application Publication No. 2004/0118681, the entire contents of which are incorporated herein by reference); (iv) an E. coli, histidine binding protein having a E167C, K229C, V163C, Y230C, F231C, Y88C mutation (e.g., comprising 1, 2, 3, 4, 5 or 6 of these mutations) (see, e.g., U.S. Patent Application Publication No. 2004/0118681, the entire contents of which are incorporated herein by reference); (v) an E. coli ribose binding protein having a T135C, D165C, E192C, A234C, L236C, or L265C mutation (e.g., comprising 1, 2, 3, 4, 5 or 6 of these mutations) (see, e.g., U.S. Patent Application Publication No. 2004/0118681, the entire contents of which are incorporated herein by reference); (vi) an E. coli sulfate binding protein having a L65C, N70C, Q294C, R134C, W290C, or Y67C mutation (e.g., comprising 1, 2, 3, 4, 5 or 6 of these mutations) (see, e.g., U.S. Patent Application Publication No. 2004/0118681 the entire content of which is incorporated herein by reference); (vii) an E. coli maltose binding protein having a D95C, F92C, E163C, G174C, I329C, or S233C mutation (e.g., comprising 1, 2, 3, 4, 5 or 6 of these mutations) (see, e.g., U.S. Patent Application Publication No. 2004/0118681 the entire content of which is incorporated herein by reference); (viii) an E. coli glutamine binding protein having a N160C, F221C, K219C, L162C, W220C, Y163C, or Y86C mutation (e.g., comprising 1, 2, 3, 4, 5 or more of these mutations) (see, e.g., U.S. Patent Application Publication No. 2004/0118681 the entire content of which is incorporated herein by reference); (ix) an E. coli glutamate/aspartate binding protein having a A207C, A210C, E119C, F126C, F131C, F270C, G211C, K268C, Q123C, or T129C mutation (e.g., comprising 1, 2, 3, 4, 5 or more of these mutations) (see, e.g., U.S. Patent Application Publication No. 2004/0118681 the entire content of which is incorporated herein by reference); (x) an E. coli phosphate binding protein having a A225C, N223C, N226C, S164C, or S39C mutation (e.g., comprising 1, 2, 3, 4, or 5 of these mutations) (see, e.g., U.S. Patent Application Publication No. 2004/0118681 the entire content of which is incorporated herein by reference); or (xi) a Haemophilus influenza (H. influenzae) iron binding protein having a E203C, K202C, K85C, or V287C mutation (e.g., comprising 1, 2, 3, or 4 of these mutations) (see, e.g., U.S. Patent Application Publication No. 2004/0118681 the entire content of which is incorporated herein by reference). In various embodiments, the sample is suspected of comprising glucose or galactose.

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crystal structure

bPBP
open form
closed form
DNA sequence
ligand affinity

arabinose BP

Quiocho and
Scripture et al.,
Clark et al.,

Vyas, 1984 1ABE
1987
1982; Miller et

al., 1983

dipeptide BP
Nickitenko et
Dunten &
Abouhamad et
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al., 1995 1DPE
Mowbray, 1995
al., 1991
1986; Smith et

1DPP

al., 1999

Glu/Asp BP

Barash Halpern,

1975; Willis

Furlong, 1975

Fe(III) BP
Bruns et al.,
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Sanders et al.,
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2001 1D9V
1MRP
1994
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glucose BP

Vyas et al., 1988;
Scholle et al.,
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Vyas et al., 1994
1987

1GLG

histidine BP

Yao et al., 1994
Joshi & Ames
Miller et al.,

1HSL
1996
1983

maltose BP
Sharff et al.,
Spurlino el al.,
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1992 1OMP
1991; Quiocho et al.,
1984
1976

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phosphate BP
Ledvina et al.,
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Medveczky &

1996 1OIB
Quiocho, 1990
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Rosenberg, 1969

1IXH

glutamine BP
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1996 1GGG
1WDN
1986
1971

ribose BP
Bjorkman &
Mowbray & Cole,
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Mowbray, 1998
1992 2DRI
1983
Furlong, 1974

1URP

sulfate BP

Pflugrath &
Hellinga &
Jacobson &

Quiocho, 1985;
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Various types of samples may be used in methods provided herein. In non-limiting examples, a sample may comprise a reaction product, a buffer, and/or a solvent. In some embodiments, the solvent is an aqueous solvent. In some embodiments, the solvent comprises a non-polar solvent, a polar aprotic solvent, and/or a polar protic solvent. For example, a sample may comprise water, liquid ammonia, liquid sulfur dioxide, sulfuryl chloride, sulfuryl chloride fluoride, phosphoryl chloride, dinitrogen tetroxide, antimony trichloride, bromine pentafluoride, hydrogen fluoride, dimethyl sulfoxide, hexane, benzene, toluene, 1,4-dioxane, chlorogorm, diethyl ether, dichloromethane, N-methylpyrrolidone, tetrahydrofuran, ethyl acetate, acetone, dimethylformamide, acetonitrile, tormic acid, n-butanol, isopropanol, nitromethane, ethanol, methanol, and/or acetic acid.

In embodiments, a sample comprises a Newtonian liquid, a shear thickening liquid, a shear thinning liquid, a thixotropic liquid, a rheopectic liquid, or a Bingham plastic. In some implementations, a sample has a dynamic viscosity of at least about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, or 2 pascal-seconds (Pa·s) or less than about 2, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5 Pa·s; and/or a kinematic viscosity of at least about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, or 2 centistokes (cSt) or less than about 2, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5 cSt.

In various embodiments, the sample comprises a biological sample. The sample may comprise, e.g., a clinical sample (i.e., a sample collected in a clinical or veterinary setting, e.g., by or at the request or supervision or direction of a doctor, nurse, aid worker, or medic) and/or a physiological sample (a sample collected from an organism, e.g., a mammal such as a human) In certain embodiments, the biological sample comprises or has been provided or obtained from a skin surface or a mucosal surface. In some embodiments, the biological sample comprises a biological fluid. Non-limiting examples of biological fluids include sweat, tear fluid, blood, serum, plasma, interstitial fluid, amniotic fluid, sputum, gastric lavage, skin oil, milk, fecal matter, emesis, bile, saliva, urine, mucous, semen, lymph, spinal fluid, synovial fluid, a cell lysate, venom, hemolymph, and fluid obtained from plants such as the fluid transported in xylem cells or phloem sieve tube elements of a plant (e.g. sap).

The present subject matter also provides biosensors, methods, compositions, and devices useful for measuring the level of a ligand within a liquid solution or suspension or composition comprising cultured cells or tissue or a supernatant of such a solution or suspension, e.g., a sample of conditioned media or a sample of growth media in which a polulation of cells was cultured. In some embodiments, the sample is within a culture (e.g., inserted into a bioreactor) or provided from a media, culture, or reaction, e.g., in a bioreactor. For example, the sample may be within or provided from a fermenter such as a culture or culture supernatant from a fermentation reaction (e.g., an ongoing fermentation). Thus, the level of a ligand can be assayed at a timepoint of interest or at a series of timepoints over the duration of cell culture, e.g. continuously, in or from a reaction or culture. Bioreactors include devices or systems that support a biologically active environment. For example, a bioreactor may comprise a vessel in which a chemical process is carried out which involves organisms or biochemically active substances derived from such organisms. Such a process can either be aerobic or anaerobic. Organisms growing in bioreactors may be, e.g., submerged or suspended in liquid medium or may be attached to the surface of a solid medium. Submerged cultures may be suspended or immobilized. Suspension bioreactors can use a wider variety of organisms, since special attachment surfaces are not needed, and can operate at much larger scale than immobilized cultures. However, in a continuously operated process the organisms will be removed from the reactor with the effluent Immobilization is a general term describing a wide variety of cell or particle attachment or entrapment. It can be applied to basically all types of biocatalysis including enzymes, cellular organelles, and cells (e.g., animal cells, plant cells, fungal cells, and bacterial cells) Immobilization is useful for continuously operated processes, since the organisms will not be removed with the reactor effluent, but is limited in scale because the cells are only present on the surfaces of the vessel. A bioreactor may also refer to a device or system meant to grow cells or tissues in the context of cell culture. The interogation and/or monitoring of glucose levels in such samples permits the evaluation of the status of growth of the cells or production of secreted products by the cells to inform harvest or feeding or other modification of the culture.

Aspects of the present subject matter relate to the use of methods and biosensors provided herein to detect contamination.

In some embodiments, the sample comprises an environmental sample. Depending on context, there are instances in which a biological sample may also be, or may be within, an environmental sample. In certain embodiments, an environmental sample comprises a solute obtained from a biological composition, such as bone, nail, hair, shell, or cartilage. In various embodiments, an environmental sample comprises a solute obtained from an environmental substance and/or an environmental surface. For example, the solute may be dissolved/obtained from the environmental substance and/or an environmental surface using an aqueous or nonaqueous solution. In some embodiments, an aqueous may optionally comprise a nonaqueous solvent (e.g., mixed with an aqueous solvent). Non-limiting examples of environmental substances include rock, soil, clay, sand, meteorites, asteroids, dust, plastic, metal, mineral, fossils, sediment, and wood. Non-limiting examples of environmental surfaces include the surface of a vehicle such as a civilian vehicle (e.g., a satellite, a bike, a rocket, an automobile, a truck, a motorcycle, a yacht, a bus, or a plane) or a military vehicle (e.g., a tank, an armored personell carrier, a transport truck, a jeep, a mobile artillery unit, a mobile antiaircraft unit, a minesweeper, a Mine-Resistant Ambush Protected (MRAP) vehicle, a lightweight tactical all-terrain vehicle, a high mobility multipurpose wheeled vehicle, a mobile multiple rocket launch system, an amphibious landing vehicle, a ship, a hovercraft, a submarine, a transport plane, a fighterjet, a helicopter, a rocket, or an Unmanned Arial Vehicle), a drone, a robot, a building, furniture, or an organism other than a human. In some embodiments, the sample comprises an environmental fluid. Non-limiting examples of environmental fluids include marine water, well water, drinking well water, water at the bottom of well dug for petroleum extraction or exploration, melted ice water, pond water, aquarium water, pool water, lake water, mud, stream water, river water, brook water, waste water, treated waste water, reservoir water, rain water, and ground water. In some embodiments, waste water comprises sewage water, septic tank water, agricultural runoff, water from an area in which chemical or oil spill has or is suspected of having occurred (e.g., an oil spill into a marine environment), water from an area where a radiation leak has or is suspected of having occurred (e.g., coolant from a nuclear reactor), water within the plumbing of a building, water within or exiting a research facility, and/or water within or exiting a manufacturing facility such as a factory.

In certain embodiments, the sample comprises a food or beverage additive and/or a food or beverage composition. In some embodiments, the food or beverage composition comprises a fermented composition. In various embodiments, the sample comprises a fluid obtained from a food composition. Alternatively or in addition, the sample may comprise a solute dissolved from a food composition. In some examples, a solute is or has been dissolved from a food composition with an aqueous or nonaqueous solution. In various implementations, an aqueous solution may optionally comprise a nonaqueous solvent. In certain embodiments, a sample comprises a food composition in semisolid or liquid form. Non-limiting examples of such compositions include yogurt, soup, ice cream, a broth, a puree, a shake, a smoothie, a batter, a condiment, a sauce, and any combination thereof. In some implementations, a sample is a food engineering process (e.g., obtained from a food design, storage, transport, or production process or from equipment intended to process, transport, or store food). A food composition may comprise, e.g., a plant or a composition isolated from a plant, and/or an animal or a composition isolated from an animal. In various embodiments, a sample comprises a beverage composition. Non-limiting examples of beverage compositions include soft drinks, fountain beverages, water, coffee, tea, milk, dairy-based beverages, soy-based beverages (e.g., soy milk), almond-based beverages (e.g., almond milk), vegetable juice, fruit juice, fruit juice-flavored drinks, energy drinks, sports and fitness drinks, alcoholic products, and beverages comprising any combination thereof. Non-limiting examples of beverage compositions comprising water include purified water (e.g., filtered water, distilled water, or water purified by reverse osmosis), flavored water, mineral water, spring water, sparkling water, tonic water, and any combination thereof. In various embodiments, the sample comprises alcohol. Non-limiting examples of such samples include samples comprising or obtained/provided from beer, malt beverages, liqueur, wine, spirits, and any combination thereof.

In some embodiments, a sample comprises a nutritional or supplement composition. In certain implementations, the nutritional or supplement composition comprises an omega-3 fatty acid, a vitamin, a mineral, a protein powder, or a meal supplement.

In certain embodiments, a biosensor is implanted in a subject's body. For example, a biosensor may be implanted in a subject's blood vessel, vein, eye, natural or artificial pancreas, alimentary canal, stomach, intestine, esophagus, or skin (e.g., within the skin or under the skin). In various embodiments, the biosensor is configured within or on the surface of a contact lens. In some embodiments, the biosensor is configured to be implanted in or under the skin. In non-limiting examples, the biosensor is implanted in a subject with an optode and/or a microbead. In certain embodiments, the biosensor generates a signal transdermally.

Aspects of the present subject matter provide a method for assaying the level of glucose in a subject. The method may comprise contacting a biological sample from the subject with a biosensor for glucose under conditions such that the biosensor binds to glucose present in the biological sample. The biosensor comprises a glucose-galactose binding protein attached to a reporter group, and binding of glucose to a glucose-binding domain of the glucose-galactose binding protein causes a change in signaling by the reporter group. In various embodiments, the subject has or is suspected of having diabetes, such as Type I diabetes or Type II diabetes. In some embodiments, the biological sample comprises blood, plasma, serum, sweat, tear fluid, or urine. In certain embodiments, the biological sample is present in or on the surface of said subject. In various implementations, the biosensor is applied onto or inserted into the subject. For example, the biosensor may be tattooed into the subject or is in or on a device that is implanted into said subject. In some embodiments, the biosensor may be present in or on a contact lens that is worn by the subject. Methods for determining the level of glucose, e.g. in a subject who has or is suspected of having diabetes, may be performed without other testing related to diabetes performed as part of a battery of clinical testing.

The present subject matter includes a method for monitoring the level of a ligand, comprising periodically or continuously detecting the level of the ligand, wherein detecting the level of the ligand comprises (a) providing or obtaining a sample; (b) contacting said sample with a biosensor for said ligand according to claim 1 under conditions such that the ligand-binding protein of said biosensor binds to said ligand, and (c) detecting a signal produced by said biosensor.

Aspects of the present subject matter also provide a method for monitoring the level of a ligand (e.g., glucose and/or galactose) in a subject, comprising periodically detecting the level of the ligand in the subject. Detecting the level of the ligand in the subject may comprise (a) providing or obtaining a biological sample from said subject; (b) contacting the biological sample with a biosensor for the ligand provided herein under conditions such that the ligand-binding protein of the biosensor binds to the ligand, if the ligand is present in said biological sample, and (c) detecting (i) a signal produced by a reporter group of said biosensor, or (ii) whether a signal is produced by a reporter group of said biosensor. The level of said ligand may be detected, e.g., at least once every 1, 2, 3, 6, or 12 hours, at least once every 1, 2, 3, or 4 days, at least once every 1, 2, or three weeks, or at least once every 1, 2, 3, 4, 6, or 12 months.

The present subject matter also provides a method for monitoring the level of a ligand in a subject. The method comprises (a) administering a biosensor provided herein or a device comprising a biosensor provided herein to said subject, wherein after administration the biosensor is in contact with a bodily fluid or surface that typically comprises said ligand, and (b) detecting (i) a signal produced by a reporter group of the biosensor continuously or repeatedly at intervals less than about 30 minutes (m), 15 m, 10 m, 5 m, 1 m, 30 seconds (s), 15 s, 10 s, 5 s, 1 s, 0.1 s, 0.001 s, 0.0001 s, or 0.00001 apart, and/or (ii) whether a signal is produced by a reporter group of the biosensor continuously or repeatedly at intervals less than about 30 m, 15 m, 10 m, 5 m, 1 m, 30 s, 15 s, 10 s, 5 s, 1 s, 0.1 s, 0.001 s, 0.0001 s, or 0.00001 apart.

Non-limiting aspects of continuously monitoring glucose levels are described in Weidemaier et al. (2011) Biosensors and Bioelectronics 26, 4117-4123 and Judge et al. (2011) Diabetes Technology & Therapeutics, 13(3):309-317, the entire contents of each of which are hereby incorporated herein by reference.

Also within the invention is a composition comprising a purified thermostable, glucose-binding fluorescently-responsive sensor protein and a solid substrate, e.g., a particle, a bead such as a magnetic bead, or a planar surface such as a chip or slide, wherein the sensor protein is immobilized onto the solid substrate. An exemplary solid substrate solid substrate comprises a cyclic olefin copolymer.

The sensor protein contains a single cysteine residue, with the single cysteine residue being located in a glucose-contacting site of protein. A thermostable glucose sensor protein is one in which the activity (glucose binding) is unaffected by relatively high temperatures. For example, the glucose sensor protein comprises a mid-point thermal melt transition greater than 50° C., greater than 60° C., greater than 70° C., greater than 80° C., greater than 90° C., or greater than 100° C. In some examples, the protein comprises the amino acid sequence of SEQ ID NO: 298, and in some examples, the single cysteine is conjugated to Badan, Acrylodan, or a derivative thereof. For example, the derivative comprises a replacement of the two-ring naphthalene of Acrylodan or Badan with a three-ring anthracene, a fluorene, or a styrene. A reporter group is covalently bound to the single cysteine. In some situations, the solid substrate comprises a plurality of sensor proteins, each of which comprises a different dissociation constant (K_D) for glucose, e.g., for detecting and quantifying glucose levels across many ranges of concentrations.

The invention also includes a composition comprising purified glucose sensor protein with less than 65% identity and greater than 27% identity (e.g., 44-48% sequence identity) to SEQ ID NO: 1, 16, or 17, wherein the sensor protein comprises a single cysteine residue, said residue being located in a glucose-contacting site of the protein, and a solid substrate, such that the sensor protein is immobilized onto said solid substrate. As described above, a reporter group is covalently bound to the single cysteine. In some example, the solid substrate comprises a plurality of sensor proteins, each of which comprises a different dissociation constant (K_D) for glucose for sensing over a wide range or ranges of glucose concentrations.

A method of detecting the presence of or the quantity of glucose in a test sample is carried out using the following steps: contacting the testsample with the biosensor or sensor protein/solid support construct to yield a complex of glucose and the ligand-binding protein or biosensor protein; contacting the complex with an excitation light; measuring an emission intensity of the reporter group from at least two wavelengths; computing a ratiometric signal from the two (or more) wavelengths; and comparing the signal to a known glucose binding curve of signals to identify the presence of or calculate the quantity of glucose in the test sample. The test sample may be obtained from a variety of sources. For example, the test sample is selected from a bodily fluid, a food, a beverage, or a bioreactor culture broth. The testing method may be carried out in vivo, e.g., using an implantable device or dermal patch, or ex vivo.

In various embodiments, the subject to be tested is a mammal, e.g., a primate (such as a human, a monkey, a chimpanzee, or a gorilla), a fish, a bird, a reptile, an amphibian, or an arthropod. In some embodiments, the subject is a fish, a cow, a pig, a camel, a llama, a horse, a race horse, a work horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a wolf, a dog (e.g., a pet dog, a work dog, a police dog, or a military dog), a rat, a mouse, a seal, a whale, a manatee, a lizard, a snake, a chicken, a goose, a swan, a duck, or a penguin.

Exemplary Devices and Compositions Comprising Biosensors

Aspects of the present subject matter provide a device comprising one or more biosensors provided herein. Such devices may be, e.g., wearable, implantable, portable, or fixed.

In some embodiments, the device is a nanoparticle or a microparticle comprising said biosensor. Non-limiting examples of devices include devices comprising a test strip, patch, plate, bead, or chip comprising a biosensor provided herein. In certain embodiments, a device may comprise a desiccated biosensor.

The present subject matter also provides a contact lens or a skin patch comprising a biosensor provided herein. In some embodiments, the biosensor is throughout the contact lens or skin patch or within a particular region or zone of a contact lens or skin patch (e.g., in one or more shapes (e.g., a square, circle, or star), dots, lines, or zones, located at the periphery or a portion of the periphery of a contact lens or patch). In some embodiments, the skin patch comprises an adhesive that facilitates attachment of the patch to the surface of skin.

Devices provided herein may include a variety of structural compositions. For example, many polymers (including copolymers), and plastics may be used. Non-limiting examples of compositions useful in certain devices include glass, polystyrene, polypropylene, cyclic olefin copolymers, ethylene-norbornene copolymers, polyethylene, dextran, nylon, amylase, paper, a natural cellulose, a modified cellulose, a polyacrylamide, gabbros, gold, and magnetite (as well as combinations thereof). In some embodiments, the device comprises a hydrogel, a cryogel, or a soluble gel. For example, the biosensor may be incorporated into or onto the hydrogel, cryogel, or soluble gel. In various embodiments, the device comprises a matrix comprising nanopores, micropores, and/or macropores. In certain embodiments, the surface of a device comprises a polymer. In an embodiment, the surface comprises the surface of a particle or a bead having a diameter of about 0.001-1, 0.001-0.1, 0.01-0.1, 0.001-0.01, 0.1-1, 0.1-0.5, or 0.01-0.5 centimeters (cm). For example, the particle comprises a nanoparticle or a microparticle.

Non-limiting examples of polymers include cyclic olefin copolymers, ethylene-norbornene copolymers, polylactic acid, polyglycolic acid, agarose, alginate, poly(lactide-co-glycolide), gelatin, collagen, agarose, natural and synthetic polysaccharides, polyamino acids, poly(lysine), polyesters, polyhydroxybutyrates, polyanhydrides, polyphosphazines, polyvinyl alcohol, polyalkylene oxide, polyethylene oxide, polyallylamines, polyacrylates, modified styrene polymers, poly(4-aminomethylstyrene), pluronic polyols, polyoxamers, polyuronic acid, and polyvinylpyrrolidone.

In some embodiments, the device comprises a plastic polymer comprising cyclic olefin copolymer (COC), such as e.g. TOPAS® COC. Several types of cyclic olefin copolymers are available based on different types of cyclic monomers and polymerization methods. Cyclic olefin copolymers are produced by chain copolymerization of cyclic monomers such as 8,9,10-trinorborn-2-ene (norbornene) or 1,2,3,4,4a,5,8,8a-octahydro-1,4:5,8-dimethanonaphthalene (tetracyclododecene) with ethene (such as TOPAS Advanced Polymer's TOPAS, Mitsui Chemical's APEL), or by ring-opening metathesis polymerization of various cyclic monomers followed by hydrogenation (Japan Synthetic Rubber's ARTON, Zeon Chemical's Zeonex and Zeonor). See, e.g., International Union of Pure and Applied Chemistry (2005) Purr. Appl. Chem. 77(5):801-814. These later materials using a single type of monomer may be referred to as cyclic olefin polymers (COPs). A CAS Registry number for COC is 26007-43-2.

In certain embodiments, the device is attached to a surface of a device or is not attached to a surface of said device (e.g., the biosensor is present loosely within the device as a component of a solution or powder).

A biosensor may be attached to a device via a variety or means, e.g., via attachment motif. In some embodiments, the attachment motif is attached to the N-terminus or the C-terminus of the biosensor. In certain embodiments, the biosensor is linked to an attachment motif via a covalent bond. In various embodiments, the biosensor is linked to said attachment motif via a linker. A non-limiting example of a linker is a polyglycine comprising 2, 3, 4, 5, or more glycines and optionally further comprising a serine. In some embodiments, the attachment motif comprises a polypeptide. Non-limiting examples of polypeptides useful in attachment moieties include hexahistidine peptides, hexalysine peptides, zinc-finger domains (ZF-QNKs), and disulfide-containing truncated zinc fingers (βZifs). An example of a hexalysine peptide comprises amino acids in the sequence of SEQ ID NO: 296, an example of a ZF-QNK comprises amino acids in the sequence of SEQ ID NO: 294, and an example of a βZif comprises amino acids in the sequence of SEQ ID NO: 293. In some embodiments, the attachment motif comprises a polypeptide that binds to plastic or cellulose.

The hexahistidine, hexalysine, βZif and QNK-ZF fusions (SEQ ID NOS: 173 and 175-181) enable FRSs to be immobilized onto chemically functionalized surfaces. Non-limiting aspects of chemically functionalized surfaces are discussed in Biju, V. (2014) Chem Soc Rev, 43, 744-64 and McDonagh (2008) Chem Rev, 108, 400-422, the entire contents of which are incorporated herein by reference. Directed evolution methods have been used to develop peptides that bind directly to non-functionalized surfaces (Care, Bergquist and Sunna 2015 Trends Biotechnol, 33, 259-68; Baneyx 2007 Curr. Opin. Biotechnol., 18, 312-317; Gunay and Klok 2015 Bioconjug Chem, 26, 2002-15), including various plastics (Adey et al. 1995 Gene, 156, 27-31; Serizawa et al. 2005 J Am Chem Soc, 127, 13780-1; Serizawa, Sawada and Kitayama 2007a Angew Chem Int Ed Engl, 46, 723-6; Serizawa, Sawada and Matsuno 2007b Langmuir, 23, 11127-33; Serizawa, Techawanitchai and Matsuno 2007c Chembiochem, 8, 989-93; Matsuno et al. 2008 Langmuir, 24, 6399-403; Chen, Serizawa and Komiyama 2011 J Pept Sci, 17, 163-8; Kumada 2010 J. Biosci. and BioEng., 109, 583-587; Date et al. 2011 ACS Appl Mater Interfaces, 3, 351-9; Kumada 2012, Vodnik, Strukelj and Lunder 2012 J. Biotech., 160, 222-228; Kumada 2014 Biochem. et Biophys. Acta, 1844, 1960-1969; Ejima, Matsuno and Serizawa 2010 Langmuir, 26, 17278-85), inorganic materials(Hnilova 2012 Soft Matter, 8, 4327-4334; Care et al. 2015 Trends Biotechnol, 33, 259-68), nanoparticles (Avvakumova et al. 2014 Trends Biotechnol, 32, 11-20), and cellulosic paper (Guo et al. 2013 Biomacromolecules, 14, 1795-805). Such peptides, or natural material-binding domains (Oliveira et al. 2015 Biotechnol Adv, 33, 358-69), also can be fused to FRSs to direct site-specific, oriented immobilization on their target materials while preserving FRS function. For instance, plastic-binding peptides have been developed that direct immobilization on polystyrene (Adey et al. 1995 Gene, 156, 27-31; Serizawa et al. 2007c Chembiochem, 8, 989-93; Kumada 2010 Biochem. et Biophys. Acta, 1844, 1960-1969; Vodnik et al. 2012 Anal Biochem, 424, 83-6), polymethyl acrylate (Serizawa et al. 2005 J Am Chem Soc, 127, 13780-1; Serizawa et al. 2007a Angew Chem Int Ed Engl, 46, 723-6; Serizawa et al. 2007b Langmuir, 23, 11127-33; Kumada 2014 Biochem. et Biophys. Acta, 1844, 1960-1969), polycarbonate (Kumada 2012 J. Biotech., 160, 222-228), polylactide (Matsuno et al. 2008 Langmuir, 24, 6399-403), and polyphenylene vinylene (Ejima et al. 2010 Langmuir, 26, 17278-85). Cellulose-binding peptides (Guo et al. 2013 Biomacromolecules, 14, 1795-805) and natural domains (Oliveira et al. 2015 Biotechnol Adv, 33, 358-69; Shoseyov, Shani and Levy 2006 Microbiol Mol Biol Rev, 70, 283-95) can be used to immobilize fusion proteins on paper. Inorganic material include noble metals (Hnilova 2012 Soft Matter, 8, 4327-4334), semi-conductors (Care et al. 2015 Trends Biotechnol, 33, 259-68), and fluorescent quantum dots(Medintz et al. 2005 Nat Mater, 4, 435-46; Lee et al. 2002 Science, 296, 892-5). The entire contents of each of the references above (and all other references herein) is incorporated herein by reference.

In some embodiments, the attachment motif is attached to a device surface and/or within a matrix of the device. In some embodiments, a biosensor is attached to an attachment motif via a covalent bond and the attachment motif is attached to a device via a covalent bond. Non-limiting examples of covalent bonds include disulfide bonds, ester bonds, thioester bonds, amide bonds, and bonds that have been formed by click reactions. Non-limiting examples of a click reaction include a reaction between an azide and an alkyne; an azide and an alkyne in the presence of Cu(I); an azide and a strained cyclooctyne; an azide and a dibenzylcyclooctyne, a difluorooctyne, or a biarylazacyclooctynone; a diaryl-strained-cyclooctyne and a 1,3-nitron; an azide, a tetrazine, or a tetrazole and a strained alkene; an azide, a tetrazine, or a tretrazole and a oxanorbornadiene, a cyclooctene, or a trans-cycloalkene; a tetrazole and an alkene; or a tetrazole with an amino or styryl group that is activated by ultraviolet light and an alkene.

Alternatively or in addition, a surface of a device may be modified to contain a moiety (e.g. a reactive group) what facilitates the attachment of a biosensor and/or binds to the biosensor. In some embodiments, the biosensor is attached to a surface via a biotin-avidin interaction.

In various implementations, the device comprises a first region for receiving a sample and second a region that comprises the biosensor, wherein said first region is separated from said second region by a filter. In some examples, the filter is impermeable to compounds greater than about 1, 2, 3, 4, 5, 10, 50, 200, or 250 kiloDalton (kDa) in size. The sample may comprise, e.g., a tube, such as a tube that is configured for centrifugation. When sample is placed into the first region and the device is centrifuged, then a portion of the sample comprising a ligand flows through the filter into the second region where the biosensor is contacted.

Non-limiting examples of devices provided herein include endoscopy probes and colonoscopy probes.

In some embodiments, the device comprises an optode. In non-limiting examples, the optode comprises an optical fiber and a single biosensor or composite biosensor. In certain embodiments, the single biosensor or composite biosensor is immobilized on the surface or at an end of the optical fiber. In some embodiments, the optode is configured for implantation into a subject. Alternatively or in addition, the optode is configured for insertion into a sample.

The devices provided herein may optionally comprise a biosensor panel, a composite sensor, a sensor array, and/or a composition comprising a plurality of biosensors. In various embodiments, a device comprises multiple glucose and/or galactose biosensors that detect a range of different glucose and/or galactose concentrations in a single sample and/or assay run (i.e., each biosensor has a different affinity for glucose and/or galactose). Devices may provide spatial localization of multiple biosensors to provide the necessary addressability of different elements in a multi-sensor array comprising sensors that differ in their engineered affinities for coverage of a wide range of glucose concentrations, or sensors that each detects distinct analytes.

Aspects of the present subject matter provide a biosensor panel comprising a plurality of biosensors, wherein said plurality of biosensors comprises at least one biosensor disclosed herein. In some embodiments, the plurality comprises at least about 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 biosensors.

The present subject matter also provides a composite sensor. The composite sensor may comprise a sensor element, wherein the sensor element comprises 2 or more biosensors, wherein at least 1 of said 2 or more biosensors is a biosensor disclosed herein. In some embodiments, the biosensors are not spatially separated in the sensor element, e.g., the biosensors are mixed within a solution or on a surface of the sensor element. In various embodiments, the composite sensor comprises a plurality of sensor elements, wherein each sensor element of the plurality of sensor elements comprises 2 or more biosensors, wherein at least 1 of the 2 or more biosensors is a biosensor provided herein. In some embodiments, the plurality of sensor elements comprises at least about 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 sensor elements.

Also included herein is a sensor array comprising a plurality of biosensors of the present subject matter. The sensor array may include, e.g., multichannel array or a multiplexed array. In some embodiments, the biosensors of the plurality of biosensors are spatially separated from each other. In certain embodiments, the biosensors are arranged linearly or in a grid on a surface of said array.

The present subject matter provides a composition comprising a plurality of biosensors including at least one biosensor disclosed herein. Also provided is a non-human mammal comprising a biosensor or device disclosed herein.

Exemplary Polypeptides and Polynucleotides

The present subject matter provides polynucleotides encoding any one of the polypeptides disclosed herein. The polypeptides are also provided. In various embodiments, the polynucleotides are codon-optimized for expression in a desired host cell, such as bacterial cells (e.g., E. coli), yeast, insect cells, plant cells, algal cells, or mammalian cells. The polypeptides provided herein include polypeptides comprising the amino acid sequence of any one of SEQ ID NOS: 1-103, 157-292, 197, or 198. The polynucleotides provided herein include polynucleotides encoding a polypeptide comprising the amino acid sequence of any one of SEQ ID NOS: 1-103, 157-292, 197, or 198.

The polypeptides and biosensors provided herein may be in a variety of forms, e.g., purified in solution, dried (e.g. lyophilized) such as in the form of a powder, and in the form of a crystal (e.g., a crystal suitable for x-ray crystallography). Thus, aspects of the present subject matter provide crystal structures and crystalized forms of the ligand-binding proteins and biosensors disclosed herein. Such crystal structures and crystalized proteins are useful for designing and optimizing biosensors using principles and methods discussed herein.

Also provided are expression vectors comprising a polynucleotide of the present subject matter and/or encoding a polypeptide disclosed herein. Non-limiting examples of expression vectors include viral vectors and plasmid vectors. In some embodiments, an expression vector comprises nucleotides in the sequence set forth as SEQ ID NO: 105-156. In various embodiments, a polynucleotide encoding a ligand-binding protein and/or biosensor is operably linked to a promoter. The promoter may be expressed, e.g., in a prokaryotic and/or a eukaryotic cell.

The subject matter further includes an isolated cell comprising an expression vector provided herein. The isolated cell may be, e.g., a bacterial cell, a yeast cell, an algal cell, a plant cell, an insect cell, or a mammalian cell. Also included is a non-human multicellular organism such as a plant or an animal (e.g., an insect, a mammal, a worm, a fish, a bird, or a reptile) comprising an expression vector disclosed herein.

Exemplary Methods for Designing Biosensors

Aspects of the present subject matter provide method of identifying a candidate ligand-binding protein for use in a biosensor, comprising: (a) selecting a first protein having a known amino acid sequence (seed sequence), wherein the first protein is a glucose-galactose binding protein; (b) identifying a second protein having an amino acid sequence (hit sequence) with at least 15% sequence identity to the seed sequence; (c) aligning the seed amino acid sequence and the hit sequence, and comparing the hit sequence with the seed sequence at positions of the seed sequence that correspond to at least 5 primary complementary surface (PCS) amino acids, wherein each of the at least 5 PCS amino acids has a hydrogen bond interaction or a van der Waals interaction with glucose when glucose is bound to the first protein; and (d) identifying the second protein to be a candidate ligand-binding protein if the hit sequence comprises at least 5 amino acids that are consistent with the PCS.

The present subject matter also includes a method for constructing a candidate biosensor, comprising: (a) providing a candidate ligand-binding protein; (b) generating a structure of the second protein; (c) identifying at least one putative allosteric, endosteric, or peristeric site of the second protein based on the structure; (d) mutating the second protein to substitute an amino acid at said at least one putative allosteric, endosteric, or peristeric site of the second protein with a cysteine; and (e) conjugating a fluorescent compound to the cysteine. In some embodiments, the structure comprises a homology model of the second protein generated using a structure of the first protein. In some embodiments, the structure comprises a structure experimentally determined by nuclear magnetic resonance spectroscopy or X-ray crystallography.

Aspects of the present subject matter further provide a method for constructing a biosensor comprising a desired dissociation constant (KD) for glucose, comprising: (a) providing an initial biosensor that does not comprise the desired KD for glucose, wherein said initial biosensor is a biosensor provided herein; (b) mutating the initial biosensor to (i) alter a direct interaction in the PCS between the initial biosensor and bound glucose; (ii) manipulate the equilibrium between open and closed states of the initial biosensor; (iii) alter an interaction between the ligand-binding protein and the reporter group of the initial biosensor; or (iv) alter an indirect interaction that alters the geometry of the binding site of the biosensor, to produce a modified biosensor; and (c) selecting the modified biosensor if the modified biosensor comprises the desired KD for glucose. In some embodiments, the reporter comprises Acrylodan, Badan, or a derivative thereof, and mutating the initial biosensor in (b) comprises altering an interaction between the ligand-binding protein and a carbonyl group of the Acrylodan, Badan, or derivative thereof. In some embodiments, the reporter group comprises Acrylodan, Badan, or a derivative thereof, and mutating the initial biosensor in (b) comprises altering an interaction between the ligand-binding protein and a naphthalene ring of the Acrylodan, Badan, or derivative thereof. In some embodiments, mutating the initial biosensor comprises introducing a substitution mutation into said initial biosensor. In some embodiments, the method further comprises immobilizing said affinity-tuned biosensor on a substrate.

In some embodiments, the second protein comprises (i) amino acids in the sequence of SEQ ID NO: 1-15 or 17-31; (ii) a stretch of amino acids in a sequence that is least about 95, 96, 97, 98, or 99% identical to the sequence of SEQ ID NO: 1-31; (iii) a stretch of at least about 50, 100, 150, 200, 250, 300, or 350 amino acids in a sequence that is at least about 95, 96, 97, 98, or 99% identical to a sequence within SEQ ID NO: 1-31; or (iv) a stretch of at least about 50, 100, 150, 200, 250, 300, or 350 amino acids in a sequence that is identical to a sequence within SEQ ID NO: 1-31. In various embodiments, attaching the reporter group to the putative allosteric, endosteric, or peristeric site of the first protein comprises substituting a cysteine at the site with a cysteine. For example, the reporter group is conjugated to the cysteine. Preferably, attaching a reporter group to the corresponding amino acid of the second protein produces a functional biosensor.

The selected first protein (e.g., the amino acid sequence thereof) may be novel or known. However, in many instances, the function of the first protein will not be known. In a non-limiting example, identifying a protein not previously known to have glucose binding activity may comprise a structurally assisted functional evaluation (SAFE) homolog search method comprising the following steps:

(1) Collecting a sequence homology set using a BLAST sequence alignment tool starting with glucose-galactose binding protein (GGBP) sequence disclosed herein as a seed. Permissive settings are used, such that pairwise hits are required to have a minimum of only, e.g., 20%, 25%, 30%, 35% or 40% sequence identity with the seed sequence. The lengths of the hit and seed are mutually constrained such that the alignment covers at least, e.g., 60%, 65%, 70%, 85%, or 90% within each partner.

(2) Structure-based encoding of biological function: A primary complementary surface (PCS) comprising the protein residues that form hydrogen bonds and van der Waals contacts with a bound glucose is defined using computer-assisted, visual inspection of the three-dimensional structure of the GGBP-glucose complex. This definition specifies residue positions and their permitted amino acid identity. Multiple amino acid identities are permitted at each position to encode functionally equivalent residues. This definition establishes a search filter for the accurate prediction of glucose-binding proteins within the universe of sequence homologs collected in (1). For example, a candidate's residue corresponding to position 14 of ecGGBP may be D or N, a candidate's residue corresponding to position 16 of ecGGBP may be F, Y, or W, a candidate's residue corresponding to position 91 of ecGGBP may be N or D, a candidate's residue corresponding to position 152 of ecGGBP may be H, N, or Q, a candidate's residue corresponding to position 154 of ecGGBP may be D or N, a candidate's residue corresponding to position 158 of ecGGBP may be R, a candidate's residue corresponding to position 183 of ecGGBP may be W, F, or Y, a candidate's residue corresponding to position 211 of ecGGBP may be N or D, a candidate's residue corresponding to position 236 of ecGGBP may be D or N, and a candidate's residue corresponding to position 256 of ecGGBP may be N or D.

(3) Accurate sequence alignment: Tools such as ClustalW are used to construct an accurate alignment of all the sequence homologs. The GGBP seed sequence is included in the alignment. This multiple sequence alignment establishes the equivalent positions of the seed GGBP (primary complementary surface) PCS in each sequence homolog.

(4) Function evaluation: The glucose-binding properties of each of the aligned sequence homologs is determined by measuring their compliance with the PCS sequence filter. A “Hamming distance”, H, is assigned for each homolog, which specifies the degree of sequence identity of all the residues at the aligned PCS positions. A value of H=0 indicates that the identities of all the residues at the aligned PCS positions match the amino acid(s) allowed in the PCS search filter; H>0, indicates that one or more aligned positions have disallowed residues. Sequences for which H=0 are predicted to encode glucose-binding proteins.

(5) Selection of representative SAFE homologs: The sequence homologs are ordered by (a) identity with the seed PCS, as measured by the Hamming distance, (b) fractional overall sequence identity with the seed sequence. A subset for sequences with H=0, sampling the fractional overall sequence identity is selected for experimental verification.

In a non-limiting example, identifying a protein not previously known to have glucose binding activity may comprises the following steps:

(1) performing a computational search of sequence databases to define a broad group of simple sequence or structural homologs of any known, glucose-galactose binding protein;

(2) using the list from step (1), deriving a search profile containing common sequence and/or structural motifs shared by the members of the list [e.g. by using computer programs such as MEME (Multiple Em for Motif Elicitation available at meme.sdsc.edu/meme/cgi-bin/meme.cgi) or BLAST];

(3) searching sequence/structural databases, using a derived search profile based on the common sequence or structural motif from step (2) as query (e.g., using computer programs such as BLAST, or MAST (Motif Alignment Search Tool available at meme.sdsc.edu/meme/cgi-bin/mast.cgi), and identifying a candidate sequence, wherein a sequence homology and/or structural similarity to a reference glucose-galactose binding protein is a predetermined percentage threshold;

(4) compiling a list of candidate sequences to generate a list of candidate glucose-galactose binding proteins;

(5) expressing the candidate glucose-binding proteins in a host organism; and

(6) testing for glucose and/orgalactose binding activity, wherein detection of glucose and/or galactose binding in said organism (or the media thereof) indicates that the candidate sequence comprises a novel glucose and/or galactose binding protein.

In non-limiting examples, the MEME suite of sequence analysis tools (meme.sdsc.edu/meme/cgi-bin/meme.cgi) can also be used as an alternative to BLAST. Sequence motifs are discovered using the program “MEME”. These motifs can then be used to search sequence databases using the program “MAST.” The BLAST search algorithm is well-known.

Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the invention.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C are a cartoon and graphs illustrating emission spectra changes upon ligand binding to FRSs. (A) FRSs can be constructed by site-specifically attaching a fluorophore to a protein that undergoes a conformational change upon binding ligand (triangle), such that the shape and intensities of the fluorescent conjugate emission spectra changes. (B) In the absence of ligand, the emitted fluorescence color is predominantly blue, whereas the ligand complex fluoresces green. Arrows indicate the direction of change upon ligand addition. (C) Ligand binding is determined by measuring the ratio of blue to green emission intensities.

FIGS. 2A-C are structural illustrations and a table showing exemplary features of the E. coli glucose-galactose binding protein (ecGGBP). (A) The glucose and calcium complex [PDB identifier 2gbp (Vyas et al. 1988)], glucose is sandwiched by van der Waals contacts with the rings of Phe16 and Trp183. (B) Residues that form hydrogen bonds with the bound glucose. The primary complementary surface (PCS) comprises the aromatic sandwich and the hydrogen-bonding residues. (C) The PCS sequence filter used to identify the subset of glucose-binding proteins within a family of sequence ecGGBP homologs. Note redundancies in the allowed residues at each position (the first amino acid listed corresponds to the wild-type ecGGBP sequence).

FIG. 3 is an alignment of the homologs predicted to be glucose-binding proteins (alignment generated by ClustalW; ordered by fractional sequence identity to the ecGGBP seed sequence). Sequences taken from Table 1 (name, line number in Table 1, accession code, species, fractional identity to ecGGBP):

ecGGBP, 2, NC_013654|YP_003350022.1, Escherichia coli, 100%;

ttGGBP, 96, NC_014410|YP_003852930.1, Thermoanearobacterium thermosaccharolyticum, 48%;

cobGGBP, 94, NC_014392|YP_003839461.1, Caldicellosiruptor obsidiansis, 48%;

chyGGBP, 101, NC_014652|YP_003991244.1, Caldicellulosiruptor hydrothermalis, 47%;

pspGGBP, 112, NC_013406|YP_003243743.1, Paenibacillus sp., 44%;

ereGGBP, 123, NC_012781|YP_002936409.1, Eubacterium rectale, 37%;

cauGGBP, 127, NC_022592|CAETHG_2982, Clostridium autoethanogenum, 36%;

erhGGBP, 124, N_015601|YP_004561181.1, Erysipelthrix rhusiopathiae, 36%;

rinGGBP_B, 126, NC_021012|YP_00777811241, Roseburia intestinalis, 36%;

cljGGBP, 128, NC_014328|CLJU_c08950, Clostridium ljunghaldii, 35%;

fprGGBP, 129, NC_021020|YP_007799070.1, Faecalibacterium prausnitzii, 34%;

rinGGBP_A, 132, NC_021012|YP_007778116.1, Roseburia intestinalis, 33%;

bprGGBP, 137, NC_014387|YP_003830205.1, Butyvibrio proteoclasticus, 30%;

csaGGBP, 138, NC_014376|YP_003822565.1, Clostridium saccharolyticum, 29%.

Numbering according to ecGGBP. Light grey: leader peptides; dark grey, primary complementary surface (PCS) residues; —, position of insertions. Positions of the α helices (α_x), β sheets (β_x), inter-domain hinge segments (h_x), and calcium binding site EF hand (EF) observed in the ecGGBP structure are indicated.

FIGS. 4A and 4B are graphs relating to the analysis of glucose binding by ligand-mediated increases in protein thermostability. Protein (un)folding is monitored by binding of SYPRO to the unfolded state as a function of temperature. Fluorescence intensity increases upon binding of SYPRO by the unfolded state. (A) Temperature dependence of fluorescence emission intensity of csaGGBP (see Tables 1 and 2) in the presence of varying amounts of glucose. (B) The mid-point denaturation temperature, T_m, increases upon addition of glucose, indicative of glucose binding to the folded state of csaGGBP, as predicted

FIG. 5 is a series of graphs showing temperature- and glucose-dependent ratiometric fluorescent landscapes of the Acrylodan conjugates of GGBP homologs. Rows correspond to the E. coli (ecGGBP), T. thermosaccharolyticum (ttGGBP), C. obsidiansis (cobGGBP), and C. hydrothermalis (chyGGBP) glucose-galactose binding proteins site-specifically labeled with Acrylodan at the endosteric cysteine mutation replacing the tryptophan that contacts the glucose pyranose ring (W183 in ecGGPB). Column I: Three-dimensional landscapes representing the ratio of fluorescence emission intensities (Z axis) at 488 nm and 510 nm as a function of temperature and glucose concentration. Indicated are the main equilibrium states: N, native apo-protein; D, denatured protein; S, saturated glucose complex. Subscripts indicate temperature-dependent conformational sub-states (see main text). Dotted lines indicate approximate mid-points (i.e. ΔG=0) for the equilibria of the underlying reactions: black lines ( - - - ), binding; red (-⋅-⋅-), thermal denaturation; purple ( ⋅ ⋅ ⋅), thermal conformational changes. Column II: Examples of isothermal glucose-binding curves, determined at 310 K (37° C.). For each protein, two panels are shown: top, fit to the ratiometric signal (red line, fit to the ratiometric data only; blue line, joint fit to ratiometric and monochromatic emission intensities); bottom, fit to monochromatic emission intensities (red, 488 nm; blue, 510 nm).

FIG. 6A is a structure showing exemplary positions for introducing cysteine mutations to which fluorophores can be covalently coupled for reagentless biosensor construction are indicated: positions 157, 159, and 300, endosteric; positions 11, 16, 42, 67, 92, 111, 148, 181, and 183, peristeric; positions 17, 91, 151, and 182, allosteric. FIG. 6B shows a structure of Acrylodan. FIG. 6C shows a structure of Badan.

FIG. 7A-F is a series of graphs for glucose-dependent emission spectra of Acrylodan and Badan conjugates of Y11C, F17C and W182C mutants of ttGGBP. Corrected spectra (apo-protein, red; saturated glucose, purple; intermediate glucose concentrations, black): left column, Acrylodan; right column, Badan. Insets, fit of the ratiometric signal (equation 1 and 2; 20 nm integration bandwidth): blue circles, experimentally observed ratios; black line, calculated fit (baselines; apo-protein, constant; saturated glucose complex, linear). (A) Y11C⋅Acrylodan (isochromic; λ₁, 458 nm; λ₂, 511 nm; ^appK_d, 0.14 mM); (B) Y11C⋅Badan (hypsochromic; λ₁, 492 nm; λ₂, 528 nm; ^appK_d, 0.28 mM); (C) F17C⋅Acrylodan (hypsochromic; λ₁, 482 nm; λ₂, 545 nm; ^appK_d, 0.08 mM); (D) F17C⋅Badan (hypsochromic; λ₁, 470 nm; λ₂, 542 nm; ^appK_d, 0.18 mM); (E) F182C⋅Acrylodan (bathochromic; λ₁, 475 nm; λ₂, 545 nm; ^appK_d, 2.2 mM); (F) F182C⋅Badan (isochromic; λ_d, 474 nm; λ₂, 518 nm; ^appK_d, 26 mM).

FIGS. 8A-P are illustrations of fluorophore structures. Naphthalene family: (A) Acrylodan; (B) Badan; (C) IAEDANS. Xanthene family: (D) Fluorescein (5-IAF and 6-IAF); (E) Oregon Green; (F) Alexa 432; (G) Alexa 532; (H) Alexa 546; (I) Texas Red. Coumarin family: (J) Pacific Blue; (K) CPM. Benzoxadiazole family: (L) IANBD. Boradiazaindacine (BODIPY) family: (M) BODIPY 499/508; (N) BODIPY 507/545. Cyanine family: (O) CyS. Miscellaneous: (P) PyMPO.

FIGS. 9A-H are illustrations of structures of ttGGBP conjugates determined by X-ray crystallography. (A) Overview of the ttGGBP F17C⋅Badan conjugate, viewed from the back of the protein looking through the three hinge strands (h₁-h₃). W182 (magenta) forms extensive van der Waals contacts (translucent surface) with the bound glucose (cyan). The F17C mutation removes the wild-type contacts (translucent surface) between the benzene ring and glucose. The Badan conjugate (blue) points out of the glucose-binding site into the solvent. (B) Overview of the ttGGBP182C2.0⋅Acrylodan conjugate (note that the view point is different from (A). The conjugated Acrylodan also points out into solution. (C) Cutaway illustrating the channels surrounding the bound glucose within which Badan, Acrylodan, and the galactoside R-group (Sooriyaarachchi et al. 2009) (dark gray) are placed. The view is down the long axis of ttGGBP, looking at the glucose-binding surface of the N-terminal domain. The C-terminal domain (with Ca²⁺ site) has been cut away (bisecting the hinge). (D) The electronic densities of the Badan fluorophore and bound sugar are well defined (gray lines, 2F_o-F_celectron density at 1 σ contour levels). (E) Close-up side-view of Badan illustrates that the fluorophore carbonyl is twisted out the of the naphthalene ring plane. (F) End view, looking down the linker towards the exterior of the binding site shows that the carbonyl torsion is ˜28° (gray lines, 2F_o-F_celectron density at 1 σ contour levels; arrow, cysteine thiol). (G) The same view of the ecGGBP W183C⋅Acrylodan shows that in this fluorophore the carbonyl is co-planar with the naphthalene ring (blue lines, 2F_o-F_celectron density at 1 σ contour levels; arrow, cysteine thiol). (H) Close-up view of the fit between the fluorophore carbonyl knob into the hole (translucent surface) formed by van der Waals contacts with D238 and N258.

FIGS. 10A and B are structures relating to structure-guided design of affinity mutants. (A) The ttGGBP182C2.0 Acrylodan conjugate structure reveals the presence of a cavity filled with water and the cryoprotectant ethylene glycol, replacing the indole ring of the mutated tryptophan. (B) Inter-domain hydrogen bonds that have been mutated.

FIGS. 11A-C are graphs relating to the determination of ^trueK_dand ^appK_dvalues for glucose in ttGGBP182C.2.0 (see Table 5). (A) Glucose dependence of the fluorescence emission intensities for the ttGGBP182C.2.0⋅Acrylodan mutant conjugate (see Table 4). Arrows indicate direction of change with increased glucose concentrations. (B) Determination of ^appK_dusing ratiometry the emission intensities (R) at 475 nm and 545 nm (20 nm bandwidth). (C) Determination of ^trueK_dby monochromatic fits the two ratiometric intensities (I(λ): green, 475 nm; red, 545 nm). Values for ^appK_d(4.5 mM) and ^trueK_d(6.0 mM) were simultaneously fit to the three experimental binding isotherms using equations 1 and 5 using constant baselines for both the apo- and saturated protein, with the two monochromatic isotherms sharing the same ^trueK_dvalue.

FIGS. 12A and B are graphs showing that a sensor arrays that combine several ttGGBP mutants (Table 6) could span the full pathophysiological range at better than 90% of maximal possible precision. (A) Mutants in the ttGGBP182C Acrylodan background. The responses are shown as relative precision, calculated from the fits to the experimental data according to equation 8, from left to right: ttGBP182C, hypoglycemia; ttGGBP182C.2.0, euglycemia; ttGGBP182C.2.3, mild hyperglycemia; ttGGBP182C.8, severe hyperglycemia; ttGGBP182C.9, hyperosmolar hyperglycemic state. (B) Mutants in the ttGGBP17C Badan background. Ratiometric signals are shown from left to right: ttGGBP 17C⋅Acrylodan (^appK_d=0.06 mM), ttGGBP 17C.1⋅Badan (^appK_d=1.2 mM), ttGGBP 17C.3⋅Badan (^appK_d=3.9 mM), ttGGBP 17C.5⋅Badan (^appK_d=19.3 mM). Black circles: experimental data points

FIG. 13 is a set of cartoons showing fusion constructs for sensor immobilization. Light gray, ttGGBP182C.2.0; diagonal striped, hexa-lysine immobilization tag; dark gray, hexa-histidine affinity purification tag; horizontal striped, ZF-QNK zinc finger domain; vertical striped, truncated zinc finger βZif domain; wavy line, Gly-Gly-Ser linker (two segments, indicate Gly-Gly-Ser-Gly-Gly-Ser). Left column, names of constructs.

FIGS. 14A-F are graphs showing that the immobilization of ttGGBP182C2.0⋅Acrylodan does not affect its thermostability. Thermostabilities were determined by measuring the ratio fluorescence emission intensities through 488 nm and 510 nm filters as a function of temperature in a Roche LightCycler. Panels A-C: ttGBP182C.2.0-Imm0; Panels D-F: ttGBP182C.2.0-Imm1 (see FIG. 13). (A) Solution (T_m=347.6K). (B) Immobilized on Nickel-nitriloacetic acid (Ni-NTA) beads (T_m=346.7K). (C) Reconstituted, desiccated Ni-NTA beads (T_m=347.6K). (D) Fluorescence response of ttGGBP5 2 Imm1 immobilized on NHS-functionalized agarose beads. Data was collected on a Roche LightCycler real-time PCS instrument, recording emission intensities at 488 nm and 510 nm as a function of temperature in a 384-well microtiter plate. Wells contain beads with labelled, immobilized protein in hydrogel at different glucose concentrations. Temperature melts of twelve different wells. Apo protein thermostability as reported by the denaturation mid-point temperature is 349 K, similar to the protein free in solution. (E) The three-dimensional landscape showing ratiometric signal as a function of glucose concentration and temperature is essentially indistinguishable from the protein free in solution. (F) Representative Langmuir ligand-binding curve extracted from the three-dimensional data. The K_d(glucose) value at 25° C. is 4.8 mM, which is similar to the protein free in solution.

FIG. 15 is a graph of a glucose titration curve determined for magnetic Ni-NTA beads coated with immobilized ttGGBP182C2.0⋅Acrylodan. Data were highly precise, being reproducible<0.2% coefficient of variance.

FIGS. 16A-F are grapgs showing the singular value decomposition of the glucose-dependent corrected spectra of Acrylodan and Badan conjugates of Y11C, F17C and W182C mutants of ttGGBP. (A) Y11C⋅Acrylodan; (B) Y11C⋅Badan; (C) F17C⋅Acrylodan; (D) F17C⋅Badan; (E) F182C⋅Acrylodan; (F) F182C⋅Badan. Frequency transformations of the spectra (equation 11) were decomposed into principal components (equation 12). The contribution of the first component, C₁(black), is largely invariant with glucose concentration, whereas the second component, C₂(gray), encode accounts for the glucose-dependent changes in the spectra (component contributions are given in Table 1). Inserts, glucose dependence of the fractional contribution of each component (equation 13).

FIGS. 17A-F are graphs showing glucose dependence of electronic transitions in the fluorescence emission intensity spectra of Acrylodan and Badan conjugates of Y11C, F17C and W182C mutants of ttGGBP. (A) Y11C⋅Acrylodan; (B) Y11C⋅Badan; (C) F17C⋅Acrylodan; (D) F17C⋅Badan; (E) F182C⋅Acrylodan; (F) F182C⋅Badan. The emission intensities can be accounted for to a first approximation by two excited state electronic transitions (FIG. 16): a low-energy, green, S₁(Acrylodan: 521±9 nm; Badan: 530±14 nm) and a high-energy, blue, S₂(Acrylodan: 477±14 nm; Badan: 477±16 nm) transition. The S₁transition is present in all spectra; S₂is present in all the glucose-responsive and 36% of the non-responsive conjugates (Table 7 and 8). In conjugates that exhibit glucose-responsive changes in fluorescence emission intensities, glucose binding shifts the population of these excited states. At each titration point, the experimentally observed emission intensities were modeled with Gaussians fits (equation 14) for S₁and S₂electronic transition (model parameter values in Table 3). Experimental emission spectra and Gaussian fits are shown only for the apo-protein, and saturated glucose complex. Emission spectra: dashed black line, apo-protein; solid black line, glucose complex. Gaussians (S₁, green lines; S₂, blue lines): dashed lines, apo-protein; solid lines, glucose complex. Thin black lines: residuals (equation 16) at each titration point. Inserts show the population fractions (equation 15) of the S₁and S₂transitions extracted from the spectra at each titration point (black circles) fit to Langmuir binding isotherms (solid lines) with ^appK_dvalues constrained to be the same for both populations. As a first approximation, the wavelengths of S₁or S₂transitions are the same in apo-protein and the saturated glucose complex. The residuals indicate that this approximation does not hold for all conjugates, and a more extensive treatment is required in which the S₁and S₂are split into multiple transitions (see discussion below). Wavelength shifts occur if there is a significant redistribution of the two excited state populations in the apo-protein and the saturated ligand complexes. In the bathochromic ratiometric 182C⋅Acrylodan conjugate, the S₁state dominates in the glucose complex (panel E); in the hypsochromic conjugates 17C⋅Badan (panel D) and 17C⋅Acrylodan (panel C), apo-proteins comprise a mixture of the two states, whereas their glucose complexes contain almost exclusively the S₂state. In the hypsochromic 11C⋅Badan sensor (panel B), the system shifts from a predominantly S₁in the absence of glucose to almost equally populated S₁and S₂states in the glucose complex. Shifts in the fraction of S₁and S₂populations also occur in conjugates that did not exhibit overt shifts in the wavelengths of their emission intensity maxima. For instance, neither 11C⋅Acrylodan nor 182C⋅Badan exhibited significant shifts in emission maxima upon binding glucose. Nevertheless, SVD analysis revealed similar changes in the variable C₂component as observed for the overtly ratiometric sensors, but with a smaller contribution (FIG. 16A and F). Gaussian analysis revealed small shifts in the S₁and S₂populations in response to glucose (panel A and F). This pattern also was observed in other non-ratiometrically responsive conjugates (Table 7 and 8). Wavelength shifts were observed if the C₂component constitutes>5% of the total contribution.

FIGS. 18A-H is a series of graphs showing Glucose dependence of the S₁and S₂electronic transition contributions to the fluorescence emission intensity spectra of E. coli GGBP Acrylodan and Badan conjugates. Color schemes, symbols and line representation as in FIGS. 2-4. 16C⋅Acrylodan: (A) emission spectra (hypsochromic) and ratiometric glucose-binding curve (inset: λ₁, 454 nm; λ₂, 540 nm; ^appK_d, 1.0 mM); (C) singular value decomposition; (E) Gausssian fits and glucose dependence of the S₁and S₂populations (inset). 16C⋅Badan: (B) emission spectra (hypsochromic) and ratiometric glucose-binding curve (inset: λ₁, 465 nm; λ₂, 555 nm; ^appK_d, 0.4 mM); (D) singular value decomposition; (F) Gausssian fits and glucose dependence of the S₁and S₂populations (inset). 182C⋅Acrylodan: (G) singular value decomposition; (H) Gausssian fits and glucose dependence of the S₁and S₂populations (inset).

FIGS. 19A-F are graphs showing glucose dependence of the absorption spectra of ttGGBP Acrylodan and Badan conjugates that undergo wavelength shifts in their fluorescence emission intensities in response to ligand binding. ttGGBP F17C⋅Badan: (A) Absorption spectra (red, no glucose; purple, saturating glucose; black, intermediate glucose concentrations). Hypsochromic response was modeled by fit of a Langmuir ligand-binding isotherm (equation 1) to ratiometric analysis of the absorption intensities (inset: λ₁, 390 nm; λ₂, 340 nm; ^appK_d, 1.7 mM); (C) singular value decomposition (components: black, C₁; gray, C₂); (E) Fit of Gaussian representations of electronic transitions to absorption spectra (dashed black line, apo-protein; solid black line, glucose complex) of G₁(386 nm green; apo-protein, dashed; glucose complex, solid) and G₂(359 nm, blue; apo-protein, dashed; glucose complex, solid) ground state electronic transitions. Inset, glucose dependence of population fractions at each glucose titration (black circles) of the G₁and G₂transitions fit to Langmuir binding isotherms (solid lines) with ^appK_dvalues constrained to be the same for both populations (residuals, thin lines). ttGGBP F182C⋅Acrylodan (color scheme, line representations, symbols, as above): (B) Absorption spectra (bathochromic response) and glucose binding (inset: λ₁, 375 nm; λ₂, 430 nm; ^appK_d, 1.1 mM; ^trueK_d, 1.9 mM); (D) singular value decomposition; (F) G₁and G₂electronic transitions and populations. The residuals indicate that the maximal wavelength of the G₁and G₂transitions is different in the apo-protein and glucose (also see legend to FIG. 17).

FIG. 20 shows the sequence of an exemplary csaGGBP (Table 2) expression construct (SEQ ID NO: 105).

FIG. 21 shows the sequence of an exemplary bprGGBP (Table 2) expression construct (SEQ ID NO: 106).

FIG. 22 shows the sequence of an exemplary rinGGBP_A (Table 2) expression construct (SEQ ID NO: 107).

FIG. 23 shows the sequence of an exemplary fprGGBP (Table 2) expression construct (SEQ ID NO: 108).

FIG. 24 shows the sequence of an exemplary cljGGBP (Table 2) expression construct (SEQ ID NO: 109).

FIG. 25 shows the sequence of an exemplary cauGGBP (Table 2) expression construct (SEQ ID NO: 110).

FIG. 26 shows the sequence of an exemplary rinGGBP_B (Table 2) expression construct (SEQ ID NO: 111).

FIG. 27 shows the sequence of an exemplary erhGGBP (Table 2) expression construct (SEQ ID NO: 112).

FIG. 28 shows the sequence of an exemplary ereGGBP (Table 2) expression construct (SEQ ID NO: 113).

FIG. 29 shows the sequence of an exemplary ttGGBP (Table 2) expression construct (SEQ ID NO: 114).

FIG. 30 shows the sequence of an exemplary cobGGBP (Table 2) expression construct (SEQ ID NO: 115).

FIG. 31 shows the sequence of an exemplary chyGGBP (Table 2) expression construct (SEQ ID NO: 116).

FIG. 32 shows the sequence of an exemplary pspGGBP (Table 2) expression construct (SEQ ID NO: 117).

FIG. 33 shows the sequence of an exemplary ttGGBP11C Cysteine Scan Mutant (Table 3) expression construct (SEQ ID NO: 118).

FIG. 34 shows the sequence of an exemplary ttGGBP16C Cysteine Scan Mutant (Table 3) expression construct (SEQ ID NO: 119).

FIG. 35 shows the sequence of an exemplary ttGGBP17C Cysteine Scan Mutant (Table 3) expression construct (SEQ ID NO: 120).

FIG. 36 shows the sequence of an exemplary ttGGBP42C Cysteine Scan Mutant (Table 3) expression construct (SEQ ID NO: 121).

FIG. 37 shows the sequence of an exemplary ttGGBP67C Cysteine Scan Mutant (Table 3) expression construct (SEQ ID NO: 122).

FIG. 38 shows the sequence of an exemplary ttGGBP91C Cysteine Scan Mutant (Table 3) expression construct (SEQ ID NO: 123).

FIG. 39 shows the sequence of an exemplary ttGGBP92C Cysteine Scan Mutant (Table 3) expression construct (SEQ ID NO: 124).

FIG. 40 shows the sequence of an exemplary ttGGBP111C Cysteine Scan Mutant (Table 3) expression construct (SEQ ID NO: 125).

FIG. 41 shows the sequence of an exemplary ttGGBP148C Cysteine Scan Mutant (Table 3) expression construct (SEQ ID NO: 126).

FIG. 42 shows the sequence of an exemplary ttGGBP151C Cysteine Scan Mutant (Table 3) expression construct (SEQ ID NO: 127).

FIG. 43 shows the sequence of an exemplary ttGGBP152C Cysteine Scan Mutant (Table 3) expression construct (SEQ ID NO: 128).

FIG. 44 shows the sequence of an exemplary ttGGBP181C Cysteine Scan Mutant (Table 3) expression construct (SEQ ID NO: 129).

FIG. 45 shows the sequence of an exemplary ttGGBP182C Cysteine Scan Mutant (Table 3) expression construct (SEQ ID NO: 130).

FIG. 46 shows the sequence of an exemplary ttGGBP183C Cysteine Scan Mutant (Table 3) expression construct (SEQ ID NO: 131).

FIG. 47 shows the sequence of an exemplary ttGGBP257C Cysteine Scan Mutant (Table 3) expression construct (SEQ ID NO: 132).

FIG. 48 shows the sequence of an exemplary ttGGBP259C Cysteine Scan Mutant (Table 3) expression construct (SEQ ID NO: 133).

FIG. 49 shows the sequence of an exemplary ttGGBP300C Cysteine Scan Mutant (Table 3) expression construct (SEQ ID NO: 134).

FIG. 50 shows the sequence of an exemplary ttGGBP17C.1 Affinity-Tuning Mutant (17C background) (Table 6) expression construct (SEQ ID NO: 135).

FIG. 51 shows the sequence of an exemplary ttGGBP17C.2 Affinity-Tuning Mutant (17C background) (Table 6) expression construct (SEQ ID NO: 136).

FIG. 52 shows the sequence of an exemplary ttGGBP17C.3 Affinity-Tuning Mutant (17C background) (Table 6) expression construct (SEQ ID NO: 137).

FIG. 53 shows the sequence of an exemplary ttGGBP17C.4 Affinity-Tuning Mutant (17C background) (Table 6) expression construct (SEQ ID NO: 138).

FIG. 54 shows the sequence of an exemplary ttGGBP17C.5 Affinity-Tuning Mutant (17C background) (Table 6) expression construct (SEQ ID NO: 139).

FIG. 55 shows the sequence of an exemplary ttGGBP17C.6 Affinity-Tuning Mutant (17C background) (Table 6) expression construct (SEQ ID NO: 140).

FIG. 56 shows the sequence of an exemplary ttGGBP17C.7 Affinity-Tuning Mutant (17C background) (Table 6) expression construct (SEQ ID NO: 141).

FIG. 57 shows the sequence of an exemplary ttGGBP17C.8 Affinity-Tuning Mutant (17C background) (Table 6) expression construct (SEQ ID NO: 142).

FIG. 58 shows the sequence of an exemplary ttGGBP17C.9 Affinity-Tuning Mutant (17C background) (Table 6) expression construct (SEQ ID NO: 143).

FIG. 59 shows the sequence of an exemplary ttGGBP17C.10 Affinity-Tuning Mutant (17C background) (Table 6) expression construct (SEQ ID NO: 144).

FIG. 60 shows the sequence of an exemplary ttGGBP17C.11 Affinity-Tuning Mutant (17C background) (Table 6) expression construct (SEQ ID NO: 145).

FIG. 61 shows the sequence of an exemplary ttGGBP17C.19 Affinity-Tuning Mutant (17C background) (Table 6) expression construct (SEQ ID NO: 146).

FIG. 62 shows the sequence of an exemplary ttGGBP17C.20 Affinity-Tuning Mutant (17C background) (Table 6) expression construct (SEQ ID NO: 147).

FIG. 63 shows the sequence of an exemplary ttGGBP17C.21 Affinity-Tuning Mutant (17C background) (Table 6) expression construct (SEQ ID NO: 148).

FIG. 64 shows the sequence of an exemplary ttGGBP17C.22 Affinity-Tuning Mutant (17C background) (Table 6) expression construct (SEQ ID NO: 149).

FIG. 65 shows the sequence of an exemplary ttGGBP17C.23 Affinity-Tuning Mutant (17C background) (Table 6) expression construct (SEQ ID NO: 150).

FIG. 66 shows the sequence of an exemplary ttGGBP17C.24 Affinity-Tuning Mutant (17C background) (Table 6) expression construct (SEQ ID NO: 151).

FIG. 67 shows the sequence of an exemplary ttGGBP17C.25 Affinity-Tuning Mutant (17C background) (Table 6) expression construct (SEQ ID NO: 152).

FIG. 68 shows the sequence of an exemplary ttGGBP17C.26 Affinity-Tuning Mutant (17C background) (Table 6) expression construct (SEQ ID NO: 153).

FIG. 69 shows the sequence of an exemplary ttGGBP17C.27 Affinity-Tuning Mutant (17C background) (Table 6) expression construct (SEQ ID NO: 154).

FIG. 70 shows the sequence of an exemplary ttGGBP17C.28 Affinity-Tuning Mutant (17C background) (Table 6) expression construct (SEQ ID NO: 155).

FIG. 71 shows the sequence of an exemplary ttGGBP17C.29 Affinity-Tuning Mutant (17C background) (Table 6) expression construct (SEQ ID NO: 156).

FIG. 72 shows the sequence of an exemplary ttGGBP182C.2.0 Affinity-Tuning Mutant (182C background) (Table 6) expression construct (SEQ ID NO: 157).

FIG. 73 shows the sequence of an exemplary ttGGBP182C.2.1 Affinity-Tuning Mutant (182C background) (Table 6) expression construct (SEQ ID NO: 158).

FIG. 74 shows the sequence of an exemplary ttGGBP182C.2.3 Affinity-Tuning Mutant (182C background) (Table 6) expression construct (SEQ ID NO: 159).

FIG. 75 shows the sequence of an exemplary ttGGBP182C.2.4 Affinity-Tuning Mutant (182C background) (Table 6) expression construct (SEQ ID NO: 160).

FIG. 76 shows the sequence of an exemplary ttGGBP182C.2.5 Affinity-Tuning Mutant (182C background) (Table 6) expression construct (SEQ ID NO: 161).

FIG. 77 shows the sequence of an exemplary ttGGBP182C.2.6 Affinity-Tuning Mutant (182C background) (Table 6) expression construct (SEQ ID NO: 162).

FIG. 78 shows the sequence of an exemplary ttGGBP182C.2.7 Affinity-Tuning Mutant (182C background) (Table 6) expression construct (SEQ ID NO: 163).

FIG. 79 shows the sequence of an exemplary ttGGBP182C.2.8 Affinity-Tuning Mutant (182C background) (Table 6) expression construct (SEQ ID NO: 164).

FIG. 80 shows the sequence of an exemplary ttGGBP182C.2.9 Affinity-Tuning Mutant (182C background) (Table 6) expression construct (SEQ ID NO: 165).

FIG. 81 shows the sequence of an exemplary ttGGBP182C.3 Affinity-Tuning Mutant (182C background) (Table 6) expression construct (SEQ ID NO: 166).

FIG. 82 shows the sequence of an exemplary ttGGBP182C.4 Affinity-Tuning Mutant (182C background) (Table 6) expression construct (SEQ ID NO: 167).

FIG. 83 shows the sequence of an exemplary ttGGBP182C.5 Affinity-Tuning Mutant (182C background) (Table 6) expression construct (SEQ ID NO: 168).

FIG. 84 shows the sequence of an exemplary ttGGBP182C.6 Affinity-Tuning Mutant (182C background) (Table 6) expression construct (SEQ ID NO: 169).

FIG. 85 shows the sequence of an exemplary ttGGBP182C.7 Affinity-Tuning Mutant (182C background) (Table 6) expression construct (SEQ ID NO: 170).

FIG. 86 shows the sequence of an exemplary ttGGBP182C.8 Affinity-Tuning Mutant (182C background) (Table 6) expression construct (SEQ ID NO: 171).

FIG. 87 shows the sequence of an exemplary ttGGBP182C.9 Affinity-Tuning Mutant (182C background) (Table 6) expression construct (SEQ ID NO: 172).

FIG. 88 is a cartoon illustrating non-limiting aspects of continuous glucose monitoring.

FIG. 89 is a cartoon illustrating a non-limiting example of sample analysis with a biosensor.

DETAILED DESCRIPTION

Fluorescently responsive sensors (FRSs) based on engineered proteins that couple ligand-binding events to changes in the emission properties of intrinsic or semi-synthetically incorporated chromophores have wide-ranging applications in cell biology and analytical chemistry. If the fluorescence emission spectrum of an engineered FRS changes shape in response to ligand binding such that the ratio of intensities at two appropriately chosen wavelengths reports on ligand concentration (dichromatic response), then ratiometric measurements can be used to monitor analyte concentrations. Ratiometry Advantage is essential for devices that rely on changes in fluorescence emission intensities, because it provides an internally consistent reference. The self-calibrating nature of a ratiometric measurement removes the necessity for carrying out on-board calibration tests prior to each measurement, obviating the need for multiple components and fluidic circuitry. Accordingly, reagentless, ratiometric fluorescent sensors have many uses in process engineering, environmental or clinical chemistry, including single-use point-of-care applications, wearable devices, or implanted “tattoos” that are interrogated transdermally.

The periplasmic binding protein (PBP) superfamily provide a rich source of FRSs, because PBPs combine a large diversity of ligand specificities with a common structural mechanism that is well suited to the construction of fluorescence signal transduction schemes. The three-dimensional PBP monomer structure comprises two α/β domains linked by a β-strand hinge FIG. 1A. Binding of ligand is accompanied by a large hinge-bending motion that transitions the protein from an open to a closed state in which the ligand is enveloped within a cleft between the two domains. Semi-synthetic FRSs can be engineered with PBPs by site-specifically attaching single, thiol-reactive, environmentally sensitive fluorophores that respond to the ligand-mediated conformational change (FIGS. 1A-C). Here we describe the construction of robust, ratiometric, fluorescently responsive glucose sensors derived from thermostable members of a family of glucose-binding PBPs.

Glucose monitoring is essential for the management of diabetes mellitus, a disease that affects at least 366 million people world-wide, increasing every year. The majority of current glucose-monitoring technologies rely on enzymes for which glucose is one of the substrates. Glucose concentration measurements are therefore subject to variations in second substrate concentrations consumed in the enzyme reaction, such as oxygen in the case of glucose oxidase. Additional complications arise in systems where reaction rates are measured for enzymes immobilized on electrodes. In such arrangements, accuracy is compromised by factors that alter the rate at which glucose arrives at the electrode surface interfere with accuracy, such as hematocrit levels, or surface “fouling” by deposition of proteins and cells in the foreign body response. Ratiometric fluorescent glucose sensors obviate these problems, and accordingly have been incorporated successfully in optodes for continuous glucose monitoring in animals and humans.

In FRS-based sensors, signals arise from reversible binding equilibria of the analyte (ligand) to a receptor. These signals are most precise at ligand concentrations that match the receptor ligand-disassociation constant. Precision is maintained to within ˜80% of this maximal level over a concentration range approximately 3-fold above or below this point. Construction of effective FRS therefore requires matching of ligand-binding affinities to the relevant analyte concentrations. Arrays of multiple sensors may have to be used in concert to cover wide concentration ranges. Clinically relevant glucose levels vary approximately 100-fold (from ˜1 mM in extreme hypoglycemia, to ˜100 mM for the hyperosmolar, hyperglycemic condition, with healthy, euglycemic levels at ˜6 mM(Association 2000 Clinical Diabetes, 18; Pasquel 2014 Diabetes Care, 37, 3124-3131), requiring an array of multiple FRS sensors with distinct glucose affinities to report directly on the full range of clinically relevant glucose concentrations with high precision. Here we report a set of appropriately tuned thermostable, glucose-responsive FRSs, constructed by mutating their glucose-binding site.

Immobilization of FRSs on solid surfaces with minimal perturbation of the molecular sensing mechanism is an important step for incorporating biosensors into devices. Immobilization enables retention of the sensor within the sampling element (e.g. optode surface or implanted bead for in vivo sensing applications; or in a sample-handling cartridge for ex vivo sensing) Immobilization also may provide spatial localization to provide the necessary addressability of different elements in a multi-sensor array comprising sensors that differ in their engineered affinities for coverage of a wide range of glucose concentrations, or sensors that each detect distinct analytes.

Ex vivo clinical chemistries such as point-of-care applications require that the FRS is incorporated into a cartridge into which a sample is introduced at the time of measurement. Such “disposables” need to have a long shelf life that preferably does not require temperature control (e.g. refrigeration) for storage or distribution. It is preferable to incorporate immobilized protein in a stable, dried form in such disposables. The inherent resistance to denaturation of thermostable proteins minimizes the need for temperature control during manufacturing and storage, and may extend to the long-term stability of a desiccated state. The protein sensors described herein meet these requirements.

The spectral response, binding affinity, and thermostability of the robust thermostable glucose FRSs reported here are maintained following site-specific immobilization on a substrate such as beads. Furthermore, these properties are recovered rapidly upon reconstitution following drying and prolonged storage under accelerated aging conditions. These engineered proteins therefore are useful for high-precision, wide-dynamic range glucose sensing applications, including continuous monitoring, point-of-care, wearable sensor systems.

Biosensors

Biosensors are molecular recognition elements that transduce ligand-binding events into physical signals. Biosensors as detailed herein bind at least one ligand and emit a signal. A ligand-bound biosensor results in a signal that is different from the unbound biosensor. This difference facilitates detection of the at least one ligand and/or determination of ligand concentration. The biosensors may be used without the assistance of other reagents.

Described herein are novel engineered biosensors. These biosensors may have altered ligand-binding affinities, tailored ligand-binding specificities, and/or temperature dependencies of ligand binding or stability. For example, the herein described engineered glucose and galactose biosensors provide high-accuracy information related to extended glucose concentration ranges.

Binding of ligand mediates conformational changes in the biosensor, such as hinge-bending motions of the polypeptide. The conformational changes affect the environment of the reporter such that a change in the reporter-generated signal occurs. That is, without ligand bound, the biosensor results in signal generated from the reporter, and when ligand is bound, the signal generated from the reporter changes. The ligand-bound biosensor results in a reporter-generated signal that is different from the unbound biosensor.

In some embodiments, the methods and compositions include a plurality of a single type of biosensor. The biosensors may be identical in structure and function. For example, the biosensors of a single type may have the same polypeptide, the same reporter, and the same ligand affinity.

In other embodiments, the methods and compositions include a plurality of different types of biosensors. A plurality of these different types of biosensors may be arranged or incorporated in a panel. As used herein, a “panel” refers to two or more biosensors. The two or more biosensors may be different from each other. The biosensors may differ in structure and/or function. Biosensors may differ in polypeptide sequence, reporter, ligand affinities, or a combination thereof. Accordingly, there may be different types of biosensors. In some embodiments, each biosensor in the panel comprises the same reporter group. In some embodiments, each biosensor in the panel comprises a different reporter group. The panel may include at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 biosensors.

The panel of biosensors includes at least one sensor element. “Sensor element” refers to a single spot, site, location, or well for the at least one biosensor, to which a sample or aliquot thereof may be applied. The panel may be a composite sensor or an array.

In some embodiments, the panel is a composite sensor. In a composite sensor, each sensor element includes a mixture of two or more different biosensors. In some embodiments, the composite sensor includes one sensor element. In some embodiments, the composite sensor includes two or more sensor elements. In some embodiments, signals are measured from a composite sensor in which the signals arise from one or more biosensors in the sensor element. For example, signals may be measured from a composite sensor in which the signals arise from a subset of the total number of biosensors in the sensor element. For example, signals may be measured from a composite sensor in which the signals arise from two of five biosensors in the sensor element.

In some embodiments, the panel is an array. In an array, each sensor element includes a single type of biosensor. An array comprises a plurality of individually and spatially localized sensor elements. Each sensor element includes a biosensor that is different than or the same as the biosensor of a different sensor element. In some embodiments, signals are measured from an array in which the signals arise separately from two or more selected biosensors in separate sensor elements. An array may comprise a plurality of sensor elements of a variety of sizes and configurations. An array may comprise a plurality of sensor elements arranged linearly. For example, an array may comprise a plurality of micrometer-sized sensor elements arranged in a single row. An array may comprise a plurality of sensor elements arranged in a grid. The grid may be two- or three-dimensional. In some embodiments, the grid is a spatially addressable grid. In some embodiments, the biosensors are incorporated into an array, such as a multichannel or multiplexed array.

The biosensors of the present disclosure can be used in any setting where glucose detection is required or desired, such a medical setting (e.g., determining the level of blood glucose in a subject), environmental setting (e.g., determining the level of glucose in an environmental sample), biological setting (e.g., determining the presence or amount of glucose in a reaction), or in process engineering, such as monitoring the amount of glucose in a fermentation reaction (e.g., beer/wine production, etc.). Other examples include, but are not limited to, uses in the food industry (Suleiman et al, In: Biosensor Design and Application: Mathewson and Finley Eds; American Chemical Society, Washington, D.C. 1992, vol. 511); in clinical chemistry (Wilkins et al., Med. Eng. Phys. 1996, 18, 273-288; Pickup, Tr. Biotech. 1993, 11, 285-291; Meyerhoff et al., Endricon 1966, 6, 51-58; Riklin et al., Nature 1995, 376, 672-675); Willner et al., J. Am. Chem. Soc. 1996, 118, 10321-10322); as the basis for the construction of a fluorescent flow cell containing immobilized GGBP-FAST conjugates (see, e.g., Wilkins et al., Med. Eng. Phys. 1966, 18, 273-288; Pickup, Tr. Biotech. 1993, 11, 285-291; Meyerhoff et al., Endricon. 1966, 6, 51; Group, New Engl. J. Med. 1993, 329, 977-986; Gough et al., Diabetes 1995, 44, 1005-1009); and in an implantable devices, such as those suitable for use as an artificial pancreas.

The biosensors as detailed herein may be administered in a variety of ways known by those of skill in the art, as appropriate for each application. Biosensors may be provided in a solution. The solution may be buffered. Biosensors may be provided in a solution and mixed directly with a sample. In some embodiments, a biosensor is immobilized onto a surface. Biosensors may be immobilized within a disposable cartridge into which a sample may be introduced or applied. Biosensors may be implanted or incorporated in a wearable device. The biosensor may be provided as an optode.

The biosensor may be attached to or incorporated in a wearable device. Wearable devices may include, for example, adhesive strips, patches, and contact lenses. The biosensor may be configured for placement in contact with a subject's skin or mucosal surface. In some embodiments, the biosensor is configured as an adhesive strip. In some embodiments, the biosensor is configured within or on the surface of a contact lens. In some embodiments, the contact lens is formed from a transparent substrate shaped to be worn directly over a subject's eye, as described in, for example, U.S. Pat. No. 8,608,310.

The biosensor may be implanted. The biosensor may be implanted in a subject's body. The biosensor may be implanted in a subject's blood vessel, vein, eye, natural or artificial pancreas, skin, or anywhere in the alimentary canal including the stomach, intestine and esophagus. The biosensor may be implanted in a subject with a microbead. In some embodiments, the biosensor is configured to be implanted in the skin. The biosensor may be implanted in a subject sub-dermally. The biosensor may generate the signal trans-dermally. In some embodiments, the biosensor may be implanted in a subject with transdermal microbeads, wherein the optical signals can be transmitted remotely between the biosensor and detecting device.

In some embodiments, the biosensor is administered as an optode. As used herein, “optode” refers to an optical fiber with a single biosensor, or a composite biosensor, immobilized at the surface or at the end. An “optode” may also be referred to as an “optrode.” In some embodiments, the biosensor is implanted in a subject as an optode. The optode may be incorporated with or into a needle. The optode may be incorporated with a probe such as endoscopy or colonoscopy probes. The optode may be used in a tumor, near a tumor, or at the periphery of a tumor. In some embodiments, the biosensor may be implanted in a subject as an optode, wherein the optical signals can be transmitted between the biosensor and detecting device using physical links. In some embodiments, the biosensor is administered as an optode to a sample or reaction. The optode may be contacted with a sample or reaction. In some embodiments, an optode is used to continuously or episodically monitor a ligand in a sample or reaction.

Methods of Detecting the Presence of a Ligand

Provided herein is a method of detecting the presence of a ligand in a sample. The method may include contacting the biosensor with the sample; measuring a signal from the biosensor; and comparing the signal to a ligand-free control. A difference in signal indicates the presence of ligand in the sample.

Also provided herein is a method of detecting the presence of glucose in a sample. The method may include (a) providing a glucose biosensor disclosed herein in which the reporter group is attached the GGBP so that a signal transduced by the reporter group when the GGBP is bound to glucose differs from a signal transduced by the reporter group when the GGBP is not bound to glucose; (b) contacting the biosensor with the test sample under conditions such that the biosensor can bind to glucose present in the test sample; and (c) comparing the signal transduced by the reporter group when the biosensor is contacted with the test sample with the signal transduced by the reporter group when the biosensor is contacted with a glucose-free control sample, wherein a difference in the signal transduced by the reporter group when the biosensor is contacted with the test sample, as compared to when the biosensor is contacted with the control sample, indicates that the test sample contains glucose.

Methods of Determining the Concentration of a Ligand

Provided herein is a method of determining the concentration of a ligand in a sample. The method may include contacting the biosensor with the sample; measuring a signal from the biosensor; and comparing the signal to a standard hyperbolic ligand binding curve to determine the concentration of ligand in the test sample. The standard hyperbolic ligand binding curve may be prepared by measuring the signal transduced by the biosensor when contacted with control samples containing known concentrations of ligand.

Another aspect of the present disclosure provides a method of determining the concentration of glucose in a test sample comprising, consisting of, or consisting essentially of: (a) providing a glucose biosensor comprising a glucose biosensor as described herein in which the reporter group is attached the GGBP so that a signal transduced by the reporter group when the GGBP is bound to glucose differs from a signal transduced by the reporter group when the GGBP is not bound to glucose; (b) contacting the biosensor with the test sample under conditions such that the biosensor can bind to glucose present in the test sample; and (c) comparing the signal transduced by the reporter group when the biosensor is contacted with the test sample with a standard hyperbolic glucose binding curve prepared by measuring the signal transduced by the reporter group when the biosensor is contacted with control samples containing known quantities of glucose to determine the concentration of glucose in the test sample.

Methods of Monitoring the Presence of a Ligand

The present invention is directed to a method of episodically or continuously monitoring the presence of a ligand in a reaction. In certain embodiments, the biosensors may be used in the continuous monitoring of glucose in a reaction. In certain embodiments, the glucose sensors may be used in episodic monitoring of sample aliquots.

The method of episodically or continuously monitoring the presence of a ligand in a reaction may include contacting the biosensor with the reaction; maintaining the reaction under conditions such that the polypeptide is capable of binding ligand present in the reaction; episodically or continuously monitoring the signal from the biosensor in the reaction; and comparing the signal to a standard hyperbolic ligand binding curve to determine the concentration of ligand in the test sample. The standard hyperbolic ligand binding curve may be prepared by measuring the signal transduced by the biosensor when contacted with control samples containing known concentrations of ligand.

In some embodiments, the method further includes comparing the signal to a ligand-free control, wherein a difference in signal indicates the presence of ligand in the reaction.

In some embodiments, the method further includes comparing the signal to a standard hyperbolic ligand binding curve to determine the concentration of ligand in the test sample. The standard hyperbolic ligand binding curve may be prepared by measuring the signal transduced by the biosensor when contacted with control samples containing known concentrations of ligand.

Another aspect of the present disclosure provides a method of continuously monitoring the presence of glucose in a reaction comprising, consisting of, or consisting essentially of: (a) providing a glucose biosensor as described herein in which the reporter group is attached the GGBP so that a signal transduced by the reporter group when the GGBP is bound to glucose differs from a signal transduced by the reporter group when the GGBP is not bound to glucose; (b) maintaining the biosensor within the reaction and under conditions such that the biosensor can bind to glucose present in the reaction; (c) continuously monitoring the signal transduced by the reporter group when the biosensor is contacted with the glucose present in the reaction; and optionally (d) comparing the signal transduced by the reporter group when the biosensor is contacted with the glucose present in the reaction with the signal transduced by the reporter group when the biosensor is contacted with a glucose-free control sample, wherein a difference in the signal transduced by the reporter group when the biosensor is contacted with the glucose present in the reaction, as compared to when the biosensor is contacted with the control sample, indicates glucose is present in the reaction.

Yet another aspect of the present disclosure provides a method of continuously monitoring the concentration of glucose in a reaction comprising, consisting of, or consisting essentially of: (a) providing a glucose biosensor comprising a glucose biosensor as described herein in which the reporter group is attached the GGBP so that a signal transduced by the reporter group when the GGBP is bound to glucose differs from a signal transduced by the reporter group when the GGBP is not bound to glucose; (b) maintaining the biosensor within the reaction under conditions such that the biosensor can bind to glucose present in the reaction; and (c) continuously monitoring the signal transduced by the reporter group when the biosensor is contacted with the glucose present in the reaction; and (d) comparing the signal transduced by the reporter group when the biosensor is contacted with the glucose present in the reaction with a standard hyperbolic glucose binding curve prepared by measuring the signal transduced by the reporter group when the biosensor is contacted with control samples containing known quantities of glucose to determine the concentration of glucose in the reaction.

General Definitions

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, and biochemistry).

As used herein, the term “about” in the context of a numerical value or range means ±10% of the numerical value or range recited or claimed, unless the context requires a more limited range.

In the descriptions above and in the claims, phrases such as “at least one of” or “one or more of” may occur followed by a conjunctive list of elements or features. The term “and/or” may also occur in a list of two or more elements or features. Unless otherwise implicitly or explicitly contradicted by the context in which it is used, such a phrase is intended to mean any of the listed elements or features individually or any of the recited elements or features in combination with any of the other recited elements or features. For example, the phrases “at least one of A and B;” “one or more of A and B;” and “A and/or B” are each intended to mean “A alone, B alone, or A and B together.” A similar interpretation is also intended for lists including three or more items. For example, the phrases “at least one of A, B, and C;” “one or more of A, B, and C;” and “A, B, and/or C” are each intended to mean “A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together.” In addition, use of the term “based on,” above and in the claims is intended to mean, “based at least in part on,” such that an unrecited feature or element is also permissible

It is understood that where a parameter range is provided, all integers within that range, and tenths thereof, are also provided by the invention. For example, “0.2-5 mg” is a disclosure of 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg etc. up to and including 5.0 mg.

A small molecule is a compound that is less than 2000 daltons in mass. The molecular mass of the small molecule is preferably less than 1000 daltons, more preferably less than 600 daltons, e.g., the compound is less than 500 daltons, 400 daltons, 300 daltons, 200 daltons, or 100 daltons.

As used herein, an “isolated” or “purified” nucleic acid molecule, polynucleotide, polypeptide, or protein, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. Purified compounds are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. For example, a purified compound is one that is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the desired compound by weight. Purity is measured by any appropriate standard method, for example, by column chromatography, thin layer chromatography, or high-performance liquid chromatography (HPLC) analysis. A purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) is free of the genes/nucleic acids or sequences/amino acids that flank it in its naturally-occurring state. Purified also defines a degree of sterility that is safe for administration to a human subject, e.g., lacking infectious or toxic agents. In the case of tumor antigens, the antigen may be purified or a processed preparation such as a tumor cell lysate.

Similarly, by “substantially pure” is meant a nucleotide or polypeptide that has been separated from the components that naturally accompany it. Typically, the nucleotides and polypeptides are substantially pure when they are at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, free from the proteins and naturally-occurring organic molecules with they are naturally associated.

The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention.

“Subject” as used herein refers to any organism from which a biological sample is obtained. For example, the sample is a biological fluid or tissue. For example, a subject is one who wants or is in need of detecting ligand or determining the concentration of ligand with the herein described biosensors. The subject may be a human or a non-human animal. The subject may be a mammal. The mammal may be a primate or a non-primate. The mammal can be a primate such as a human; a non-primate such as, for example, dog, cat, horse, cow, pig, mouse, rat, camel, llama, goat, rabbit, sheep, hamster, and guinea pig; or non-human primate such as, for example, monkey, chimpanzee, gorilla, orangutan, and gibbon. The subject may be of any age or stage of development, such as, for example, an adult, an adolescent, or an infant.

As used herein, an “expression vector” is a DNA or RNA vector that is capable of effecting expression of one or more polynucleotides. Preferably, the expression vector is also capable of replicating within the host cell. Expression vectors can be either prokaryotic or eukaryotic, and are typically include plasmids. Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in host cells of the present invention, including in one of the prokaryotic or eukaryotic cells described herein, e.g., gram-positive, gram-negative, pathogenic, non-pathogenic, commensal, cocci, bacillus, or spiral-shaped bacterial cells; archaeal cells; or protozoan, algal, fungi, yeast, plant, animal, vertebrate, invertebrate, arthropod, mammalian, rodent, primate, or human cells. Expression vectors of the present invention contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the host cell and that control the expression of a polynucleotide. In particular, expression vectors of the present invention include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art.

As used herein, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a disease,” “a disease state”, or “a nucleic acid” is a reference to one or more such embodiments, and includes equivalents thereof known to those skilled in the art and so forth.

As used herein, “pharmaceutically acceptable” carrier or excipient refers to a carrier or excipient that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio. It can be, e.g., a pharmaceutically acceptable solvent, suspending agent or vehicle, for delivering the instant compounds to the subject.

The term “diagnosis” refers to a determination that a disease is present in the subject. Similarly, the term “prognosis” refers to a relative probability that a certain future outcome may occur in the subject. For example, in the context of the present disclosure, prognosis can refer to the likelihood that an individual will develop a disease, or the likely severity of the disease (e.g., severity of symptoms, rate of functional decline, survival, etc.).

Depending on context, the terms “polypeptide” and “protein” may be used interchangeably.

A polypeptide or class of polypeptides may be defined by the extent of identity (% identity) of its amino acid sequence to a reference amino acid sequence, or by having a greater % identity to one reference amino acid sequence than to another. A variant of any of genes or gene products disclosed herein may have, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid or amino acid sequences described herein. The term “% identity,” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. For example, % identity is relative to the entire length of the coding regions of the sequences being compared, or the length of a particular fragment or functional domain thereof. Variants as disclosed herein also include homologs, orthologs, or paralogs of the genes or gene products described herein. In some embodiments, variants may demonstrate a percentage of homology or identity, for example, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity conserved domains important for biological function, e.g., in a functional domain, e.g. a ligand-binding or catalytic domain.

For sequence comparison, one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Percent identity is determined using BLAST. For the BLAST searches, the following parameters were employed: (1) Expect threshold is 10; (2) Gap cost is Existence:11 and Extension:1; (3) The Matrix employed is BLOSUM62; (4) The filter for low complexity regions is “on.”

The present invention also provides for functional fragments of the genes or gene products described herein. A fragment of a protein is characterized by a length (number of amino acids) that is less than the length of the full length mature form of the protein. A fragment, in the case of these sequences and all others provided herein, may be a part of the whole that is less than the whole. Moreover, a fragment ranges in size from a single nucleotide or amino acid within a polynucleotide or polypeptide sequence to one fewer nucleotide or amino acid than the entire polynucleotide or polypeptide sequence. Finally, a fragment is defined as any portion of a complete polynucleotide or polypeptide sequence that is intermediate between the extremes defined above.

For example, fragments of any of the proteins or enzymes disclosed herein or encoded by any of the genes disclosed herein can be 10 to 20 amino acids, 10 to 30 amino acids, 10 to 40 amino acids, 10 to 50 amino acids, 10 to 60 amino acids, 10 to 70 amino acids, 10 to 80 amino acids, 10 to 90 amino acids, 10 to 100 amino acids, 50 to 100 amino acids, 75 to 125 amino acids, 100 to 150 amino acids, 150 to 200 amino acids, 200 to 250 amino acids, 250 to 300 amino acids, or 300 to 350 amino acids. The fragments encompassed in the present subject matter comprise fragments that retain functional fragments. As such, the fragments preferably retain the binding domains that are required or are important for functional activity. Fragments can be determined or generated by using the sequence information herein, and the fragments can be tested for functional activity using standard methods known in the art. For example, the encoded protein can be expressed by any recombinant technology known in the art and the binding activity of the protein can be determined.

As used herein a “biologically active” fragment is a portion of a polypeptide which maintains an activity of a full-length reference polypeptide. Biologically active fragments as used herein exclude the full-length polypeptide. Biologically active fragments can be any size as long as they maintain the defined activity. Preferably, the biologically active fragment maintains at least 10%, at least 50%, at least 75% or at least 90%, of the activity of the full length protein.

Amino acid sequence variants/mutants of the polypeptides of the defined herein can be prepared by introducing appropriate nucleotide changes into a nucleic acid defined herein, or by in vitro synthesis of the desired polypeptide. Such variants/mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence. A combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final peptide product possesses the desired activity and/or specificity.

Mutant (altered) peptides can be prepared using any technique known in the art. For example, a polynucleotide defined herein can be subjected to in vitro mutagenesis or DNA shuffling techniques as broadly described by Harayama (1998). Products derived from mutated/altered DNA can readily be screened using techniques described herein to determine if they possess, for example, glucose and/or galactose binding activity.

In designing amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified. The sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site.

Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues. In some embodiments, a mutated or modified protein does not comprise any deletions or insertions. In various embodiments, a mutated or modified protein has less than about 10, 9, 8, 7, 6, 5, 4, 3, or 2 deleted or inserted amino acids.

Substitution mutants have at least one amino acid residue in the polypeptide molecule removed and a different residue inserted in its place. Sites may be substituted in a relatively conservative manner in order to maintain activity and/or specificity. Such conservative substitutions are shown in the table below under the heading of “exemplary substitutions.”

In certain embodiments, a mutant/variant polypeptide has only, or not more than, one or two or three or four conservative amino acid changes when compared to a naturally occurring polypeptide. Details of conservative amino acid changes are provided in the table below. As the skilled person would be aware, such minor changes can reasonably be predicted not to alter the activity of the polypeptide when expressed in a recombinant cell.

Exemplary Substitutions

Original Residue
Exemplary Substitutions

Alanine (Ala)
Val; Leu; Ile; Gly

Arginine (Arg)
Lys

Asparagine (Asn)
Gln; His

Cysteine (Cys)
Ser

Glutamine (Gln)
Asn; His

Glutamic Acid (Glu)
Asp

Glycine (Gly)
Pro; Ala

Histidine (His)
Asn; Gln

Isoleucine (Ile)
Leu; Val; Ala

Leucine (Leu)
Ile; Val; Met; Ala; Phe

Lysine (Lys)
Arg

Methionine (Met)
Leu; Phe

Phenylalanine (Phe)
Leu; Val; Ala

Proline (Pro)
Gly

Serine (Ser)
Thr

Threonine (Thr)
Ser

Tryptophan (Trp)
Tyr

Tyrosine (Tyr)
Trp; Phe

Valine (Val)
Ile; Leu; Met; Phe; Ala

Mutations can be introduced into a nucleic acid sequence such that the encoded amino acid sequence is altered by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Certain amino acids have side chains with more than one classifiable characteristic. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, tryptophan, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tyrosine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a given polypeptide is replaced with another amino acid residue from the same side chain family Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a given coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for given polypeptide biological activity to identify mutants that retain activity. Conversely, the invention also provides for variants with mutations that enhance or increase the endogenous biological activity. Following mutagenesis of the nucleic acid sequence, the encoded protein can be expressed by any recombinant technology known in the art and the activity/specificity of the protein can be determined. An increase, decrease, or elimination of a given biological activity of the variants disclosed herein can be readily measured by the ordinary person skilled in the art, i.e. , by measuring the capability for binding a ligand and/or signal transduction.

In various embodiments, a polypeptide comprises mutations such that 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or less than about 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acids is substituted with a cysteine and/or a lysine.

Polypeptides can be produced in a variety of ways, including production and recovery of natural polypeptides or recombinant polypeptides according to methods known in the art. In one embodiment, a recombinant polypeptide is produced by culturing a cell capable of expressing the polypeptide under conditions effective to produce the polypeptide, such as a host cell defined herein.

Key to the Sequence Listing

SEQ

ID NO
Sequence Name

1
ecGGBP [U.S. National Center for Biotechnology Information (NCBI)

Accession No. WP_032329053]

2
ttGGBP (NCBI Accession Nos. YP_003852930.1 and WP_013298803.1)

3
stGGBP (NCBI Accession No. WP_001036943)

4
chyGGBP (NCBI Accession Nos. WP_013402088.1 and

YP_003991244.1)

5
cobGGBP (NCBI Accession Nos. WP_013289482.1 and

YP_003839461.1)

6
pspGGBP (NCBI Accession Nos. WP_015735911.1 and

YP_003243743.1)

7
csaGGBP (NCBI Accession Nos. WP_013273028.1 and

YP_003822565.1)

8
bprGGBP (NCBI Accession Nos. WP_013280279.1 and

YP_003830205.1)

9
rinGGBP_A (NCBI Accession Nos. WP_006855636.1 and

YP_007778116.1)

10
fprGGBP (NCBI Accession Nos. WP_015536639.1 and

YP_007799070.1)

11
cljGGBP (NCBI Accession No. CLJU_c08950)

12
cauGGBP (NCBI Accession No. CAETHG_2989)

13
rinGGBP_B (NCBI Accession Nos. WP_006855628.1 and

YP_007778124.1)

14
erhGGBP (NCBI Accession Nos. WP_003775352.1 and

YP_004561181.1)

15
ereGGBP (NCBI Accession Nos. WP_012741392.1 and

YP_002936409.1)

16
ecGGBP (with signal peptide replaced with M)

17
ecGGBP (with signal sequence removed)

18
ttGGBP (with signal peptide replaced with M)

19
stGGBP (with signal peptide replaced with M)

20
chyGGBP (with signal peptide replaced with M)

21
cobGGBP (with signal peptide replaced with M)

22
pspGGBP (with signal peptide replaced with M)

23
csaGGBP (with signal peptide replaced with M)

24
bprGGBP (with signal peptide replaced with M)

25
rinGGBP_A (with signal peptide replaced with M)

26
fprGGBP (with signal peptide replaced with M)

27
cljGGBP (with signal peptide replaced with M)

28
cauGGBP (with signal peptide replaced with M)

29
rinGGBP_b (with signal peptide replaced with M)

30
erhGGBP (with signal peptide replaced with M)

31
ereGGBP (with signal peptide replaced with M)

32
ecGGBPW183C (with signal peptide replaced with M; a W183C

mutation; and a GGSHHHHHH at C-terminus)

33
ecGGBP (with signal peptide replaced with M and a GGSHHHHHH at C-

terminus)

34
csaGGBP (with signal peptide replaced with M and a GGSHHHHHH at

C-terminus)

35
bprGGBP (with signal peptide replaced with M and a GGSHHHHHH at

C-terminus)

36
rinGGBP_A (with signal peptide replaced with M and a GGSHHHHHH

at C-terminus)

37
fprGGBP (with signal peptide replaced with M and a GGSHHHHHH at

C-terminus)

38
cljGGBP (with signal peptide replaced with M and a GGSHHHHHH at C-

terminus)

39
cauGGBP (with signal peptide replaced with M and a GGSHHHHHH at

C-terminus)

40
rinGGBP_B (with signal peptide replaced with M and a GGSHHHHHH at

C-terminus)

41
erhGGBP (with signal peptide replaced with M and a GGSHHHHHH at

C-terminus)

42
ereGGBP (with signal peptide replaced with M and a GGSHHHHHH at

C-terminus)

43
ttGGBP (with signal peptide replaced with M and a GGSHHHHHH at C-

terminus)

44
cobGGBP (with signal peptide replaced with M and a GGSHHHHHH at

C-terminus)

45
chyGGBP (with signal peptide replaced with M and a GGSHHHHHH at

C-terminus)

46
pspGGBP (with signal peptide replaced with M and a GGSHHHHHH at

C-terminus)

47
cobGGBP.W181C (Signaling cys mutant of cobGGBP with signal peptide

replaced with M and GGSHHHHHH at C-terminus)

48
chyGGBP.W181C (Signaling cys mutants of chyGGBP with signal

peptide replaced with M and GGSHHHHHH at C-terminus)

49
ttGGBP11C (ttGGBP cys mutant with signal peptide replaced with M and

GGSHHHHHH at C-terminus)

50
ttGGBP16C (ttGGBP cys mutant with signal peptide replaced with M and

GGSHHHHHH at C-terminus)

51
ttGGBP17C (ttGGBP cys mutant with signal peptide replaced with M and

GGSHHHHHH at C-terminus)

52
ttGGBP42C (ttGGBP cys mutant with signal peptide replaced with M and

GGSHHHHHH at C-terminus)

53
ttGGBP67C (ttGGBP cys mutant with signal peptide replaced with M and

GGSHHHHHH at C-terminus)

54
ttGGBP91C (ttGGBP cys mutant with signal peptide replaced with M and

GGSHHHHHH at C-terminus)

55
ttGGBP92C (ttGGBP cys mutant with signal peptide replaced with M and

GGSHHHHHH at C-terminus)

56
ttGGBP111C (ttGGBP cys mutant with signal peptide replaced with M

and GGSHHHHHH at C-terminus)

57
ttGGBP148C (ttGGBP cys mutant with signal peptide replaced with M

and GGSHHHHHH at C-terminus)

58
ttGGBP151C (ttGGBP cys mutant with signal peptide replaced with M

and GGSHHHHHH at C-terminus)

59
ttGGBP152C (ttGGBP cys mutant with signal peptide replaced with M

and GGSHHHHHH at C-terminus)

60
ttGGBP181C (ttGGBP cys mutant with signal peptide replaced with M

and GGSHHHHHH at C-terminus)

61
ttGGBP182C (ttGGBP cys mutant with signal peptide replaced with M

and GGSHHHHHH at C-terminus)

62
ttGGBP183C (ttGGBP cys mutant with signal peptide replaced with M

and GGSHHHHHH at C-terminus)

63
ttGGBP257C (ttGGBP cys mutant with signal peptide replaced with M

and GGSHHHHHH at C-terminus)

64
ttGGBP259C (ttGGBP cys mutant with signal peptide replaced with M

and GGSHHHHHH at C-terminus)

65
ttGGBP300C (ttGGBP cys mutant with signal peptide replaced with M

and GGSHHHHHH at C-terminus)

66
ttGGBP17C.1 (affinity-tuning mutant, 17C background (Table 6) with

signal peptide replaced with M and GGSHHHHHH at C-terminus)

67
ttGGBP17C.2 (affinity-tuning mutant, 17C background (Table 6) with

signal peptide replaced with M and GGSHHHHHH at C-terminus)

68
ttGGBP17C.3 (affinity-tuning mutant, 17C background (Table 6) with

signal peptide replaced with M and GGSHHHHHH at C-terminus)

69
ttGGBP17C.4 (affinity-tuning mutant, 17C background (Table 6) with

signal peptide replaced with M and GGSHHHHHH at C-terminus)

70
ttGGBP17C.5 (affinity-tuning mutant, 17C background (Table 6) with

signal peptide replaced with M and GGSHHHHHH at C-terminus)

71
ttGGBP17C.6 (affinity-tuning mutant, 17C background (Table 6) with

signal peptide replaced with M and GGSHHHHHH at C-terminus)

72
ttGGBP17C.7 (affinity-tuning mutant, 17C background (Table 6) with

signal peptide replaced with M and GGSHHHHHH at C-terminus)

73
ttGGBP17C.8 (affinity-tuning mutant, 17C background (Table 6) with

signal peptide replaced with M and GGSHHHHHH at C-terminus)

74
ttGGBP17C.9 (affinity-tuning mutant, 17C background (Table 6) with

signal peptide replaced with M and GGSHHHHHH at C-terminus)

75
ttGGBP17C.10 (affinity-tuning mutant, 17C background (Table 6) with

signal peptide replaced with M and GGSHHHHHH at C-terminus)

76
ttGGBP17C.11 (affinity-tuning mutant, 17C background (Table 6) with

signal peptide replaced with M and GGSHHHHHH at C-terminus)

77
ttGGBP17C.19 (affinity-tuning mutant, 17C background (Table 6) with

signal peptide replaced with M and GGSHHHHHH at C-terminus)

78
ttGGBP17C.20 (affinity-tuning mutant, 17C background (Table 6) with

signal peptide replaced with M and GGSHHHHHH at C-terminus)

79
ttGGBP17C.21 (affinity-tuning mutant, 17C background (Table 6) with

signal peptide replaced with M and GGSHHHHHH at C-terminus)

80
ttGGBP17C.22 (affinity-tuning mutant, 17C background (Table 6) with

signal peptide replaced with M and GGSHHHHHH at C-terminus)

81
ttGGBP17C.23 (affinity-tuning mutant, 17C background (Table 6) with

signal peptide replaced with M and GGSHHHHHH at C-terminus)

82
ttGGBP17C.24 (affinity-tuning mutant, 17C background (Table 6) with

signal peptide replaced with M and GGSHHHHHH at C-terminus)

83
ttGGBP17C.25 (affinity-tuning mutant, 17C background (Table 6) with

signal peptide replaced with M and GGSHHHHHH at C-terminus)

84
ttGGBP17C.26 (affinity-tuning mutant, 17C background (Table 6) with

signal peptide replaced with M and GGSHHHHHH at C-terminus)

85
ttGGBP17C.27 (affinity-tuning mutant, 17C background (Table 6) with

signal peptide replaced with M and GGSHHHHHH at C-terminus)

86
ttGGBP17C.28 (affinity-tuning mutant, 17C background (Table 6) with

signal peptide replaced with M and GGSHHHHHH at C-terminus)

87
ttGGBP17C.29 (affinity-tuning mutant, 17C background (Table 6) with

signal peptide replaced with M and GGSHHHHHH at C-terminus)

88
ttGGBP182C.2.0 (affinity-tuning mutant, 182C background (Table 6)

with signal peptide replaced with M and GGSHHHHHH at C-terminus)

89
ttGGBP182C.2.1 (affinity-tuning mutant, 182C background (Table 6)

with signal peptide replaced with M and GGSHHHHHH at C-terminus)

90
ttGGBP182C.2.3 (affinity-tuning mutant, 182C background (Table 6)

with signal peptide replaced with M and GGSHHHHHH at C-terminus)

91
ttGGBP182C.2.4 (affinity-tuning mutant, 182C background (Table 6)

with signal peptide replaced with M and GGSHHHHHH at C-terminus)

92
ttGGBP182C.2.5 (affinity-tuning mutant, 182C background (Table 6)

with signal peptide replaced with M and GGSHHHHHH at C-terminus)

93
ttGGBP182C.2.6 (affinity-tuning mutant, 182C background (Table 6)

with signal peptide replaced with M and GGSHHHHHH at C-terminus)

94
ttGGBP182C.2.7 (affinity-tuning mutant, 182C background (Table 6)

with signal peptide replaced with M and GGSHHHHHH at C-terminus)

95
ttGGBP182C.2.8 (affinity-tuning mutant, 182C background (Table 6)

with signal peptide replaced with M and GGSHHHHHH at C-terminus)

96
ttGGBP182C.2.9 (affinity-tuning mutant, 182C background (Table 6)

with signal peptide replaced with M and GGSHHHHHH at C-terminus)

97
ttGGBP182C.3 (affinity-tuning mutant, 182C background (Table 6) with

signal peptide replaced with M and GGSHHHHHH at C-terminus)

98
ttGGBP182C.4 (affinity-tuning mutant, 182C background (Table 6) with

signal peptide replaced with M and GGSHHHHHH at C-terminus)

99
ttGGBP182C.5 (affinity-tuning mutant, 182C background (Table 6) with

signal peptide replaced with M and GGSHHHHHH at C-terminus)

100
ttGGBP182C.6 (affinity-tuning mutant, 182C background (Table 6) with

signal peptide replaced with M and GGSHHHHHH at C-terminus)

101
ttGGBP182C.7 (affinity-tuning mutant, 182C background (Table 6) with

signal peptide replaced with M and GGSHHHHHH at C-terminus)

102
ttGGBP182C.8 (affinity-tuning mutant, 182C background (Table 6) with

signal peptide replaced with M and GGSHHHHHH at C-terminus)

103
ttGGBP182C.9 (affinity-tuning mutant, 182C background (Table 6) with

signal peptide replaced with M and GGSHHHHHH at C-terminus)

104
GGSHHHHHH

105
csaGGBP Exemplary Expression Construct (Table 2)

106
bprGGBP Exemplary Expression Construct (Table 2)

107
rinGGBP_A Exemplary Expression Construct (Table 2)

108
fprGGBP Exemplary Expression Construct (Table 2)

109
clj GGBP Exemplary Expression Construct (Table 2)

110
cauGGBP Exemplary Expression Construct (Table 2)

111
rinGGBP_B Exemplary Expression Construct (Table 2)

112
erhGGBP Exemplary Expression Construct (Table 2)

113
ereGGBP Exemplary Expression Construct (Table 2)

114
ttGGBP Exemplary Expression Construct (Table 2)

115
cobGGBP Exemplary Expression Construct (Table 2)

116
chyGGBP Exemplary Expression Construct (Table 2)

117
pspGGBP23 Exemplary Expression Construct (Table 2)

118
ttGGBP11C Exemplary Cysteine Scan Mutant Expression Construct

(Table 3)

119
ttGGBP16C Exemplary Cysteine Scan Mutant Expression Construct

(Table 3)

120
ttGGBP17C Exemplary Cysteine Scan Mutant Expression Construct

(Table 3)

121
ttGGBP42C Exemplary Cysteine Scan Mutant Expression Construct

(Table 3)

122
ttGGBP67C Exemplary Cysteine Scan Mutant Expression Construct

(Table 3)

123
ttGGBP91C Exemplary Cysteine Scan Mutant Expression Construct

(Table 3)

124
ttGGBP92C Exemplary Cysteine Scan Mutant Expression Construct

(Table 3)

125
ttGGBP111C Exemplary Cysteine Scan Mutant Expression Construct

(Table 3)

126
ttGGBP148C Exemplary Cysteine Scan Mutant Expression Construct

(Table 3)

127
ttGGBP151C Exemplary Cysteine Scan Mutant Expression Construct

(Table 3)

128
ttGGBP152C Exemplary Cysteine Scan Mutant Expression Construct

(Table 3)

129
ttGGBP181C Exemplary Cysteine Scan Mutant Expression Construct

(Table 3)

130
ttGGBP182C Exemplary Cysteine Scan Mutant Expression Construct

(Table 3)

131
ttGGBP183C Exemplary Cysteine Scan Mutant Expression Construct

(Table 3)

132
ttGGBP257C Exemplary Cysteine Scan Mutant Expression Construct

(Table 3)

133
ttGGBP259C Exemplary Cysteine Scan Mutant Expression Construct

(Table 3)

134
ttGGBP300C Exemplary Cysteine Scan Mutant Expression Construct

(Table 3)

135
ttGGBP17C.1 Exemplary Affinity-Tuning mutant (17C Background)

Expression Construct (Table 6)

136
ttGGBP17C.2 Exemplary Affinity-Tuning mutant (17C Background)

Expression Construct (Table 6)

137
ttGGBP17C.3 Exemplary Affinity-Tuning mutant (17C Background)

Expression Construct (Table 6)

138
ttGGBP17C.4 Exemplary Affinity-Tuning mutant (17C Background)

Expression Construct (Table 6)

139
ttGGBP17C.5 Exemplary Affinity-Tuning mutant (17C Background)

Expression Construct (Table 6)

140
ttGGBP17C.6 Exemplary Affinity-Tuning mutant (17C Background)

Expression Construct (Table 6)

141
ttGGBP17C.7 Exemplary Affinity-Tuning mutant (17C Background)

Expression Construct (Table 6)

142
ttGGBP17C.8 Exemplary Affinity-Tuning mutant (17C Background)

Expression Construct (Table 6)

143
ttGGBP17C.9 Exemplary Affinity-Tuning mutant (17C Background)

Expression Construct (Table 6)

144
ttGGBP17C.10 Exemplary Affinity-Tuning mutant (17C Background)

Expression Construct (Table 6)

145
ttGGBP17C.11 Exemplary Affinity-Tuning mutant (17C Background)

Expression Construct (Table 6)

146
ttGGBP17C.19 Exemplary Affinity-Tuning mutant (17C Background)

Expression Construct (Table 6)

147
ttGGBP17C.20 Exemplary Affinity-Tuning mutant (17C Background)

Expression Construct (Table 6)

148
ttGGBP17C.21 Exemplary Affinity-Tuning mutant (17C Background)

Expression Construct (Table 6)

149
ttGGBP17C.22 Exemplary Affinity-Tuning mutant (17C Background)

Expression Construct (Table 6)

150
ttGGBP17C.23 Exemplary Affinity-Tuning mutant (17C Background)

Expression Construct (Table 6)

151
ttGGBP17C.24 Exemplary Affinity-Tuning mutant (17C Background)

Expression Construct (Table 6)

152
ttGGBP17C.25 Exemplary Affinity-Tuning mutant (17C Background)

Expression Construct (Table 6)

153
ttGGBP17C.26 Exemplary Affinity-Tuning mutant (17C Background)

Expression Construct (Table 6)

154
ttGGBP17C.27 Exemplary Affinity-Tuning mutant (17C Background)

Expression Construct (Table 6)

155
ttGGBP17C.28 Exemplary Affinity-Tuning mutant (17C Background)

Expression Construct (Table 6)

156
ttGGBP17C.29 Exemplary Affinity-Tuning mutant (17C Background)

Expression Construct (Table 6)

157
ttGGBP182C.2.0 Exemplary Affinity-Tuning mutant (182C Background)

Expression Construct (Table 6)

158
ttGGBP182C.2.1 Exemplary Affinity-Tuning mutant (182C Background)

Expression Construct (Table 6)

159
ttGGBP182C.2.3 Exemplary Affinity-Tuning mutant (182C Background)

Expression Construct (Table 6)

160
ttGGBP182C.2.4 Exemplary Affinity-Tuning mutant (182C Background)

Expression Construct (Table 6)

161
ttGGBP182C.2.5 Exemplary Affinity-Tuning mutant (182C Background)

Expression Construct (Table 6)

162
ttGGBP182C.2.6 Exemplary Affinity-Tuning mutant (182C Background)

Expression Construct (Table 6)

163
ttGGBP182C.2.7 Exemplary Affinity-Tuning mutant (182C Background)

Expression Construct (Table 6)

164
ttGGBP182C.2.8 Exemplary Affinity-Tuning mutant (182C Background)

Expression Construct (Table 6)

165
ttGGBP182C.2.9 Exemplary Affinity-Tuning mutant (182C Background)

Expression Construct (Table 6)

166
ttGGBP182C.3 Exemplary Affinity-Tuning mutant (182C Background)

Expression Construct (Table 6)

167
ttGGBP182C.4 Exemplary Affinity-Tuning mutant (182C Background)

Expression Construct (Table 6)

168
ttGGBP182C.5 Exemplary Affinity-Tuning mutant (182C Background)

Expression Construct (Table 6)

169
ttGGBP182C.6 Exemplary Affinity-Tuning mutant (182C Background)

Expression Construct (Table 6)

170
ttGGBP182C.7 Exemplary Affinity-Tuning mutant (182C Background)

Expression Construct (Table 6)

171
ttGGBP182C.8 Exemplary Affinity-Tuning mutant (182C Background)

Expression Construct (Table 6)

172
ttGGBP182C.9 Exemplary Affinity-Tuning mutant (182C Background)

Expression Construct (Table 6)

173
ttGGBP182C.2.0_Imm0

174
ttGGBP, R91K,Q148E

175
ttGGBP182C.2.0_Imm1

176
ttGGBP182C.2.0_Imm2

177
ttGGBP182C.2.0_Imm3

178
ttGGBP182C.2.0_Imm4

179
ttGGBP182C.2.0_Imm5

180
ttGGBP182C.2.0_Imm6

181
ttGGBP182C.2.0_Imm7

182
IYKXDDXFM (Conserved Sequence)

183
YKXDDXFM (Conserved Sequence)

184
YKXDDXF (Conserved Sequence)

185
KXDDXF (Conserved Sequence)

186
YKXDD (Conserved Sequence)

187
KXDD (Conserved Sequence)

188
DDXF (Conserved Sequence)

189
DXFMXXVR (Conserved Sequence)

190
DXFMXXV (Conserved Sequence)

191
DXFM (Conserved Sequence)

192
IYKXDNXFM (Conserved Sequence)

193
YKXDNXFM (Conserved Sequence)

194
YKXDNXF (Conserved Sequence)

195
KXDNXF (Conserved Sequence)

196
YKXDN (Conserved Sequence)

197
KXDN (Conserved Sequence)

198
DNXF (Conserved Sequence)

199
NXFMXXVR (Conserved Sequence)

200
NXFMXXV (Conserved Sequence)

201
NXFM (Conserved Sequence)

202
PVVFFNKEP (Conserved Sequence)

203
PVVFXNKEP (Conserved Sequence)

204
PVVFFNXEP (Conserved Sequence)

205
PVVFXNXEP (Conserved Sequence)

206
VVFXNXEP (Conserved Sequence)

207
VFXNXEP (Conserved Sequence)

208
PVVFXNXE (Conserved Sequence)

209
PVVFXN (Conserved Sequence)

210
PXVFXN (Conserved Sequence)

211
PVXFXN (Conserved Sequence)

212
FXNXEP (Conserved Sequence)

213
FXNXE (Conserved Sequence)

214
PGHPDAEART (Conserved Sequence)

215
PGHXDAXXRT (Conserved Sequence)

216
GHXDAXXRT (Conserved Sequence)

217
HXDAXXRT (Conserved Sequence)

218
DA)OCRT (Conserved Sequence)

219
PGHXDAXXR (Conserved Sequence)

220
PGHXDA (Conserved Sequence)

221
PGHXD (Conserved Sequence)

222
PGNPDAEART (Conserved Sequence)

223
PGNXDAXXRT (Conserved Sequence)

224
GNXDAXXRT (Conserved Sequence)

225
NXDAXXRT (Conserved Sequence)

226
PGNXDAXXR (Conserved Sequence)

227
PGNXDA (Conserved Sequence)

228
PGNXD (Conserved Sequence)

229
DTAMWD (Conserved Sequence)

230
DTAMCD (Conserved Sequence)

231
DTAMW (Conserved Sequence)

232
DTAMC (Conserved Sequence)

233
TAMWD (Conserved Sequence)

234
TAMCD (Conserved Sequence)

235
AXWXX (Conserved Sequence)

236
AXCXX (Conserved Sequence)

237
IEVVIANND (Conserved Sequence)

238
EVVIANND (Conserved Sequence)

239
IEVVIANN (Conserved Sequence)

240
EVVIANN (Conserved Sequence)

241
IEXVXXNND (Conserved Sequence)

242
IEXVXXNN (Conserved Sequence)

243
EXVXXNND (Conserved Sequence)

244
EXVXXNN (Conserved Sequence)

245
PVFGVDA (Conserved Sequence)

246
VFGVDA (Conserved Sequence)

247
PVFGVD (Conserved Sequence)

248
FGVDA (Conserved Sequence)

249
PVXGVDA (Conserved Sequence)

250
VXGVDA (Conserved Sequence)

251
PVXGVD (Conserved Sequence)

252
VXGVD (Conserved Sequence)

253
GTVLNDA (Conserved Sequence)

254
GTVLND (Conserved Sequence)

255
GTVLN (Conserved Sequence)

256
TVLND (Conserved Sequence)

257
chyGGBP_F12C (chGGBP with signal sequence replaced with M; F12C,

C39A, and C206A mutations; and GGSHHHHHH at C-terminus)

258
chyGGBP_F16C (chGGBP with signal sequence replaced with M; F16C,

C39A, and C206A mutations; and GGSHHHHHH at C-terminus)

259
chyGGBP_W181C (chGGBP with signal sequence replaced with M;

W181C, C39A, and C206A mutations; and GGSHHHHHH at C-terminus)

cobGGBP_F12C (cobGGBP with signal sequence replaced with M;

260
F12C, C39A, C173A, and C206A mutations; and GGSHHHHHH at C-

terminus)

261
cobGGBP_F16C (cobGGBP with signal sequence replaced with M;

F16C, C39A, C173A, and C206A mutations; and GGSHHHHHH at C-

terminus)

262
cobGGBP_W181C (cobGGBP with signal sequence replaced with M,

W181C, C39A, C173A, and C206A mutations; and GGSHHHHHH at C-

terminus)

263
pspGGPB_F13C (pspGGBP with signal sequence replaced with M; F13C

mutation; and GGSHHHHHH at C-terminus)

264
pspGGPB_F9C (pspGGBP with signal sequence replaced with M; F9C

mutation; and GGSHHHHHH at C-terminus)

265
pspGGPB_W178C (pspGGBP with signal sequence replaced with M;

W178C mutation; and GGSHHHHHH at C-terminus)

266
csaGGBP_F18C (csaGGBP with signal sequence replaced with M; F18C,

C62A, C82A, C113A, and C211A mutations; and GGSHHHHHH at C-

terminus)

267
csaGGBP_W186C (csaGGBP with signal sequence replaced with M;

W186C, C62A,C82A,C113A, and C211A mutations; and GGSHHHHHH

at C-terminus)

268
csaGGBP_Y14C (csaGGBP with signal sequence replaced with M;

Y14C, C62A,C82A,C113A, and C211A mutations; and GGSHHHHHH at

C-terminus)

269
bprGGBP_F16C (bprGGBP with signal sequence replaced with M;

F16C, C8A, C112A, C116A, C179A, C211A, and C289A mutations; and

GGSHHHHHH at C-terminus)

270
bprGGBP_K12C (bprGGBP with signal sequence replaced with M;

K12C, C8A, C112A, C116A, C179A, C211A, and C289A mutations; and

GGSHHHHHH at C-terminus)

271
bprGGBP_W187C (bprGGBP with signal sequence replaced with M;

W187C, C8A, C112A, C116A, C179A, C211A, and C289A mutations;

and GGSHHHHHH at C-terminus)

272
rinGGBP_A_F10C (rinGGBP_A with signal sequence replaced with M;

F10C, C6A, C114A, C177A, C210A, and C288A mutations; and

GGSHHHHHH at C-terminus)

273
rinGGBP_A_F14C (rinGGBP_A with signal sequence replaced with M;

F14C, C6A, C114A, C177A, C210A, and C288A mutations; and

GGSHHHHHH at C-terminus)

274
rinGGBP_A_W185C (rinGGBP_A with signal sequence replaced with

M; W185C, C6A, C114A, C177A, C210A, and C288A mutations; and

GGSHHHHHH at C-terminus)

275
rinGGBP_B_F17C (rinGGBP_B with signal sequence replaced with M;

F17C, C66A, C70A, and C306A; and GGSHHHHHH at C-terminus)

276
rinGGBP_B_Q13C (rinGGBP_B with signal sequence replaced with M;

Q13C, C66A, C70A, and C306A mutations; and GGSHHHHHH at C-

terminus)

277
rinGGBP_B_W189C (rinGGBP_B with signal sequence replaced with M;

W189C, C66A, C70A, and C306A mutations; and GGSHHHHHH at C-

terminus)

278
fprGGBP_F12C (fprGGBP with signal sequence replaced with M; F12C,

C8A, C105A, C106A, C143A, and C205A mutations; and GGSHHHHHH

at C-terminus)

279
fprGGBP_F16C (fprGGBP with signal sequence replaced with M; F16C,

C8A, C105A, C106A, C143A, and C205A mutations; and GGSHHHHHH

at C-terminus)

280
fprGGBP_W180C (fprGGBP with signal sequence replaced with M;

W180C, C8A, C105A, C106A, C143A, and C205A mutations; and

GGSHHHHHH at C-terminus)

281
cljGGBP_Fl1C (cljGGBP with signal sequence replaced with M; F11C,

C77A, and C198A mutations; and GGSHHHHHH at C-terminus)

282
cljGGBP_W15C (cljGGBP with signal sequence replaced with M;

W15C, C77A, and C198A mutations; and GGSHHHHHH at C-terminus)

283
cljGGBP_W176C (cljGGBP with signal sequence replaced with M;

W176C, C77A, and C198A mutations; and GGSHHHHHH at C-terminus)

284
cauGGBP_F12C (cauGGBP with signal sequence replaced with M;

F12C, C78A, and C199A mutations; and GGSHHHHHH at C-terminus)

285
cauGGBP_W16C (cauGGBP with signal sequence replaced with M;

W16C, C78A, and C199A mutations; and GGSHHHHHH at C-terminus)

286
cauGGBP_W177C (cauGGBP with signal sequence replaced with M;

W177C, C78A, and C199A mutations; and GGSHHHHHH at C-terminus)

287
erhGGBP_F13C (erhGGBP with signal sequence replaced with M; F13C

mutation; and GGSHHHHHH at C-terminus)

288
erhGGBP_F17C (erhGGBP with signal sequence replaced with M; F17C

mutation; and GGSHHHHHH at C-terminus)

289
erhGGBP_W188C (erhGGBP with signal sequence replaced with M;

W188C mutation; and GGSHHHHHH at C-terminus)

290
ereGGBP_F17C (ereGGBP with signal sequence replaced with M; F17C,

C10A, C29A, C65A, C69A, and C183A mutations; and GGSHHHHHH at

C-terminus)

291
ereGGBP_Q13C (ereGGBP with signal sequence replaced with M;

Q13C, ClOA, C29A, C65A, C69A, and C183A mutations; and

GGSHHHHHH at C-terminus)

292
ereGGBP_W188C (ereGGBP with signal sequence replaced with M;

W188C, C10A, C29A, C65A, C69A, and C183A mutations; and

GGSHHHHHH at C-terminus)

293
r3Zif

294
ZF-QNK

295
Hexahistidine Tag

296
Hexalysine Tag

297
ecGGBP.F16C (with signal peptide replaced with M; a F16C mutation;

and a GGSHHHHHH at C-terminus)

298
ttGGBP182C.2.0 (affinity-tuning mutant, 182C background (Table 6)

with signal peptide replaced with M)

Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.

EXAMPLE 1
Fluorescently Responsive Sensor Engineering Phases

The engineering of FRSs can be divided into five phases:

- 1. Binding protein discovery. A set of glucose-binding protein sequence homologs is identified. Accurate assignment of their ligand-binding function utilizes application of a prediction method that incorporates information encoded in the experimentally determined three-dimensional structure of periplasmic glucose-binding proteins.
- 2. Experimental lead validation. Synthetic genes are constructed, which are optimized for heterologous expression in Escherichia coli of one or more predicted glucose-binding protein sequences. The glucose-binding properties and thermostabilities of the corresponding expressed, purified proteins are evaluated.
- 3. Engineering of fluorescent responses. Semisynthetic fluorescent conjugates of the experimentally validated leads are constructed by attaching fluorophores to single cysteine mutants. The effect of glucose binding on the fluorescence emission properties of those conjugates is evaluated. Those conjugates that evince strong, ratiometric responses are selected as FRSs.
- 4. Affinity tuning. Single or multiple mutations are introduced by site-directed mutagenesis to alter the glucose-binding affinities of glucose-responsive FRSs. A set of FRS variants is selected that together cover the clinical glucose concentration range with high accuracy.
- 5. Device integration. FRSs are immobilized in the sampling component of the analytical device in a manner that preserves their fluorescent response and glucose affinity. Long-term storage conditions are established.

EXAMPLE 2
Identification of a Family of Periplasmic Glucose-Binding Proteins Homologs Using Structurally Assisted Function Evaluation (Phase Step 1)

As a first step in constructing robust glucose sensor candidates, we examined bacterial genomic sequences to identify periplasmic glucose-binding protein sequences in known thermophiles. Homologs from such organisms are likely to encode thermostable proteins. Analysis of enzyme families has shown that overall sequence identity below ˜60% is a weak predictor of function conservation (Todd 2001 J. Mol. Biol., 307, 1113-1143; Tian 2003 J. Mol. Biol., 333, 863-882). Furthermore, functional assignments based on sequence homology alone are known to be particularly problematic in the PBP superfamily For instance, PBPs that by overall sequence identity are predicted to bind oligopeptides were found to bind oligosaccharides. Enzyme functional assignments are improved greatly if a sequence selection filter based on conservation of catalytic residues identified from protein structures is included. Such catalytic residues comprise a subset of all the residues that contact an enzyme substrate or inhibitor. In the case of the PBPs, functional selection filters need to take into account all the protein-ligand contacts that encode the ligand-binding function. Accordingly, we have developed a structurally assisted functional evaluation (SAFE) method to identify PBP sequence homologs with accurately predicted function. The SAFE homolog search method consists of five steps encoded in the ProteinHunter software package:

- 1. Sequence homolog set is collected using the BLAST sequence alignment tool(Altschul et al. 1990) with standard settings (expect threshold: 10.0; gap cost existence: 11; gap extension: 1; substitution matrix: Blosum62; filter for low complexity regions on), starting with Escherichia coli periplasmic glucose-galactose binding protein (ecGGBP) sequence (SEQ ID NO: 17) as a seed (Vyas, Vyas and Quiocho 1988 Science, 242, 1290-5). The ProteinHunter program selects only those pairwise BLAST alignments that (a) share a minimum of 20% sequence identity, and (b) have 70% coverage of their entire sequence length by the aligned region. This set of sequences defines a universe of possible glucose-binding proteins without assigning function.
- 2. Structure-based encoding of biological function. A PCS comprising the protein residues that form hydrogen bonds and van der Waals contacts with the bound glucose is defined using computer-assisted, visual inspection of the three-dimensional structure of the ecGGBP-glucose complex. ProteinHunter identifies if the calculated distance between any of their atoms and any glucose atom is less than 5 Å, and the distances between their backbone C_α and any atom in glucose is greater than that of their C_β atom and any atom in glucose. Secondary shell residues that do not form hydrogen bonds or van der Waals contacts are removed by inspection from the resulting set. The choice of allowed residues is guided by the identity of the residue in the structure, and mutants that are likely to maintain that interaction. The resulting definition specifies residue positions and their permitted amino acid identity. Multiple amino acid identities are permitted at each position to encode functionally equivalent residues. This definition establishes a search filter for the accurate prediction of glucose-binding proteins within the universe of sequence homologs collected in (1).
- 3. Accurate sequence alignment. Tools such as ClustalW (Chenna et al. 2003 Nucleic Acids Res, 31, 3497-500) are used to construct an accurate alignment of all the sequence homologs. The ecGGBP seed sequence is included in this alignment. This multiple sequence alignment establishes the equivalent positions of the ecGGBP PCS in each sequence homolog.
- 4. Function evaluation. The glucose-binding properties of each of the aligned sequence homologs is determined by measuring their compliance with the PCS sequence filter. A “Hamming distance”, H, is assigned for each homolog by the ProteinHunter program, which specifies the degree of sequence identity of all the residues at the aligned PCS positions. A value of H=0 indicates that the identities of all the residues at the aligned PCS positions match the amino acid(s) allowed in the PCS search filter; H>0, indicates that one or more aligned positions have disallowed residues. Sequences for which H=0 are predicted to encode glucose-binding proteins.
- 5. Selection of representative SAFE homologs. The sequence homologs are ordered by (a) identity with the seed PCS, as measured by the Hamming distance, (b) fractional overall sequence identity with the seed sequence. A subset for sequences with H=0, sampling the fractional overall sequence identity is selected for experimental verification.

The ProteinHunter software tool encodes the flow of execution, applies the PCS search filter, and visualizes the results that include organism annotations such as thermophilicity, and Gram stain status.

These steps are encoded in the ProteinHunter software tool, which encodes the flow of execution, applies the PCS search filter, and visualizes the results, and handles organism annotations such as thermophilicity, and Gram stain status.

Annotated genomic and plasmid sequences of 5062 prokaryotes were downloaded from the National Center of Biotechnology Information (download date: May 17, 2015; ftp://ftp.ncbi.nih.gov/genomes/Bacteria/all.gbk.tar.gz). The protein sequence for the E. coli glucose-galactose binding protein (ecGGBP) was extracted from the protein structure file 2gbp(Vyas et al. 1988 Science, 242, 1290-5), and used as the seed sequence for the BLAST search described above. A total of 1822 sequence homologs were identified.

In ecGGBP, glucose binding is encoded by a PCS comprising ten residues. Two aromatic residues (F16, W182) sandwich the bound glucose through extensive van der Waals interactions with either surface of the pyranose ring. The other eight residues form hydrogen bonds with all the glucose hydroxyls. A PCS filter specifying multiple amino acids at these 10 positions was used to predict glucose-binding proteins (FIGS. 2A-C). A total of 139 homologs were predicted to encode glucose-binding proteins, on the basis of their Hamming distance scores (H=0). The overall sequence identities of these homologs relative to the ecGGBP seed varied from 100% to 29% (Table 1).

EXAMPLE 3
Lead Protein Validation Using Ligand-Mediated Thermostability Shifts (Phase Step 2)

Thirteen homologs that were predicted to be glucose-binding proteins (FIG. 3) were selected to probe different degrees of sequence identity to the ecGGBP seed, and their glucose-binding properties were determined experimentally (Table 2). These experiments comprise four successive steps:

- 1. Synthetic gene construction. The amino acid sequence of the homology leads are backtranslated into DNA sequences. These are optimized for directing heterologous cytoplasmic expression of the protein homologues in E. coli, using either the OrfOpt, (U.S. Patent Publication No. 2011/0171737), hereby incorporated by reference or OrfMorph (described herein) programs. These programs predict mRNA sequences that direct high-level protein expression in E. coli. The predicted gene sequences are assembled de novo from synthetic oligonucleotides.
- 2. Heterologous protein expression of the homologues in E. coli. Plasmids carrying the synthetic expression constructs (see above) were transformed into KRX (E. coli K12 Derivative Strain) competent cells (Promega, Technical Bulletin TB352). Protein production was induced in bacterial cultures of these cultures, as described in the Materials and Methods.
- 3. Purification of successfully expressed protein using immobilized metal affinity chromatography, as described in Materials and Methods.
- 4. Verification of glucose binding. Determination of the glucose-binding properties of the purified proteins using a thermal stability shift assay, as described in Materials and Methods.
  
  Seven of the thirteen synthetic expression constructs successfully directed heterologous cytoplasmic expression of a GGBP homolog in E. coli. The glucose-binding properties of five of these were confirmed directly using the thermal shift assay (FIGS. 4A-B). The thermostability of two homologs was too high (^apoT_m>90° C.) for this assay to be applicable. Their glucose-binding properties were confirmed by measuring the temperature-dependent fluorescence responses of Acrylodan-labeled single cysteine mutants (see section 5) to glucose (FIGS. 5A-B).

Surprisingly, the sequence identity of all these experimentally verified glucose-binding homologs relative the ecGGBP seed are considerably below the 60% threshold, ranging from 29% to 48%. Several use alternate amino acids in the PCS at position 152, demonstrating the predictive power of allowing functionally equivalent residues. These results therefore demonstrate that biological function can be predicted accurately with the SAFE technique, even in sequence homologs with low fractional identities to the original seed.

Three of the experimentally verified GGBP homologs exhibit mid-point thermal denaturation temperatures (T_mvalues) in the 347-350 K range, which is at least 25 K more stable than the ecGGBP seed (T_m=322 K). Of these, the homolog from Thermoanaerobacter thermosaccharolyticum (ttGGBP) was produced at the highest level by heterologous expression in E. coli. This protein was selected as the candidate for constructing robust glucose sensors.

EXAMPLE 4
Cysteine Mutant Scans and Fluorophore Screening to Identify Fluorescently Responsive Glucose Sensors (Phase Step 3)

Semi-synthetic FRSs were engineered by site-specifically attaching thiol-reactive, environmentally sensitive fluorophores that respond to ligand-mediated conformational changes. Identification of FRS candidates that are useful for sensing applications comprises three steps:

- 1. Cysteine scan. Mutant glucose-binding proteins containing single cysteines are constructed for site-specific attachment of thiol-reactive fluorophores. Positions in PBPs where attached single fluorophores are likely to exhibit ligand-dependent responses were determined (de Lorimier et al. 2002 Protein Sci, 11, 2655-75). Candidate positions fall into three classes: endosteric, replacing a residue that contacts the ligand directly; peristeric, located at the rim of the binding site; allosteric (Marvin et al. 1997 Proc Natl Acad Sci USA, 94, 4366-71; Marvin 1998 J Am Chem Soc, 120, 7-11), located outside the binding site at sites that undergo local structural changes in concert with the hinge-bending motion.
- 2. Fluorophore screening. Thiol-reactive, environmentally sensitive fluorophores are attached to each cysteine mutant prepared in step 1.
- 3. Evaluation of the glucose-mediated change of all the fluorescent conjugates prepared in step 2. Responses to ligand binding in which there is both a change in fluorescence emission intensity and spectral shape was essential for chemometric applications, because such changes enable ratiometric measurements. Changes in spectral shape typically are accompanied by a shift in the wavelength of the emission intensity maxima. Three classes of fluorescent responses are possible:
  - i. No response.
  - ii. Monochromatic response (emission intensity increases or decreases without a change in spectral shape)
  - iii. Dichromatic response (both intensity and spectral shape changes) which can be classified into two sub-classes:
    - i. Hypsochromatic: emission intensity shifts to shorter wavelengths upon binding glucose (“blue shift”).
    - ii. Bathochromatic: emission intensity shifts to longer wavelengths upon binding glucose (“red shift”).

We constructed seventeen single cysteine mutants in ttGGBP, exploring four endosteric, ten peristeric, and three allosteric positions (FIGS. 6A-B). At each position, we attached the Prodan-derived fluorophores such as Acrylodan and Badan, which differ by one methylene group in their thiol-reactive linking moiety. The fluorescence emission intensities of seven Acrylodan and five Badan conjugates responded to glucose at seven attachment positions (Table 3). At only four attachment positions were the responses of both fluorophores qualitatively similar, and never quantitatively. We also tested for glucose binding by measuring ligand-mediated shifts in protein thermal stability (Table 3). By this criterion, the majority of the non-responsive conjugates bound glucose. The two conjugates that did not bind glucose (N42C⋅Badan, V67C⋅Badan) also exhibited large losses in overall thermostability (28-30 K decrease in apo T_mvalues), indicating that the attached fluorophore adversely affected their structural integrity. By contrast, all other conjugates exhibited only small variations in apo-protein thermostability (1-4 K), indicating that the fluorophore causes minimal perturbations instability.

Endosteric attachment positions exhibited the most pronounced changes in fluorescence emissions in response to ligand binding. Conjugates at three of the ten peristeric positions were responsive to glucose. No allosteric conjugates exhibited fluorescence responses to glucose.

We observed ligand-dependent shifts in the wavelengths of emission intensity maxima at one peristeric (Y11C) and two endosteric (F17C, W182C) sites (FIGS. 7A-F), enabling dichromatic ratiometric measurements; the maximum intensity of other glucose-responsive conjugates remained the same (monochromatic responses). Wavelengths shifts occurred in both directions: in the ttGGBP W182C⋅Acrylodan conjugate the apo-protein spectrum converted from a short- to a long-wavelength emission state upon binding glucose (bathochromic shift), whereas the emissions in F17C⋅Acrylodan, F17C⋅Badan and Y11C⋅Badan conjugates shifted to shorter wavelengths (hypsochromic shift). The Badan and Acrylodan conjugates attached to F17C and W182C, respectively, exhibited the largest, wavelength-dependent changes in fluorescence emission intensities, (FIGS. 7A-C).

The responses to glucose of a number of other fluorophores (FIGS. 8A-P) were also tested at positions 182C and 17C (Table 4). Several conjugates exhibited significant changes in emission intensity upon glucose addition, but none of these were accompanied by large wavelengths shifts of their maxima. Both increases and decreases in fluorescence were observed.

The most effective sensors that were discovered are the Badan and Acrylodan conjugates attached to the endosteric cysteine mutations F17C and W182C, respectively. For example, peptides with SEQ ID No:61 (ttGGBP182C), SEQ ID NO:88 (ttGGBP182C.2.0), SEQ ID NO:90 (ttGGBP182C.2.3), and SEQ ID NO:102 (ttGGBP182C.8) are particularly useful for clinical diagnostics, because they span hypoglycemia, euglycemia, mild hyperglycemia, severe hyperglycemia, and hyperosmolar hyperglycemic glucose concentrations respectively. These two fluorophores differ only in their linker geometry, but this small difference determines whether dichromatic or monochromatic responses are observed for a particular conjugate. At position 17C, only the Badan and not the Acrylodan conjugate evinces a dichromatic response; at 182C; surprisingly, the reverse case is observed. Within a given bacterial periplasmic binding protein, some attachment sites are more effective than others, and within a given attachment site, some fluorophores are more effective than others (DeLorimier et al., 2002, Protein Science 11:2655-2675). Thus, the nature of the responses therefore is idiosyncratic, and therefore the choice of which site to mutate (e.g., substitute with a single cysteine) and which fluorophore to use at a particular site is nonobvious. Changes in linker geometry and chromophore modifications give rise to significant differences in the detailed interactions of particular fluorophores with the protein, even within families of closely related molecules, thereby significantly impacting sensor characteristics, consistent with previous observations.

EXAMPLE 5
Determination of Exemplary Fluorescent Conjugate Structures

The structures of the fluorescent conjugates with pronounced dichromatic responses, ttGGBP17C⋅Badan and ttGGBP182C.2.0⋅Acrylodan (SEQ ID NO: 88) (this is a variant in which the glucose-binding affinity has been manipulated), were determined by X-ray crystallography (Table 5; FIG. 9). The structure of the E. coli GGBP homolog conjugate, ecGGBP W183C⋅Acrylodan (ecGGBP W183C, SEQ ID NO: 32), was determined also.

Like all members of the periplasmic-binding protein (PBP) superfamily, GGBPs comprise two domains connected by a flexible hinge, with the ligand-binding site located in a cleft between the two domains. In the absence of ligand, PBPs adopt an open conformation, in which the two domains move apart and the cleft is wide. In ligand complexes, hinge-bending motions move the two domains closer together, forming a binding site that completely envelops the ligand, reminiscent of a protein interior. The overall structures of the ecGGBP and ttGGBP conjugates are similar with a backbone C_α RMSD of 0.6 Å and 0.9 Å for the N- and C-terminal domains respectively (C-terminal domain contains the Ca²⁺-binding site) as expected for proteins that are 48% identical (Chothia 1986 EMBO J., 5, 823-826). Small variations in the degree of closure can affect critical inter-domain hydrogen-bonding interactions. With the exception of mutated aromatic groups, the interactions between the protein and bound glucose are conserved. The structures of these conjugates enabled us to determine the interactions between the protein and the fluorophore and the internal conformation of the fluorophore. These sets of observations guided the choice of mutations for manipulating glucose affinities.

In both F17C⋅Badan and W182C⋅Acrylodan, the conjugated fluorophores point out into solution. A view down the long axis of ttGGBP from the C-terminal towards the N-terminal domain into the ligand-binding site reveals that three channels, each approximately 120° apart, connect the centrally located bound monosaccharide to the protein surface (FIG. 9c). One channel is occupied by the R-group of the natural galactoside ligand (this position is occupied by water in the glucose and galactose complexes), the other two are filled by Badan and Acrylodan in their respective conjugates.

In the wild-type protein, the rings of F17 and W182 form extensive van der Waals contact with the bound glucose. The outward orientation of the fluorophore in the W182C⋅Acrylodan conjugate leaves a cavity vacated by the indole ring, which is filled with water and cryoprotectant. The cavity created by the loss of the smaller benzyl ring in F17C⋅Badan is largely filled with the linker; no water or cryoprotectant was observed.

The Acrylodan and Badan fluorophores contain two important internal degrees of freedom (FIGS. 6a-c): the twist of the dimethylamino group, and the carbonyl. The state of these two angles influences the fluorescence properties of the fluorophore, by altering the extent of electronic conjugation within the system, and the degree of polarization of the excited dipole. The electron density of Badan in the ttGGBP F17C conjugate was well defined, enabling us to determine the internal fluorophore conformation with high confidence (FIG. 9d-f). The dimethyl amino group is co-planar with the naphthalene ring. The carbonyl group is twisted out of plane by approximately 30°. By contrast, in ecGGBP.183C⋅Acrylodan the carbonyl is co-planar (FIG. 9g). This group is probably also co-planar in the ttGGBP.182C⋅Acrylodan structure, but the partial occupancy of the conjugate in this structure precluded a definitive conclusion. Nevertheless, the high degree of similarity in structure and spectral properties of the ecGGBP 183C and ttGGBP 182C Acrylodan conjugates suggests that the Acrylodan carbonyl is co-planar in both homolog conjugates.

In both ttGGBP 17C⋅Badan and 182C⋅Acrylodan (and ecGGBP 183C⋅Acrylodan) conjugates, the pocket in which the fluorophore carbonyl is located comprises residues contributed by each domain, and is fully formed only in the closed, glucose-bound protein conformation. No hydrogen bonds are observed between the fluorophore carbonyl and the protein at either attachment position. In ttGGBP 17C⋅Badan the “knob” of the twisted carbonyl conformation is bound in a small “hole” formed by residues also contributed by both domains (FIG. 9h). The carbonyl is held in place by van der Waals interactions proximal to the attachment site with residues located in the domain opposite to the F17C site: the C_β carbon of N258 touches the carbonyl; the backbone atoms of residues 239-241 interact with one of the two naphthalene rings faces. The twisted ttGGBP F17C⋅Badan conformation therefore is stabilized by interactions of the protein with both the naphthalene ring and the carbonyl. These interactions are fully formed in the closed state only. By contrast, in ecGGBP 183C⋅Acrylodan the carbonyl hole is not well defined. The carbonyl is flanked by E149 (same domain as attachment site), and K92 (opposite domain), but these residues form no direct contacts in the structure. Neither the carbonyl nor the naphthalene rings of this planar conjugate are constrained by clear interactions in the protein closed conformation.

EXAMPLE 6
Affinity Tuning (Phase Step 4)

Blood glucose concentrations range from ˜3 mM (hypoglycemia) to ˜30 mM (hyperglycemia) and up to ˜100mM for the hyperosmolar hyperglycemic state (HHS), with healthy levels at around 6 mM (euglycemia). Measurements using reagentless sensors are most sensitive at analyte concentrations that match the dissociation constant. The glucose affinity of ttGGBP182C⋅Acrylodan is too high and must therefore be “tuned” by raising the K_dvalue.

The mutations that alter glucose affinities fall into four classes:

- 1. Alteration of direct interactions in the PCS between the protein and the bound glucose.
- 2. Manipulation of the equilibrium between the open and closed states.
- 3. Alteration of interactions between the protein and the fluorescent conjugate. Two sub-classes can be distinguished:
  - a. Interactions with the carbonyl group
  - b. Interactions with the naphthalene ring
- 4. Indirect interactions that alter the geometry of the binding site.

Representatives of mutant classes 1-3 were constructed in the ttGGBP182C and ttGGBP17C backgrounds, using Acrylodan and Badan conjugates to evaluate their effects on glucose binding (Table 6).

Mutations in the PCS residues (Class 1) do not afford many opportunities for manipulating affinity subtly. Mutagenesis of ecGGBP has demonstrated that most PCS positions are intolerant of mutations, with the exception of histidine that interacts with the 6-hydroxyl, and the aspartate that binds the epimeric 4-hydroxyl. In ttGGBP182C⋅Acrylodan, the H151Q mutations subtly lower the affinity into the middle of the pathophysiological concentration range, whereas in ttGGBP17C⋅Badan H151Q lowers affinity, but nearly abolishes ratiometry. In ttGGBP182C⋅Acrylodan, D15N weakens glucose binding into the HHS range and increases discrimination between glucose and galactose. In ttGGBP17C⋅Badan, D15A weakens ligand binding and enhances the dichromatic response.

Uniquely in ttGGBP182C⋅Acrylodan, the PCS also can be manipulated by mutating position 154 adjacent to the cavity vacated by the missing indole ring of W182 (FIG. 10a). Three mutations in Ala154 alter the affinity 50-fold, spanning the entire pathophysiological glucose concentration range. The A154M mutation increases the affinity ˜10-fold, presumably by (partially) restoring direct van der Waals interactions between the protein and bound glucose, which were lost upon removal of the tryptophan. The 154S mutation causes a 19 K decrease in thermostability; the other mutations exhibit negligible effects on thermostability.

The conformational equilibrium can be altered by manipulating those hydrogen bonds between the N- and C-terminal domains that are predicted to form in the closed, but not the open state (Class 2 mutations). Four locations form inter-domain hydrogen bonds in the protein closed conformation (FIG. 10b) of ttGGBP: T16-D211, R69-G150 (main-chain), R91-Q148, and E92 (main-chain)-Q152. The first of these interactions is located between the Badan and Acrylodan channels, whereas the other three are positioned between the Acrylodan and galactoside R-group channels (FIG. 9c). Disruption of inter-domain interactions alters the intrinsic equilibrium between the open and closed conformations, thereby decreasing ligand affinity. Accordingly, in the ttGGBP W182C⋅Acrylodan conjugate mutants, glucose affinities are lowered (K_dvalues raised) in the class 2 mutations. These mutations also alter the fluorescence emission spectra. In all the class 2 mutants of both ttGGBPF17C Acrylodan and Badan conjugates the spectral responses are altered, either enhancing or abolish wavelength shifts. These observations indicate that mutations of the inter-domain hydrogen bonds cause (small) changes in the structure of the closed state, which affect the fluorophore environment.

Mutations in the interactions between the protein and the conjugated fluorophore (Class 3 mutants), changing either the carbonyl hole (Class 3a) or the fluorophore channel walls (Class 3b), have large effects on glucose binding and fluorescence spectra. In ttGGBP17C⋅Badan, most of the N258 mutants in the carbonyl hole do not respond to glucose. The absence of shifts in thermal stability in the presence of glucose indicates that these mutants no longer bind ligand. The 17C⋅Acrylodan N258S mutant conjugate is the exception: it binds and responds to glucose, but binding does not evoke a fluorescent response. Mutations in residues located on either side of the channel wall strongly influence spectral properties of the fluorescent conjugates. In ttGGBP17C⋅Badan, with the exception of A260W, mutations in A260 convert dichromatic to monochromatic responses. In ttGGBP182C⋅Acrylodan, the behavior of mutations in the R91 andQ148 interaction is complex, because these potentially affect both inter-domain hydrogen bonding and interactions between the protein and the conjugated fluorophore. The fluorophore blocks the formation of R91K-Q148E in the ttGGBP182C.2.0⋅Acrylodan double mutant structure, but in ecGGBP183C⋅Acrylodan E149-K92 is present, reflecting subtle differences in fluorophore conformation and inter-domain closure angles. The two single Q148E and R91K mutants both exhibit a modest increase of glucose affinities in ttGGBP182C⋅Acrylodan, suggesting that this inter-domain is maintained, and slightly improved. By contrast, the Q148E+R91K double mutant exhibits a 2.6-fold decrease in affinity, consistent with the observed loss of interaction in the ttGGBP182C2.0⋅Acrylodan double mutant structure (SEQ ID NO: 88). The glucose affinity of the ttGGBP182C.2.0⋅Acrylodan double mutant (SEQ ID NO: 88) falls within the euglycemic concentration range (FIG. 13), which is why this variant was selected for structural analysis (FIG. 11).

Affinity-tuning mutations introduced in both the ttGGBP17C⋅Badan or ttGGBP182C⋅Acrylodan backgrounds yield a collection of dichromatic sensors that cover the wide range of glucose concentrations encountered in clinical chemistry.

EXAMPLE 7
Sensor Arrays for Detecting a Wide Range of Glucose Concentrations

The precision (reciprocal of the error) of individual sensor precision is maximal at the K_dvalue, and decreases at lower or higher glucose concentrations(Marvin et al. 1997 Proc Natl Acad Sci USA, 94, 4366-71). Construction of a high-precision sensor capable of spanning the entire 100-fold clinical concentration range from extreme hypoglycemia to the HHS therefore requires combining several sensors together to maintain a high precision level. In the ttGGBP182C⋅Acrylodan background, a suite of five affinity mutants can be combined that together provide high-precision coverage from 1-100 mM (FIG. 12). A suite of four mutants in the ttGGBP17C⋅Badan background provides similar coverage. It should be noted that the ttGGBP17C⋅Badan variants switch from dim green to bright blue upon binding glucose, whereas the ttGGBP182C⋅Acrylodan variants switch from bright blue to dim green. These opposing changes in color and brightness in response to glucose binding provide additional internal checks on the integrity of the observations in an array that combines sensors derived from both backgrounds.

EXAMPLE 8
Device Integration (Phase Step 5)

Protein immobilization on solid surfaces, e.g. a polymeric planar structure or bead structure, paper, glass, silica or metal, is an important step for incorporating biosensors into devices. Immobilization enables (i) spatial localization, (ii) control over the presentation of the sensors to the reader (e.g. by encoding geometries for optical readouts), (iii) selective retention in sample separation procedures. It is advantageous to control the geometry of the protein attachment to the solid surface, in order to minimize perturbation of the fluorescence sensing mechanism. Such constructs fuse an N- or C-terminal protein domain that can mediate site-specific attachment to an appropriately chemically activated surface. For instance, hexa-histidine peptide for metal-mediated immobilization, a hexa-lysine peptide for attachment to amine-reactive groups, or a zinc-finger domain (ZF-QNK) (Smith et al. 2005 Protein Sci, 14, 64-73), or a disulfide-containing truncated zinc finger (βZif) (Smith et al. 2005 Protein Sci, 14, 64-73) at N- or C-termini of the FRS to thiol-reactive groups (FIG. 13). Here we show that site-specific attachment of a robust glucose sensor to suitably derivatized agarose beads conserves its emission fluorescence spectral response, binding affinity, and thermostability.

The ttGGBP182C.2.0⋅Acrylodan protein was site-specifically immobilized through its C-terminal hexa-histidine tag on commercially available magnetic beads coated with Ni-NTA. The use of magnetic beads affords a straightforward means for holding the beads in place within their respective sensor patches in the sampling cartridge with a magnetic field. Site-specific immobilization minimizes perturbation of the sensing mechanism. Comparison of protein thermostabilities determined in solution and on beads shows that protein is stability is not perturbed by immobilization (FIG. 14A-F) These glucose-responsive magnetic beads also useful to measure highly precise glucose titration curves (FIG. 15). Similarly, hexa-lysine fusion domains were immobilized on agarose beads derivatized with N-hydroxysuccinimide, and the QNK- or βZif-fusion on iodacetyl-derivatized beads. These fusion proteins also maintained their spectral, glucose affinities, and thermostabilities (FIG. 14D-F).

The glucose-responsive magnetic beads were dried by incubation at 50° C. for 20 minutes, using an aqueous ammonium bicarbonate buffer. The stability properties of the sensor were recovered completely upon rehydration (FIG. 14c). The dried beads were aged in situ inside fully assembled sample-handling cartridges by incubation for up to 7 days at 25° C., ³⁷° C., and 50° C. in the dark. Fluorescence and glucose-responsive properties were tested in cartridges stored for 1, 2 and 7 days. At all temperatures, the fluorescence ratio in the absence of glucose, and the glucose affinities remained unchanged within the error of the observation. Therefore, a significant advantage of ttGGBP-based FRSs is that they are sufficiently robust to be handled at ambient temperatures in a desiccated state, greatly simplifying manufacturing, distribution, and long-term storage conditions.

EXAMPLE 9
Fluorescence Mechanism

The most effective glucose-sensing FRSs identified are based on singly labeled Acrylodan and Badan conjugates. These two dyes, and their parent, Prodan(Weber 1979 Biochemistry, 18, 3075-3078), belong to a class of fluorophores that can undergo internal rotations which change the electronic structures of excited state dipoles(Rettig 1986 Angew. Chem. Int. Ed. Engl., 25, 971-988; Grabowski et al. 2003 Chem Rev, 103, 3899-4032). The X-ray structure of Prodan shows that in the absence of external factors, the dimethyl amino (DMA) and carbonyl groups are coplanar with the naphthalene core, and the system is maximally conjugated. However, the DMA or carbonyl groups can twist out of plane, diminishing the extent of conjugation within the system, and increasing the degree of polarization of these groups. This enhanced polarization increases the magnitude of the excited state dipole, and correspondingly its sensitivity to the polarity of its surroundings, which manifests itself as general solvatochromic effects and responses to specific hydrogen-bonding interactions. There has been considerable debate regarding the magnitude of the dipole in the polarized state in Prodan (Weber 1979 Biochemistry, 18, 3075-3078; Balter 1988 Chem. Phys. Lett., 143, 565-570; Catalan 1991 Journal of Fluorescence, 1, 215-223; Nemkovich 2007 Journal of Photochemistry and Photobiology A: Chemistry, 185, 26-31; Samanta 2000 Journal of Physical Chemistry, 104, 8972-8975; Kawski 2001 Zeitschrift fur Naturforschung, 56a, 407-411; Kawski 2002 Zeitschrift fur Naturforschung, 57a, 716-722), whether the molecule undergoes twisting in the excited state, and if so, whether the carbonyl or the DMA groups twist (Nowak 1986 Journal of Molecular Structure, 139, 13-23; Heisel 1987 Chemical Physics Letters, 138, 321-326; Parusel 1997 J. Molec. Struct., 398, 341-346; Parusel 1998b Journal of Physical Chemistry, 102, 7149-7156; Parusel 1998a J. Chem. Soc., Faraday Trans., 94, 2923-2927; Mennucci 2008 Journal of Physical Chemistry, 112, 414-423; Adhikary 2009 Journal of Physical Chemistry, 113, 11999-12004; Marini 2010 Journal of Physical Chemistry, 114, 17128-17135; Pederzoli 2014 Chemical Physics Letters, 597, 57-62; Cwiklik 2011 J. Phys. Chem., 115, 11428-11437; Fukuda 2012 Chem. Phys. Lett., 552, 53-57; Barucha-Kraszewska 2010 Biochim Biophys Acta, 1798, 1724-1734; Nitschke 2012 J. Phys. Chem., 116, 2713-2721). A series of derivatives in which the DMA (Lobo 2003 Journal of Physical Chemistry, 107, 10938-10943; Davis 2005 J. Phys. Chem., 109, 1295-1298) or carbonyl (Everett 2010 Journal of Physical Chemistry, 114, 4946-4950) groups are held in planar or out-of-plane conformations have shown that it is twisting of the carbonyl and not the DMA group that alters Prodan polarization.

The collection of Acrylodan and Badan conjugates of mutant ecGGBP and ttGGBPs constructed and described herein contains several semi-synthetic glucose sensors that exhibit wavelength-dependent changes in fluorescence emission intensity in response to glucose binding. The best responses were observed for conjugates attached to ttGGBP F17C (SEQ ID NO: 51) and ttGGBP W182C (SEQ ID NO: 61) cysteines endosteric mutants that replace residues which form van der Waals contacts with the pyranose glucose ring. Remarkably, the emission intensity maxima shift in opposite directions at these two positions: glucose binding evinces a bathochromic shift for ttGGBP W182C and ecGGBP W183C Acrylodan conjugates, whereas at ttGGBP F17C and ecGGBP F16C both Acrylodan and Badan exhibit hypsochromic responses. Analysis of their ligand-mediated changes in the populations of excited state and ground state electronic transitions in combination with the X-ray structures of three different conjugates has enabled us to describe a mechanism for the fluorescence response to glucose binding.

Analysis of the emission intensity (FIGS. 16-18, Tables 7 and 8) and absorption spectra (FIG. 19) indicated that glucose binding alters the populations of two dominant electronic transitions in both the excited (S₁and S₂) and ground (G₁and G₂) states. In hypsochromic responses, the dominant excited state electronic transition shifts from S₁(green) in the apo-protein, to S₂(blue) in the glucose complex; in bathochromic responses, the opposite redistribution is observed, and the glucose complex is dominated by the S₁(green) excited state. Similarly, in the ground state, hypsochromic responses shift the electronic transitions in the absorbance spectra from a low- (G₁) to a high-energy state (G₂); for bathochromic responses, the shift is G₂→G₁. Comparison of high-resolution X-ray structures of the ttGGBP F17C⋅Badan (hypsochromic response) and ttGGBP W182C⋅Acrylodan or ecGGBP W183C⋅Acrylodan (bathochromic responses) revealed that the fluorophore structures of the glucose complexes differ in these conjugates (FIGS. 9 and 10). From these observations we deduced that the G₁ground state in ecGGBP W183C⋅Acrylodan corresponds to a planar fluorophore, whereas the G₂state of F17C⋅Badan corresponds to twisted fluorophore carbonyl in which the carbonyl has repositioned out of the plane of the naphthalene ring.

These observations indicate that the changes in fluorescence intensities of glucose-responsive Acrylodan and Badan conjugates arise as a consequence of conformational coupling between ligand-mediated shifts in the population of protein conformations and internal twisting of the fluorophore carbonyl relative to its naphthalene ring. Like all periplasmic binding proteins, GGBP undergoes a large, ligand-mediated conformational change from an open to a closed state in which the ligand is enveloped between two domains that are linked by a flexible hinge. The protein conformations of the glucose complexes of the ttGGBP F17C⋅Badan, ttGGBP W182C⋅Acrylodan and ecGGBP W183C⋅Acrylodan conjugates are as closed as the unmodified, wild-type proteins. The fluorophores do not occupy the positions of the wild-type aromatic rings that they replace, but instead point outwards into the solvent such that their dimethylamino groups make no contacts with the protein and is coplanar with naphthalene ring (e.g., in the case of ttGGBP17C⋅Badan, where the structure of this group is unambiguous). By contrast, the carbonyl is located at the end of the linker through which the fluorophore is coupled to the protein cysteine. The torsion between it and the naphthalene ring therefore is affected by a combination of interactions with residues proximal to the attachment site and distal interactions with the ring. Ligand-mediated signaling occurs if two conditions are satisfied: (i) the protein interacts with both the naphthalene and the carbonyl, stabilizing their relative twist, and (ii) these interactions differ in the open and closed states.

Analysis of the spectra of all conjugates (Tables 7 and 8) showed that these signaling conditions are satisfied most commonly if the fluorophore is planar in the open, ligand-free protein conformation and twists in the closed conformation of the glucose complex. In the case of F17C⋅Badan structure, the closed protein conformation stabilizes the twisted form by interactions with both the carbonyl and the naphthalene ring. These interactions are contributed by distal residues in the domain located opposite the fluorophore attachment point, and therefore are likely to be present in the closed but not open protein conformation. Less commonly, the fluorophore twists in the open protein conformation through proximal interactions, and becomes untwisted in the closed conformation. The ttGGBP W182C⋅Acrylodan and ecGGBP W183C⋅Acrylodan conjugates represent this case. The interactions that stabilize the twisted state in the open protein conformation have not been identified, but the structure of the closed conformation reveals an absence of interactions that twist the naphthalene ring and carbonyl relative to each other and the fluorophore adopts a low-energy planar state.

Mutations that affect ligand binding (Table 6) also can influence spectroscopic properties (Table 8). In the Class 1 PCS mutant H151Q ratiometry is abolished, by diminishing the fraction of S₂that forms in the glucose complex. In D15A the redistribution of the two excited populations enhances ratiometric responses in both conjugates. In Class 2 mutants, the intrinsic equilibrium between the open and closed conformations is manipulated. In all the Class 2 mutants of both F17C Acrylodan and Badan conjugates, the fraction of the S₁state in the apo-protein (^apof(S₁)) is altered. These effects can both enhance or abolish wavelength shifts (Table 7 and 8). For instance, shifts are strengthened in the mutant Badan conjugates, but abolished in the Acrylodan conjugates as evidenced by the magnitude of the C₂component. Mutations in the carbonyl hole (Class 3a) and the fluorophore channel walls (Class 3b) have large effects on glucose binding and fluorescence spectra. Many of the N258 mutants in the carbonyl hole do not respond to glucose. The absence of shifts in thermal stability in the presence of glucose indicates that these mutants no longer bind ligand. The 17C⋅Acrylodan N258S mutant conjugate is the exception: it binds and responds to glucose, but binding does not alter the distribution of excited state populations. Mutations in residues located on either side of the channel wall strongly influence both the distributions of the excited states in the ligand-free protein, and their ligand-mediated redistribution. Mutations in A260 all switch the 17C⋅Badan conjugate from a mixture of S₁and S₂states in the apo-protein to a predominantly S₁state that exhibit small redistributions in response to glucose. Accordingly, most mutant conjugates exhibit isochromic instead of hypsochromic responses to glucose, with exception of 17C⋅Badan A260W which retains (diminished) hypsochromicity. By contrast, the 17C⋅Acrylodan A260 mutants retain the mixed S₁and S₂population in the apo-protein, but all response to glucose binding.

Although conformational coupling between the open and closed forms of the protein and the fluorophore internal torsional equilibrium is a major factor in determining the ligand-responsive changes in fluorescence of the Acrylodan and Badan conjugates, environmental effects on excited state dipole strengths also contribute, as evidenced by the presence of fine structure in the residuals between the models and obervations (FIGS. 17 and 18). The conjugates or their mutants differ in the degree to which fine structure is present in the emission spectra and responsive to glucose. This fine structure is due to changes in the energies of the S₁and S₂excited states associated with differences of their local environment in the open and closed conformations. The analysis presented here treats these differences as an ensemble average approximation.

The mechanism described is consistent with observations of the experimental properties of Prodan derivatives in which the twisted state of either the DMA or the carbonyl groups were controlled by synthesis of conformationally constrained groups (Lobo 2003 Journal of Physical Chemistry, 107, 10938-10943; Davis 2005 J. Phys. Chem., 109, 1295-1298; Green 2012 J. Org. Chem., 78, 1784-1789; Everett 2010 Journal of Physical Chemistry, 114, 4946-4950; Naughton 2013 J. Phys. Chem., 117, 3323-3327; Nikitina 2013 J. Phys. Chem., 117, 9189-9195; Daneri 2015 Journal of Photochemistry and Photobiology A: Chemistry, 310, 106-112).

Semisynthetic, fluorescently responsive proteins are useful in the development of reagentless biosensors with a wide variety of applications. However, in the absence of design principles that guide choice and placement of fluorophores, systematic identification of suitable fluorescent conjugates remains a significant challenge in their construction. The mechanistic insights described herein have revealed three key aspects of the fluorescence signal transduction mechanism that enable, inform, and direct rational engineering of fluorescent responses:

- 1. Signaling is controlled by the twisting of the carbonyl group, and changes in the specific interactions between it and the protein matrix via ligand-mediated protein conformational changes.
- 2. The linker length and concomitant degrees of torsional freedom determine the effectiveness of the ligand-mediated conformational coupling between the protein and the carbonyl at a given attachment position.
- 3. The planar ring system which determines the wavelength absorption and emission characteristics is not constrained by the protein matrix, but points outwards into the solvent.

The first two observations enable and direct structure-based approaches for identifying attachment positions and optimizing the functionalized linker for covalent modification. In such approaches, the internal flexibility of the linker is modeled, and the resulting library of fluorophore conformations are placed in a three-dimensional model of a protein host, checked for steric compatibility with their environment, and attachment sites and possible additional mutations identified that (de)stabilize a particular conformation or hydrogen bond with the protein exclusively in the apo-protein or ligand complex. The third observation indicates that the feasibility of introducing alternative chromophore structures that alter the fluorescence characteristics of the basic Prodan framework, while maintaining ligand-responsive signaling, provided the structural characteristics of the carbonyl and linker are maintained. Of particular interest are the design and construction of chromophore variants with wavelengths in the transparent window of blood (>600 nm). These principles are applied not only to periplasmic binding proteins and Prodan derivatives, but also to establish conformational coupling of any suitably placed, internally twisting fluorophore in proteins that undergo ligand-mediated changes in protein conformations.

TABLE 1

PCS position and sequence

#
Accession code
Species
14
16
91
152

1
2GBP (seed structure)
Escherichia coli
D
F
N
H

2
NC_013654|YP_003350022.1
Escherichia coli
D
F
N
H

3
NC_016822|YP_005457115.1
Shigella sonnei
D
F
N
H

4
NC_017328|YP_005727882.1
Shigella flexneri
D
F
N
H

5
NC_011740|YP_002383354.1
Escherichia fergusonii
D
F
N
H

6
NC_010658|YP_001879517.1
Shigella boydii
D
F
N
H

7
NC_022912|Asd1617_02874
Shigella dysenteriae
D
F
N
H

8
NC_013716|YP_003365823.1
Citrobacter rodentium
D
F
N
H

9
NC_009792|YP_001452231.1
Citrobacter koseri
D
F
N
H

10
NC_021500|H650_06520
Enterobacter sp.
D
F
N
H

11
NC_014121|ECL_03459
Enterobacter cloacae
D
F
N
H

12
NC_015968|YP_004829427.1
Enterobacter asburiae
D
F
N
H

13
NC_009778|YP_001437192.1
Cronobacter sakazakii
D
F
N
H

14
NC_016514|YP_004952913.1
Enterobacter cloacae
D
F
N
H

15
NC_011283|YP_002237430.1
Klebsiella pneumoniae
D
F
N
H

16
NC_020063|YP_007339667.1
Enterobacteriaceae bacterium
D
F
N
H

17
NC_016810|YP_005182060.1
Salmonella enterica
D
F
N
H

18
NC_013850|YP_003438413.1
Klebsiella variicola
D
F
N
H

19
NC_013282|YP_003211183.1
Cronobacter turicensis
D
F
N
H

20
NC_021232|YP_007990486.1
Klebsiella pneumoniae
D
F
N
H

21
NC_015761|YP_004730836.1
Salmonella bongori
D
F
N
H

22
NC_016612|YP_005021097.1
Klebsiella oxytoca
D
F
N
H

23
NC_009436|YP_001177466.1
Enterobacter sp.
D
F
N
H

24
NC_011147|YP_002141464.1
Salmonella enterica
D
F
N
H

25
NC_021066|YP_007872566.1
Raoultella ornithinolytica
D
F
N
H

26
NC_015663|YP_004594960.1
Enterobacter aerogenes
D
F
N
H

27
NC_014618|YP_003941097.1
Enterobacter lignolyticus
D
F
N
H

28
NC_015224|YP_004297664.1
Yersinia enterocolitica
D
F
N
H

29
NC_020211|YP_007405370.1
Serratia marcescens
D
F
N
H

30
NC_021741|M495_07335
Serratia liguefaciens
D
F
N
H

31
NC_014029|YP_003567546.1
Yersinia pestis
D
F
N
H

32
NC_010634|YP_001872058.1
Yersinia pseudotuberculosis
D
F
N
H

33
NC_009832|YP_001477795.1
Serratia proteamaculans
D
F
N
H

34
NC_015567|YP_004504928.1
Serratia plymuthica
D
F
N
H

35
NC_015566|YP_004499976.1
Serratia sp.
D
F
N
H

36
NC_015061|YP_004212168.1
Rahnella sp.
D
F
N
H

37
NC_016818|YP_005199494.1
Rahnella aquatilis
D
F
N
H

38
NC_007712|YP_454643.1
Sodalis glossinidius
D
F
N
H

39
NC_020418|YP_007506508.1
Morganella morganii
D
F
N
H

40
NC_021290|YP_008045423.1
Aeromonas hydrophila
D
F
N
H

41
NC_012912|YP_003005296.1
Dickeya zeae
D
F
N
H

42
NC_014562|YP_003931657.1
Pantoea vagans
D
F
N
H

43
NC_014837|YP_004116494.1
Pantoea sp.
D
F
N
H

44
NC_014500|YP_003882024.1
Dickeya dadantii
D
F
N
H

45
NC_014306|YP_003742362.1
Erwinia billingiae
D
F
N
H

46
NC_015424|YP_004390764.1
Aeromonas veronii
D
F
N
H

47
NC_013956|YP_003520836.1
Pantoea ananatis
D
F
N
H

48
NC_022546|YP_008651638.1
Plautia stali
D
F
N
H

49
NC_009348|YP_001140155.1
Aeromonas salmonicida
D
F
N
H

50
NC_010694|YP_001907230.1
Erwinia tasmaniensis
D
F
N
H

51
NC_013961|YP_003531646.1
Erwinia amylovora
D
F
N
H

52
NC_022268|YP_008522161.1
Serratia sp.
D
F
N
H

53
NC_012691|YP_002893819.1
Tolumonas auensis
D
F
N
H

54
NC_006371|YP_133522.1
Photobacterium profundum
D
F
N
H

55
NC_020802|YP_007640992.1
Psychromonas sp.
D
F
N
H

56
NC_022223|N175_08855
Listonella anguillarum
D
F
N
H

57
NC_015633|YP_004566361.1
Vibrio anguillarum
D
F
N
H

58
NC_016445|YP_004936985.1
Vibrio cholerae
D
F
N
H

59
NC_016602|YP_004992976.1
Vibrio furnissii
D
F
N
H

60
NC_015460|YP_004420811.1
Gallibacterium anatis
D
F
N
H

61
NC_014966|YP_004190360.1
Vibrio vulnificus
D
F
N
H

62
NC_006300|YP_087835.1
Mannheimia succiniciproducens
D
F
N
H

63
NC_015964|YP_004822812.1
Haemophilus parainfluenzae
D
F
N
H

64
NC_012913|YP_003007706.1
Aggregatibacter aphrophilus
D
F
N
H

65
NC_000907|NP_438982.2
Haemophilus influenzae
D
F
N
H

66
NC_009655|YP_001345182.1
Actinobacillus succinogenes
D
F
N
H

67
NC_014920|YP_004135881.1
Haemophilus influenza
D
F
N
H

68
NC_016513|YP_004948894.1
Aggregatibacter actinomycetemc
D
F
N
H

69
NC_010939|YP_001969307.1
Actinobacillus pleuropneumonia
D
F
N
H

70
NC_016808|YP_005176051.1
Pasteurella multocida
D
F
N
H

71
NC_010519|YP_001783460.1
Haemophilus somnus
D
F
N
H

72
NC_021082|YP_007882660.1
Mannheimia haemolytica
D
F
N
H

73
NC_017846|YP_006287121.1
Aggregatibacter actinomycetemc
D
F
N
H

74
NC_018690|YP_006818133.1
Actinobacillus suis
D
F
N
H

75
NC_011852|YP_002475059.1
Haemophilus parasuis
D
F
N
H

76
NC_008312|YP_720691.1
Trichodesmium erythraeum
D
F
N
H

77
NC_020515|YP_007548075.1
Bibersteinia trehalosi
D
F
N
H

78
NC_022524|N288_24375
Bacillus infantis
D
F
N
H

79
NC_021281|YP_008019649.1
Fusobacterium nucleatum
D
F
N
H

80
NC_003454|NP_604062.1
Fusobacterium nucleatum
D
F
N
H

81
NC_003366|NP_562257.1
Clostridium perfringens
D
F
N
H

82
NC_018607|YP_006708369.1
Brachyspira pilosicoli
D
F
N
H

83
NC_019908|YP_007234804.1
Brachyspira pilosicoli
D
F
N
H

84
NC_012225|YP_002722709.1
Brachyspira hyodysenteriae
D
F
N
H

85
NC_017243|YP_005593986.1
Brachyspira intermedia
D
F
N
H

86
NC_014150|YP_003634598.1
Brachyspira murdochii
D
F
N
H

87
NC_014330|YP_003786208.1
Brachyspira pilosicoli
D
F
N
H

88
NC_012491|YP_002770161.1
Brevibacillus brevis
D
F
N
H

89
NC_015732|YP_004698509.1
Treponema caldaria
D
F
N
H

90
NC_020291|Cspa_c46680
Clostridium saccharoperbutylac
D
F
N
H

91
NC_009617|YP_001311499.1
Clostridium beijerinckii
D
F
N
H

92
NC_014364|YP_003802961.1
Spirochaeta smaragdinae
D
Y
N
H

93
NC_015152|YP_004246106.1
Sphaerochaeta globosa
D
F
N
H

94
NC_014392|YP_003839461.1
Caldicellulosiruptor obsidians
D
F
N
H

95
NC_015578|YP_004532181.1
Treponema primitia
D
F
N
H

96
NC_014410|YP_003852930.1
Thermoanaerobacterium thermosa
D
F
N
H

97
NC_012914|YP_003010600.1
Paenibacillus sp.
D
F
N
H

98
NC_010278|YP_001651502.1
Actinobacillus pleuropneumonia
D
F
N
H

99
NC_016633|YP_005062659.1
Sphaerochaeta pleomorpha
D
F
N
H

100
NC_014393|YP_003845273.1
Clostridium cellulovorans
D
F
N
H

101
NC_014652|YP_003991244.1
Caldicellulosiruptor hydrother
D
F
N
H

102
NC_022777|YP_008772951.1
Clostridium tetani
D
F
N
H

103
NC_018690|YP_006817320.1
Actinobacillus suis
D
F
N
H

104
NC_009053|YP_001053159.1
Actinobacillus pleuropneumonia
D
F
N
H

105
NC_014393|YP_003845272.1
Clostridium cellulovorans
D
F
N
N

106
NC_010939|YP_001968321.1
Actinobacillus pleuropneumonia
D
F
N
H

107
NC_021658|SCE1572_01005
Sorangium cellulosum
N
F
N
H

108
NC_015690|YP_004645360.1
Paenibacillus mucilaginosus
D
F
N
H

109
NC_016935|YP_005316341.1
Paenibacillus mucilaginosus
D
F
N
H

110
NC_012914|YP_003013039.1
Paenibacillus sp.
D
F
N
H

111
NC_015519|YP_004460241.1
Tepidanaerobacter acetatoxydan
D
F
N
H

112
NC_013406|YP_003243743.1
Paenibacillus sp.
D
F
N
H

113
NC_015519|YP_004461277.1
Tepidanaerobacter acetatoxydan
D
F
N
H

114
NC_013517|YP_003308491.1
Sebaldella termitidis
D
F
N
H

115
NC_014150|YP_003634599.1
Brachyspira murdochii
D
F
N
H

116
NC_015436|YP_004411990.1
Sphaerochaeta coccoides
D
F
N
H

117
NC_021038|YP_007827124.1
Fretibacterium fastidiosum
D
F
N
H

118
NC_020291|Cspa_c46620
Clostridium saccharoperbutylac
D
F
N
H

119
NC_009617|YP_001311493.1
Clostridium beijerinckii
D
F
N
H

120
NC_015977|YP_004837837.1
Roseburia hominis
D
F
N
H

121
NC_021018|YP_007796707.1
Coprococcus sp.
D
F
N
H

122
NC_021042|YP_007838599.1
Faecalibacterium prausnitzii
D
F
N
N

123
NC_012781|YP_002936409.1
Eubacterium rectale
D
F
N
H

124
NC_015601|YP_004561181.1
Erysipelothrix rhusiopathiae
D
F
N
N

125
NC_021354|YP_008073256.1
Erysipelothrix rhusiopathiae
D
F
N
N

126
NC_021012|YP_007778124.1
Roseburia intestinalis
D
F
N
H

127
NC_022592|CAETHG_2989
Clostridium autoethanogenum
N
W
N
N

128
NC_014328|CLIU_c08950
Clostridium ljungdahlii
N
W
N
N

129
NC_021020|YP_007799070.1
Faecalibacterium prausnitzii
D
F
N
N

130
NC_021040|YP_007833443.1
Roseburia intestinalis
D
F
N
H

131
NC_021035|YP_007824422.1
butyrate-producing bacterium
D
F
N
N

132
NC_021012|YP_007778116.1
Roseburia intestinalis
D
F
N
N

133
NC_015977|YP_004837830.1
Roseburia hominis
D
F
N
N

134
NC_021040|YP_007833436.1
Roseburia intestinalis
D
F
N
N

135
NC_014376|YP_003822569.1
Clostridium saccharolyticum
D
F
N
N

136
NC_022549|YP_008656214.1
Acholeplasma brassicae
D
F
N
H

137
NC_014387|YP_003830205.1
Butyrivibrio proteoclasticus
D
F
N
N

138
NC_014376|YP_003822565.1
Clostridium saccharolyticum
D
F
N
N

PCS position and sequence

#
154
158
183
211
236
256
Identity
Thermophilicity
Gram

1
D
R
W
N
D
N

2
D
R
W
N
D
N
1.00
Mesophilic
−

3
D
R
W
N
D
N
1.00
Mesophilic
−

4
D
R
W
N
D
N
1.00
Mesophilic
−

5
D
R
W
N
D
N
1.00
Mesophilic
−

6
D
R
W
N
D
N
1.00
Mesophilic
−

7
D
R
W
N
D
N
1.00
Mesophilic
−

8
D
R
W
N
D
N
0.97
Mesophilic
−

9
D
R
W
N
D
N
0.96
Mesophilic
−

10
D
R
W
N
D
N
0.95
Mesophilic
−

11
D
R
W
N
D
N
0.95
Mesophilic
−

12
D
R
W
N
D
N
0.95
Mesophilic
−

13
D
R
W
N
D
N
0.94
Mesophilic
−

14
D
R
W
N
D
N
0.94
Mesophilic
−

15
D
R
W
N
D
N
0.94
Mesophilic
−

16
D
R
W
N
D
N
0.94
Mesophilic
+

17
D
R
W
N
D
N
0.94
Mesophilic
−

18
D
R
W
N
D
N
0.94
Mesophilic
−

19
D
R
W
N
D
N
0.94
Mesophilic
−

20
D
R
W
N
D
N
0.94
Mesophilic
−

21
D
R
W
N
D
N
0.94
Mesophilic
−

22
D
R
W
N
D
N
0.94
Mesophilic
−

23
D
R
W
N
D
N
0.94
Mesophilic
−

24
D
R
W
N
D
N
0.94
Mesophilic
−

25
D
R
W
N
D
N
0.93
Mesophilic
+

26
D
R
W
N
D
N
0.93
Mesophilic
−

27
D
R
W
N
D
N
0.93
Mesophilic
−

28
D
R
W
N
D
N
0.90
Mesophilic
−

29
D
R
W
N
D
N
0.88
Mesophilic
+

30
D
R
W
N
D
N
0.88
Mesophilic
+

31
D
R
W
N
D
N
0.87
Mesophilic
−

32
D
R
W
N
D
N
0.87
Mesophilic
−

33
D
R
W
N
D
N
0.87
Mesophilic
+

34
D
R
W
N
D
N
0.86
Mesophilic
+

35
D
R
W
N
D
N
0.86
?
+

36
D
R
W
N
D
N
0.86
Mesophilic
+

37
D
R
W
N
D
N
0.85
Mesophilic
+

38
D
R
W
N
D
N
0.84
Mesophilic
−

39
D
R
W
N
D
N
0.82
Mesophilic
+

40
D
R
W
N
D
N
0.82
Mesophilic
−

41
D
R
W
N
D
N
0.82
Mesophilic
−

42
D
R
W
N
D
N
0.81
Mesophilic
−

43
D
R
W
N
D
N
0.81
Mesophilic
−

44
D
R
W
N
D
N
0.81
Mesophilic
−

45
D
R
W
N
D
N
0.81
Mesophilic
−

46
D
R
W
N
D
N
0.81
Mesophilic
−

47
D
R
W
N
D
N
0.81
Mesophilic
−

48
D
R
W
N
D
N
0.80
Mesophilic
+

49
D
R
W
N
D
N
0.80
Mesophilic
−

50
D
R
W
N
D
N
0.80
Mesophilic
−

51
D
R
W
N
D
N
0.79
Mesophilic
−

52
D
R
W
N
D
N
0.78
?
+

53
D
R
W
N
D
N
0.76
Mesophilic
−

54
D
R
W
N
D
N
0.74
Psychrophilic
−

55
D
R
W
N
D
N
0.72
Mesophilic
+

56
D
R
W
N
D
N
0.72
Mesophilic
+

57
D
R
W
N
D
N
0.72
Mesophilic
−

58
D
R
W
N
D
N
0.72
Mesophilic
−

59
D
R
W
N
D
N
0.71
Mesophilic
−

60
D
R
W
N
D
N
0.71
Mesophilic
−

61
D
R
W
N
D
N
0.71
Mesophilic
−

62
D
R
W
N
D
N
0.70
Mesophilic
+

63
D
R
W
N
D
N
0.70
Mesophilic
−

64
D
R
W
N
D
N
0.69
Mesophilic
−

65
D
R
W
N
D
N
0.69
Mesophilic
−

66
D
R
W
N
D
N
0.69
Mesophilic
−

67
D
R
W
N
D
N
0.69
Mesophilic
−

68
D
R
W
N
D
N
0.68
Mesophilic
−

69
D
R
W
N
D
N
0.68
Mesophilic
−

70
D
R
W
N
D
N
0.68
Mesophilic
−

71
D
R
W
N
D
N
0.68
Mesophilic
−

72
D
R
W
N
D
N
0.68
Mesophilic
+

73
D
R
W
N
D
N
0.68
Mesophilic
−

74
D
R
W
N
D
N
0.67
Mesophilic
−

75
D
R
W
N
D
N
0.66
Mesophilic
−

76
D
R
W
N
D
N
0.59
Mesophilic
−

77
D
R
W
N
D
N
0.58
Mesophilic
+

78
D
R
W
N
D
N
0.58
Mesophilic
+

79
D
R
W
N
D
N
0.58
Mesophilic
−

80
D
R
W
N
D
N
0.57
Mesophilic
−

81
D
R
W
N
D
N
0.56
Mesophilic
+

82
D
R
W
N
D
N
0.53
Mesophilic
−

83
D
R
W
N
D
N
0.52
Mesophilic
−

84
D
R
W
N
D
N
0.52
Mesophilic
−

85
D
R
W
N
D
N
0.52
?
−

86
D
R
W
N
D
N
0.52
Mesophilic
−

87
D
R
W
N
D
N
0.51
Mesophilic
−

88
D
R
W
N
D
N
0.50
Mesophilic
+

89
D
R
W
N
D
N
0.50
Mesophilic
−

90
D
R
W
N
D
N
0.49
Mesophilic
+

91
D
R
W
N
D
N
0.49
Mesophilic
+

92
D
R
W
N
D
N
0.49
Mesophilic
−

93
D
R
W
N
D
N
0.48
Mesophilic
+

94
D
R
W
N
D
N
0.48
Hyperthermophilic
+

95
D
R
W
N
D
N
0.48
Mesophilic
−

96
D
R
W
N
D
N
0.48
Thermophilic
+

97
D
R
W
N
D
N
0.48
Mesophilic
+

98
D
R
W
N
D
N
0.48
Mesophilic
−

99
D
R
W
N
D
N
0.48
Mesophilic
+

100
D
R
W
N
D
N
0.47
Mesophilic
+

101
D
R
W
N
D
N
0.47
Hyperthermophilic
+

102
D
R
W
N
D
N
0.47
Mesophilic
+

103
D
R
W
N
D
N
0.47
Mesophilic
−

104
D
R
W
N
D
N
0.47
Mesophilic
−

105
D
R
W
N
D
N
0.47
Mesophilic
+

106
D
R
W
N
D
N
0.46
Mesophilic
−

107
D
R
W
N
D
N
0.46
Mesophilic
−

108
D
R
W
N
D
N
0.46
Mesophilic
+

109
D
R
W
N
D
N
0.45
Mesophilic
+

110
D
R
W
N
D
N
0.44
Mesophilic
+

111
D
R
W
N
D
N
0.44
Mesophilic
+

112
D
R
W
N
D
N
0.44
Mesophilic
+

113
D
R
W
N
D
N
0.43
Mesophilic
+

114
D
R
W
N
D
N
0.43
Mesophilic
−

115
D
R
W
N
D
N
0.43
Mesophilic
−

116
D
R
W
N
D
N
0.41
Mesophilic
+

117
D
R
W
N
D
N
0.41
Mesophilic
+

118
D
R
W
N
D
N
0.40
Mesophilic
+

119
D
R
W
N
D
N
0.39
Mesophilic
+

120
D
R
W
N
D
N
0.38
Mesophilic
+

121
D
R
W
N
D
N
0.38
Mesophilic
+

122
D
R
W
N
D
N
0.37
Mesophilic
−

123
D
R
W
N
D
N
0.37
Mesophilic
+

124
D
R
W
N
D
N
0.36
Mesophilic
+

125
D
R
W
N
D
N
0.36
Mesophilic
+

126
D
R
W
N
D
N
0.36
Mesophilic
+

127
D
R
W
N
D
N
0.36
Mesophilic
+

128
D
R
W
N
D
N
0.35
Mesophilic
+

129
D
R
W
N
D
N
0.34
Mesophilic
−

130
D
R
W
N
D
N
0.34
Mesophilic
+

131
D
R
W
N
D
N
0.33
Mesophilic
?

132
D
R
W
N
D
N
0.33
Mesophilic
+

133
D
R
W
N
D
N
0.33
Mesophilic
+

134
D
R
W
N
D
N
0.32
Mesophilic
+

135
D
R
W
N
D
N
0.31
Mesophilic
+

136
D
R
W
N
D
N
0.31
Mesophilic
+

137
D
R
W
N
D
N
0.30
Mesophilic
+

138
D
R
W
N
D
N
0.29
Mesophilic
+

Each nucleotide and amino acid sequence associated with the accession numbers listed in Table 1 is hereby incorporated by reference in its entirety.

TABLE 2

Glucose-binding properties of E. coli GGBP SAFE sequence homologs.

T_mshift with
Percent

Accession number
Protein name
Expression^a
Glucose^a
Identity

NC_013654|YP_003350022.1
ecGGBP
Y
Y
100

NC_014376|YP_003822565.1
csaGGBP
Y
Y
29

NC_014387|YP_003830205.1
bprGGBP
N

30

NC_021012|YP_007778116.1
rinGGBP_A
N

33

NC_021020|YP_007799070.1
fprGGBP
Y
Y
34

NC_014328|CLJU_c08950
cljGGBP
N

35

NC_022592|CAETHG_2989
cauGGBP
N

36

NC_021012|YP_007778124.1
rinGGBP_B
Y
Y
36

NC_015601|YP_004561181.1
erhGGBP
Y
Y
36

NC_012781|YP_002936409.1
ereGGBP
N

37

NC_014410|YP_003852930.1
ttGGBP
Y
Y
48

NC_014392|YP_003839461.1
cobGGBP
Y
Too
48

thermostable^b

NC_014652|YP_003991244.1
chyGGBP
Y
Too
47

thermostable^b

NC_013406|YP_003243743.1
pspGGBP
N

44

^aY, Yes; N, No.

^bDetermined using fluorescent Acrylodan conjugates (see text).

TABLE 3

Glucose response of Acrylodan and Badan conjugates

in a cysteine scan of the ttGGBP scaffold.

Emission
K_d^d,e

^apoT_m
Wavelength (nm)
(mM)

Mutation
Class^a
Shape^b
Conjugate^c
(K)
λ1
λ2
^appK_d
^trueK_d

Y11C
p
m
A
351
511
470
0.12
0.16

d
B
349
492
528
0.28
0.24

T16C
p
m
A
351
519
460
8.8
12

m
B
349
—
—
nb
nb

F17C
e
d
A
349
482
542
0.08
0.06

d
B
346
467
491
0.087
0.26

N42C
p

A
350
—
—
nb
nb

B
324
—
—
nb
nb

V67C
p

A
350
—
—
nb
nb

B
322
—
—
nb
nb

R91C
e
m
A
349
491
540
0.18
0.17

B
348
—
—
nb^d
nb^d

E92C
p

A
350
—
—
nb
nb

B
346
—
—
nb
nb

A111C
p
m
A
350
515
550
0.19^d
0.11^d

m/d
B
348
523
550
0.64
0.55

Q148C
p

A
351
—
—
nb
nb

B
349
—
—
nb
nb

H151C
e
m
A
351
511
489
0.012
0.025

m
B
348
523
550
0.018^d
0.027^d

Q152C
p

A
351
—
—
nb^d
nb^d

B
349
—
—
nb
nb

N181C
p

A
350
—
—
nb
nb

B
349
—
—
nb
nb

W182C
e
d
A
347
479
526
2.3
2.3

m
B
347
515
550
27
19

D183C
p

A
348
—
—
nb^d
nb^d

B
348
—
—
nb
nb

L257C
a

A
352
—
—
nb
nb

B
348
—
—
nb
nb

D259C
a

A
349
—
—
nb
nb

B
347
—
—
nb
nb

K300C
a

A
349
—
—
nb
nb

B
348
—
—
nb
nb

^aa, allosteric; e, endosteric; p, peristeric.

^bm, monochromatic; d, dichromatic (i.e. spectral shape change).

^cA, Acrylodan; B, Badan.

^dnoisy data and or bad fit.

^enb; no binding, nd; not determined.

TABLE 4

Responses of fluorophores conjugated to F17C and W182C mutants of ttGGBP^a.

λ_ex
^apoλ_max
^apoI_max
^satλ_max
^satI_max
^trueK_d

Position
Fluorophore^b
(nm)^c
(nm)
(AU ×1000)
(nm)
(AU ×1000)
(mM)

17C
Acrylodan
391
487
15.9
487
20.0
0.2

Badan
391
519
12.8
467
52.6
0.1

5-IAF
491
523
64.3
523
70.6
61.3

Oregon green
496
523
101.9
523
91.7
—

CPM
384
471
78.5
467
90.9
17.0

IANBD
478
531
15.3
535
19.8
11.8

IAEDANS
336
467
12.5
467
14.4
—

Pacific Blue
410
451
28.6
455
108.6
60.7

182C
Acrylodan
391
479
55.0
515
17.2
6.0

Badan
391
515
16.8
515
11.9
64.9

5-IAF
491
519
255.0
519
453.2
5.0

6-IAF
491
513
50.4
513
63.1
750

Oregon green
496
519
78.2
519
186.2
20.0

CPM
384
479
49.5
483
40.3
6.8

IANBD
478
543
29.1
547
12.5
210

IAEDANS
336
487
3.7
483
7.9
0.2

Pacific Blue
410
455
115.0
455
119.4
—

BODIPY 499
499
519
40.1
515
80.2
30.7

BODIPY 507
507
531
32.9
535
31.4
11.0

Alexa 488
495
519
211.6
519
182.2
—

Alexa 532
532
551
63.0
551
61.0
—

Alexa 546
546
571
152.1
571
150.2
—

Texas Red
595
611
23.3
611
23.5
—

Cy 5
646
663
19.2
663
23.3
210

PyPMPO
415
555
3.7
559
4.2
20.6

^aλ_ex, preferred excitation wavelength (from supplier); ^apoλ_max, observed maximum emission wavelength of the apo-protein; ^apoI_max, observed intensity at ^apoλ_max; ^apoλ_max, observed maximum emission wavelength of the glucose complex; ^satI_max, observed intensity at ^satλ_max; ^trueK_d, affinity determined from fit of equation 1 to the monochromatic emission intensities. Emission spectra were measured on the Nanodrop3300, using ~10 μM protein. The observed absolute emission intensities are a rough guide to the brightness of the conjugate, because the protein concentration was approximately the same for each experiment.

^bAbbreviations, chemical names and supplier catalogue numbers as follows: Acrylodan (A433); Badan (B6057); 5-IAF (I30451); Oregon Green 488 (O6034); CPM (D346); IANBD (D2004); IAEDANS (I14); Pacific Blue (P30506); BODIPY 499 (D20350); BODIPY 507 (D6004); BODIPY 577 (D20351); Alexa 532 (A10255); Alexa 555 (A20346); Texas Red (T6008); PyMPO (M6026) from Life Technologies and Cy5 (13080) from Lumiprobe.

^cThe Nanodrop3300 fixed wavelength LED that most closely matched λ_exwas used (see Materials and Methods).

TABLE 5

Crystallographic data collection and refinement statistics.^a

ttGGBP17C-
ttGGBP182C.2.0-
ecGGBP183C-

Badan
Acrylodan
Acrylodan

X-ray source
22-ID, SER-
22-ID, SER-
22-BM, SER-

CAT, APS
CAT, APS
CAT, APS

Wavelength (Å)
1.0
1.0
1.0

Space Group
P2₁2₁2₁
P2₁2₁2₁
C121

Unit Cell parameters (Å)

a, b, c
45.49, 53.06,
45.67, 53.17,
119.58, 36.53,

134.32
133.57
79.90

90.00 (α), 124.13

(β), 90.00 (γ)

Resolution range (Å)
41.63-1.59
(1.62-1.59)
33.70-1.39
50.00-1.53
(1.56-1.53)

Completeness (%)
99.8
(99.3)
98.9
(94.9)
98.8
(94.6)

No. of unique reflections
44590
65108
(9969)
43030

Wilson B-factor (Å²)
15.50
11.50
12.60

Multiplicity
7.21
(7.22)
5.29
(5.17)
6.5
(5.1)

R-sym (%)
0.08
(0.69)
0.08
(0.76)
0.06
(0.29)

R-pim (%)
0.03
(0.25)
0.04
(0.34)
0.025
(0.131)

Mean I/σ (I)
16.40
(3.58)
13.71
(2.87)
25.54
(3.99)

Refinement statistics

R factor (%)
15.11
15.88
15.04

Free_R_factor (%)
17.07
17.74
16.75

Average B-factor (Å2)
20.10
17.90
18.60

Macromolecules
17.70
15.30
15.90

Water
34.30
34.10
34.60

Glucose/Fluorophore
9.50/39.90
8.60/39.10
22.70/64.50

Number of non-hydrogen

atoms

Macromolecule
2471
2493
2358

Glucose
24^b
24^b
72^c

Calcium
1
1
1

Fluorophore
16
17
17

Water
407
391
325

RMS deviations

RMS (bonds)
0.007
0.005
0.006

RMS (angles)
1.112
0.822
1.060

Ramachandran favoured (%)
97.81
98.44
98.71

Ramachandran allowed (%)
1.88
1.25
0.97

Ramachandran outliers (%)
0.31
0.31
0.32

Rotamer outliers (%)
0.00
0.00
0.00

Clashscore
1.60
0.79
1.03

^aValues for highest resolution shell are given in parentheses.

^bTwo conformations.

^cFive molecules.

TABLE 6

Responses of mutant HGGBP17C and ttGGBP182C conjugates^a.

Emission
Glucose^d
Galactose^d

^apoT_m
wavelength (nm)
^appK_d
^trueK_d
^trueK_d

Protein
Mutation
Class^b
Conjugate^c
(K)
λ1
λ2
(mM)
(mM)
(mM)
S

ttGBP17C

B
346
467
519
0.10
0.15
0.19
1.3

″

A
349
487
515
0.08
0.09
3.8
43

ttGGBP17C.1
R91K, Q148E
2
B

463
515
0.6
0.8
0.4
0.46

ttGGBP17C.2
R69P, Q152P
2
B
350
479
523
8.6
10.8
5.2
0.48

″
″
2
A

492
515
15
18
5.0
0.28

ttGGBP17C.3
T16N, D211A
2
B
346
471
531
3.4
4.1
2.5
0.61

″
″
2
A

495
529
1.4
1.5
0.53
0.35

ttGGBP17C.4
H151Q
1
B
347
511
457
16
6.4
14
2.2

″
″
1
A

488
550
50
48
14
0.29

ttGGBP17C.5
D15A
1
B
345
487
530
16
16
4.2
0.26

″
″
1
A
348
483
498
6
6
2.4
0.41

ttGGBP17C.6
D15E
1
B
346
467
525
3.3
3.6
3.6^e
1

ttGGBP17C.7
D15N
1
B
347
483
515
0.75
1.0
0.64
0.64

″
″
1
A
348
483
515
0.3
0.3
0.3
0.91

ttGGBP17C.8
T16N
2
A
348
487
529
0.61
0.60
0.26
0.43

ttGGBP17C.9
T16S
2
A

487
520
0.2
0.20
nd

ttGGBP17C.10
G20A
3
A
351
487
520
0.4
0.4
nd

ttGGBP17C.11
T240A
3
A
348
487
500
0.04
0.04
nd

ttGGBP17C.19
N258D
3
B
344

nb^e
nb^e
nb^e

ttGGBP17C.20
N258S
3
B
344

nb^e
nb^e
nb^e

ttGGBP17C.21
N258A
3
B
345
532
494
61
69
nb

ttGGBP17C.22
A260N
3
B
343
523
492
12
15
530^e

ttGGBP17C.23
A260Q
3
B
344
527
490
15
18
nb^c

ttGGBP17C.24
A260R
3
B
344
490
515
1.8
1.7
29
17

ttGGBP17C.25
A260K
3
B

515
493
3.2
3.7
nb^e

ttGGBP17C.26
A260W
3
B
346
523
509
0.9
0.9
14
16

ttGGBP17C.27
A260F
3
B
346
523
490
0.2
0.3
8.8
33

ttGGBP17C.28
A260Y
3
B
346
523
494
0.06
0.07
2.6
37

ttGGBP17C.29
A260S
3
B
343
527
496
2.1
2.2
250
114

ttGGBP182C

A
347
472
535
2.2
2.3
3.3
1.4

ttGGBP182C.2.0^f
R91K Q148E
2, 3
A
346
475
545
4.5
6.0
18.5
3.1

ttGGBP182C.2.1^g
A154S
1
A
328
480
552
3.0
4.1
13.1
3.2

ttGGBP182C.2.3^g
A154N
1
A
346
470
537
16.3
19.0
207
10.9

ttGGBP182C.2.4^g
A154M
1
A
346
472
540
0.4
0.4
4.5
11.2

ttGGBP182C.2.5^g
H151Q
1
A
346
477
542
10.8
13.0
20.3
1.6

ttGGBP182C.2.6^g
H151N
1
A
347
475
537
19.2
17.9
52.6
2.9

ttGGBP182C.2.7^g
H151F
1
A
346
475
545
121
124

ttGGBP182C.2.8^g
D15N
1
A
343
474
542
179
209
372
1.8

ttGGBP182C.2.9^g
A154F
1
A
345
512
477
0.4
0.3
1.6
4.0

ttGGBP182C.3^f
R91K
2
A
346
494
459
1.8
0.9
3.1
3.4

ttGGBP182C.4^f
Q148E
2
A
347
475
542
0.6
0.8
6.5
8.1

ttGGBP182C.5^f
R69P Q152P
2
A
349
477
535
7.5
8.8
7.4
0.8

ttGGBP182C.6^f
T16N D211A
2
A
345
472
530
21.6
27.6
81
2.9

ttGGBP182C.7^f
R91K Q148S
2, 3
A
346
478
550
0.3
0.4
1.1
3.7

ttGGBP182C.8^f
R91K Q148K
2, 3
A
346
475
545
28.7
44.2
256
5.8

ttGGBP182C.9^f
D15N
1
A
343
475
540
75.1
76.4
282
3.7

^aMeasured on the Nanodrop at room temperature. λ_maxis the wavelength corresponding to the maximum emission intensity. Optimal ratiometry wavelengths are determined according to the analysis described in Materials and Methods (equation 7). The ^trueK_dis determined from monochromatic titration curves; ^appK_dfrom dichromatic ratiometry (equations 5 and 6). Average relative error in the ^trueK_dvalues is 5%, in the ^appK_dvalues, 1%. S is the selectivity between glucose and galactose, S = ^trueK_d(galactose)/^trueK_d(glucose); S > 1, selective for glucose.

^b1, PCS; 2, inter-domain interaction; 3, contact between protein and fluorophore

^cA, Acrylodan; B, Badan.

^dnb, no bonding; nd, not determined.

^eNoisy data or bad fit.

^fAdditional mutation constructed in ttGGBP182C.

^gAdditional mutation constructed in ttGGBP182C.2.0.

TABLE 7

Spectral analysis of ttGGBP and ecGGBP

Acrylodan and Badan conjugates^a.

SVD component
Gaussians

Muta-
fractions
Peaks (nm)
Fraction f(S₁)

tion
Conjugate^b
C₁
C₂
S₁
S₂
Apo
Sat

Y11C
A
r (h)
0.97
0.03
515
463
1.00
0.88

B
r (h)
0.84
0.16
532
484
1.00
0.47

T16C
A
n
—
—
518
—
1.00
—

B
n
—
—
539
446
0.96
—

F17C
A
r (h)
0.90
0.09
527
486
0.56
0.16

B
r (h)
0.89
0.10
535
474
0.66
0.15

F16C
A
r (h)
0.91
0.08
509
454
0.63
0.10

(Ec)

B
r (h)
0.92
0.06
522
469
0.56
0.14

N42C
A
n
—
—
518
—
1.00
—

B
n
—
—
516
—
1.00
—

V67C
A
n
—
—
522
464
0.59
—

B
n
—
—
523
480
0.38
—

R91C
A
i
0.97
0.02
533
488
0.43
0.92

B
n
—
—
522
476
0.51
—

E92C
A
n
—
—
520
—
1.00
—

B
n
—
—
522
—
1.00
—

A111C
A
d
0.98
0.01
520
474
0.98
0.95

B
d
0.98
0.02
529
467
0.91
0.84

Q148C
A
n
—
—
537
491
0.26
—

B
n
—
—
576
519
0.15
—

H151C
A
i
0.97
0.02
520
490
1.00
0.86

B
i
0.98
0.01
533
477
0.98
1.00

Q152C
A
n
—
—
520
—
1.00
—

B
n
—
—
524
472
0.74
—

N181C
A
n
—
—
527
—
1.00
—

B
n
—
—
545
—
1.00
—

W182C
A
r (b)
0.87
0.13
500
463
0.63
0.99

B
d
0.98
0.02
521
479
0.91
1.00

W183C
A
r (b)
0.84
0.15
527
481
0.37
0.81

(Ec)

D183C
A
d

523
—
1.00
—

B
n
—
—
530
477
0.61
—

L257C
A
n
—
—
533
496
0.29
—

B
n
—
—
516
—
1.00
—

D259C
A
n
—
—
516
—
1.00
—

B
n
—
—
521
—
1.00
—

K300C
A
n
—
—
520
—
1.00
—

B
n
—
—
528
—
1.00
—

^aEmission spectra determined on Nanodrop3300. SVD analysis according to equations 11-13. Gaussian fits according to equations 14-15.

^bA, Acrylodan; B, Badan; n, no observed response to glucose; r (h), ratiometric (hypsochromic shift); r (b), ratiometric (bathochromic shift); i, intensity increase only; d, intensity decrease only.

TABLE 8

Spectral analysis of ttGGBP.17C Acrylodan and Badan conjugates^a.

SVD component
Gaussians

fractions
Peaks (nm)
Fraction f(S₁)

Protein
Mutation
Conjugate^b
C₁
C₂
S₁
S₂
Apo
Sat

ttGGBP17C.1
R91K, Q148E
A
d
0.95
0.04
518
490
0.71
0.60

″
″
B
r(h)
0.85
0.14
532
472
0.64
0.15

ttGGBP17C.2
R69P, Q152P
A
d
0.92
0.06
528
462
0.88
0.73

″
″
B
r(h)
0.86
0.13
542
476
0.64
0.36

ttGGBP17C.3
T16N, D211A
A
d
0.94
0.05
519
458
0.83
0.70

″
″
B
r(h)
0.81
0.18
540
474
0.70
0.30

ttGGBP17C.8
T16N
A
r(h)
0.93
0.07
533
496
0.30
0.00

ttGGBP17C.9
T16S
A
r(h)
0.94
0.06
510
488
1.00
0.43

ttGGBP17C.4
H151Q
A
r(h)
0.95
0.04
526
485
0.65
0.52

″
″
B
r(h)
0.95
0.04
540
486
0.50
0.39

ttGGBP17C.5
D15A
A
r(h)
0.87
0.13
491
465
0.98
0.22

″
″
B
r(h)
0.84
0.15
525
470
0.58
0.16

ttGGBP17C.6
D15E
A
n
—
—
511
448
0.72
—

″
″
B
r(h)
0.90
0.09
525
473
0.39
0.15

ttGGBP17C.7
D15N
A
d
0.97
0.02
492
464
0.86
1.00

″
″
B
d
0.92
0.05
533
480
0.22
0.16

ttGGBP17C.19
N258D
A
n
—
—
525
475
0.30
—

″
″
B
n
—
—
523
—
1.00
—

ttGGBP17C.20
N258S
A
d
0.99
0.00
530
479
0.37
0.36

″
″
B
n
—
—
524
—
1.00
—

ttGGBP17C.21
N258A
A
n
—
—
526
475
0.33
—

″
″
B
n
—
—
523
—
1.00
—

ttGGBP17C.10
G20A
A
d
0.95
0.04
534
487
0.28
0.21

ttGGBP17C.11
T240A
A
d
0.95
0.03
541
487
0.35
0.25

ttGGBP17C.22
A260N
A
n
—
—
528
475
0.33
—

″
″
B
i
0.97
0.03
531
480
0.96
0.85

ttGGBP17C.23
A260Q
A
i
—
—
526
474
0.31
—

″
″
B
i
0.97
0.03
532
475
0.95
0.85

ttGGBP17C.24
A260R
A
n
—
—
525
474
0.26
—

″
″
B
i
0.96
0.03
523
483
0.90
0.75

ttGGBP17C.25
A260K
A
n
—
—
526
473
0.24
—

″
″
B
i
0.96
0.03
519
482
0.90
0.76

ttGGBP17C.26
A260W
A
i
0.99
0.01
530
479
0.42
0.41

″
″
B
r(h)
0.96
0.04
529
478
1.00
0.87

ttGGBP17C.27
A260F
A
n
—
—
523
477
0.38
—

″
″
B
i
0.96
0.04
529
487
1.00
0.82

ttGGBP17C.28
A260Y
A
n
—
—
523
477
0.38
—

″
″
B
i
0.97
0.03
530
480
1.00
0.87

ttGGBP17C.29
A260S
A
n
—
—
526
474
0.32
—

″
″
B
i
0.96
0.03
533
478
0.95
0.83

^aEmission spectra determined on Nanodrop3300. SVD analysis according to equations 11-13. Gaussian fits according to equations 14-15.

^bA, Acrylodan; B, Badan; n, no observed response to glucose; r(h), ratiometric (hypsochromic shift); r(b), ratiometric (bathochromic shift); i, intensity increase only; d, intensity decrease only.

EXAMPLE 10
Crystal Structure Coordinates for an E. coli Glucose-Galactose Binding Protein: ECGGBP183C (Acrylodan Attached to W183C Mutant)

Naming is standard three-letter amino acid code.

Atom positions are provided as Cartesian coordinates, using standard Protein Databank (PDB) format. ATOM records refer to amino acid atoms; HETATM records refer to non-amino acid atoms.

Column 1: record type (ATOM or HETATM); column 2: atom number; column 3 atom name (standard naming scheme for amino acids); column 4: residue name (ATOM records), or component name (HETATM records); column 5: chain identifier (A, B, C, . . . ); column 6: amino acid residue sequence number (ATOM records), or component number (HETATM records); columns 7-9: x,y,z atomic Cartesian positional coordinates; column 10: fractional occupancy (set to 1.0 in this listing); column 11: B-factor (ignored in this listing); column 12: file identifier (ignored in this listing); column 13: line number (same as atom number in this listing).

For heteroatom (HETATM) records, the component name (column 4) is as follows:

CA, calcium

HOH, water

ACR, Acrylodan

K, potassium

EDO, ethylene glycol

ATOM
1
O
ALA
A
2
106.533
−15.459
75.201
1.00
0.00
xxxx
1

ATOM
2
N
ALA
A
2
107.059
−18.172
74.462
1.00
0.00
xxxx
2

ATOM
3
CA
ALA
A
2
105.803
−17.753
75.070
1.00
0.00
xxxx
3

ATOM
4
C
ALA
A
2
105.616
−16.243
74.951
1.00
0.00
xxxx
4

ATOM
5
CB
ALA
A
2
105.744
−18.181
76.527
1.00
0.00
xxxx
5

ATOM
6
N
THR
A
3
104.414
−15.847
74.566
1.00
0.00
xxxx
6

ATOM
7
CA
THR
A
3
104.079
−14.441
74.397
1.00
0.00
xxxx
7

ATOM
8
C
THR
A
3
103.547
−13.847
75.689
1.00
0.00
xxxx
8

ATOM
9
O
THR
A
3
102.489
−14.249
76.156
1.00
0.00
xxxx
9

ATOM
10
CB
THR
A
3
103.023
−14.268
73.301
1.00
0.00
xxxx
10

ATOM
11
OG1
THR
A
3
103.556
−14.752
72.060
1.00
0.00
xxxx
11

ATOM
12
CG2
THR
A
3
102.616
−12.817
73.168
1.00
0.00
xxxx
12

ATOM
13
N
ARG
A
4
104.268
−12.892
76.272
1.00
0.00
xxxx
13

ATOM
14
CA
ARG
A
4
103.781
−12.274
77.501
1.00
0.00
xxxx
14

ATOM
15
C
ARG
A
4
102.892
−11.082
77.169
1.00
0.00
xxxx
15

ATOM
16
O
ARG
A
4
103.286
−10.224
76.378
1.00
0.00
xxxx
16

ATOM
17
CB
ARG
A
4
104.947
−11.827
78.392
1.00
0.00
xxxx
17

ATOM
18
CG
ARG
A
4
105.839
−12.971
78.910
1.00
0.00
xxxx
18

ATOM
19
CD
ARG
A
4
106.999
−12.408
79.742
1.00
0.00
xxxx
19

ATOM
20
NE
ARG
A
4
107.840
−13.464
80.300
1.00
0.00
xxxx
20

ATOM
21
CZ
ARG
A
4
108.891
−13.259
81.091
1.00
0.00
xxxx
21

ATOM
22
NH1
ARG
A
4
109.247
−12.027
81.435
1.00
0.00
xxxx
22

ATOM
23
NH2
ARG
A
4
109.589
−14.293
81.541
1.00
0.00
xxxx
23

ATOM
24
N
ILE
A
5
101.699
−11.040
77.760
1.00
0.00
xxxx
24

ATOM
25
CA
ILE
A
5
100.798
−9.892
77.638
1.00
0.00
xxxx
25

ATOM
26
C
ILE
A
5
100.536
−9.303
79.016
1.00
0.00
xxxx
26

ATOM
27
O
ILE
A
5
100.148
−10.025
79.938
1.00
0.00
xxxx
27

ATOM
28
CB
ILE
A
5
99.484
−10.290
76.931
1.00
0.00
xxxx
28

ATOM
29
CG2
ILE
A
5
98.491
−9.115
76.932
1.00
0.00
xxxx
29

ATOM
30
CG1
ILE
A
5
99.787
−10.790
75.514
1.00
0.00
xxxx
30

ATOM
31
CD1
ILE
A
5
98.552
−11.286
74.772
1.00
0.00
xxxx
31

ATOM
32
N
GLY
A
6
100.775
−8.001
79.158
1.00
0.00
xxxx
32

ATOM
33
CA
GLY
A
6
100.613
−7.324
80.436
1.00
0.00
xxxx
33

ATOM
34
C
GLY
A
6
99.242
−6.689
80.534
1.00
0.00
xxxx
34

ATOM
35
O
GLY
A
6
98.785
−6.074
79.579
1.00
0.00
xxxx
35

ATOM
36
N
VAL
A
7
98.583
−6.847
81.678
1.00
0.00
xxxx
36

ATOM
37
CA
VAL
A
7
97.232
−6.313
81.871
1.00
0.00
xxxx
37

ATOM
38
C
VAL
A
7
97.186
−5.551
83.186
1.00
0.00
xxxx
38

ATOM
39
O
VAL
A
7
97.627
−6.074
84.212
1.00
0.00
xxxx
39

ATOM
40
CB
VAL
A
7
96.160
−7.449
81.885
1.00
0.00
xxxx
40

ATOM
41
CG1
VAL
A
7
94.781
−6.891
82.114
1.00
0.00
xxxx
41

ATOM
42
CG2
VAL
A
7
96.199
−8.257
80.605
1.00
0.00
xxxx
42

ATOM
43
N
THR
A
8
96.645
−4.333
83.183
1.00
0.00
xxxx
43

ATOM
44
CA
THR
A
8
96.381
−3.648
84.448
1.00
0.00
xxxx
44

ATOM
45
C
THR
A
8
94.872
−3.426
84.569
1.00
0.00
xxxx
45

ATOM
46
O
THR
A
8
94.217
−2.977
83.620
1.00
0.00
xxxx
46

ATOM
47
CB
THR
A
8
97.147
−2.294
84.570
1.00
0.00
xxxx
47

ATOM
48
OG1
THR
A
8
96.693
−1.384
83.556
1.00
0.00
xxxx
48

ATOM
49
CG2
THR
A
8
98.676
−2.510
84.446
1.00
0.00
xxxx
49

ATOM
50
N
ILE
A
9
94.335
−3.761
85.739
1.00
0.00
xxxx
50

ATOM
51
CA
ILE
A
9
92.933
−3.546
86.095
1.00
0.00
xxxx
51

ATOM
52
C
ILE
A
9
92.893
−2.377
87.059
1.00
0.00
xxxx
52

ATOM
53
O
ILE
A
9
93.694
−2.340
87.986
1.00
0.00
xxxx
53

ATOM
54
CB
ILE
A
9
92.334
−4.809
86.743
1.00
0.00
xxxx
54

ATOM
55
CG1
ILE
A
9
92.474
−6.004
85.801
1.00
0.00
xxxx
55

ATOM
56
CG2
ILE
A
9
90.886
−4.563
87.149
1.00
0.00
xxxx
56

ATOM
57
CD1
ILE
A
9
91.634
−5.899
84.523
1.00
0.00
xxxx
57

ATOM
58
N
TYR
A
10
91.994
−1.411
86.871
1.00
0.00
xxxx
58

ATOM
59
CA
TYR
A
10
92.119
−0.166
87.630
1.00
0.00
xxxx
59

ATOM
60
C
TYR
A
10
91.894
−0.426
89.119
1.00
0.00
xxxx
60

ATOM
61
O
TYR
A
10
92.540
0.190
89.952
1.00
0.00
xxxx
61

ATOM
62
CB
TYR
A
10
91.159
0.920
87.078
1.00
0.00
xxxx
62

ATOM
63
CG
TYR
A
10
89.811
1.015
87.771
1.00
0.00
xxxx
63

ATOM
64
CD1
TYR
A
10
89.599
1.932
88.799
1.00
0.00
xxxx
64

ATOM
65
CD2
TYR
A
10
88.745
0.201
87.388
1.00
0.00
xxxx
65

ATOM
66
CE1
TYR
A
10
88.382
2.034
89.441
1.00
0.00
xxxx
66

ATOM
67
CE2
TYR
A
10
87.514
0.286
88.034
1.00
0.00
xxxx
67

ATOM
68
CZ
TYR
A
10
87.340
1.210
89.055
1.00
0.00
xxxx
68

ATOM
69
OH
TYR
A
10
86.139
1.297
89.724
1.00
0.00
xxxx
69

ATOM
70
N
LYS
A
11
91.014
−1.375
89.430
1.00
0.00
xxxx
70

ATOM
71
CA
LYS
A
11
90.672
−1.735
90.799
1.00
0.00
xxxx
71

ATOM
72
C
LYS
A
11
90.059
−3.128
90.779
1.00
0.00
xxxx
72

ATOM
73
O
LYS
A
11
88.993
−3.327
90.195
1.00
0.00
xxxx
73

ATOM
74
CB
LYS
A
11
89.705
−0.687
91.376
1.00
0.00
xxxx
74

ATOM
75
CG
LYS
A
11
88.958
−1.080
92.621
1.00
0.00
xxxx
75

ATOM
76
CD
LYS
A
11
88.090
0.094
93.073
1.00
0.00
xxxx
76

ATOM
77
CE
LYS
A
11
87.341
−0.224
94.359
1.00
0.00
xxxx
77

ATOM
78
NZ
LYS
A
11
88.210
−0.910
95.353
1.00
0.00
xxxx
78

ATOM
79
N
TYR
A
12
90.728
−4.107
91.389
1.00
0.00
xxxx
79

ATOM
80
CA
TYR
A
12
90.288
−5.500
91.239
1.00
0.00
xxxx
80

ATOM
81
C
TYR
A
12
88.895
−5.769
91.789
1.00
0.00
xxxx
81

ATOM
82
O
TYR
A
12
88.194
−6.643
91.273
1.00
0.00
xxxx
82

ATOM
83
CB
TYR
A
12
91.252
−6.472
91.934
1.00
0.00
xxxx
83

ATOM
84
CG
TYR
A
12
92.199
−7.239
91.032
1.00
0.00
xxxx
84

ATOM
85
CD1
TYR
A
12
92.817
−6.627
89.945
1.00
0.00
xxxx
85

ATOM
86
CD2
TYR
A
12
92.527
−8.558
91.317
1.00
0.00
xxxx
86

ATOM
87
CE1
TYR
A
12
93.724
−7.315
89.146
1.00
0.00
xxxx
87

ATOM
88
CE2
TYR
A
12
93.421
−9.258
90.532
1.00
0.00
xxxx
88

ATOM
89
CZ
TYR
A
12
94.012
−8.626
89.443
1.00
0.00
xxxx
89

ATOM
90
OH
TYR
A
12
94.917
−9.315
88.675
1.00
0.00
xxxx
90

ATOM
91
N
ASP
A
13
88.491
−5.051
92.831
1.00
0.00
xxxx
91

ATOM
92
CA
ASP
A
13
87.206
−5.393
93.433
1.00
0.00
xxxx
92

ATOM
93
C
ASP
A
13
86.059
−4.494
92.973
1.00
0.00
xxxx
93

ATOM
94
O
ASP
A
13
84.973
−4.560
93.535
1.00
0.00
xxxx
94

ATOM
95
CB
ASP
A
13
87.311
−5.409
94.960
1.00
0.00
xxxx
95

ATOM
96
CG
ASP
A
13
87.822
−4.115
95.535
1.00
0.00
xxxx
96

ATOM
97
OD1
ASP
A
13
88.328
−3.272
94.778
1.00
0.00
xxxx
97

ATOM
98
OD2
ASP
A
13
87.738
−3.962
96.775
1.00
0.00
xxxx
98

ATOM
99
N
ASP
A
14
86.279
−3.693
91.933
1.00
0.00
xxxx
99

ATOM
100
CA
ASP
A
14
85.161
−3.118
91.169
1.00
0.00
xxxx
100

ATOM
101
C
ASP
A
14
84.313
−4.302
90.691
1.00
0.00
xxxx
101

ATOM
102
O
ASP
A
14
84.858
−5.221
90.091
1.00
0.00
xxxx
102

ATOM
103
CB
ASP
A
14
85.698
−2.279
90.001
1.00
0.00
xxxx
103

ATOM
104
CG
ASP
A
14
84.601
−1.711
89.124
1.00
0.00
xxxx
104

ATOM
105
OD1
ASP
A
14
84.259
−0.523
89.295
1.00
0.00
xxxx
105

ATOM
106
OD2
ASP
A
14
84.090
−2.449
88.260
1.00
0.00
xxxx
106

ATOM
107
N
ASN
A
15
83.007
−4.316
90.980
1.00
0.00
xxxx
107

ATOM
108
CA
ASN
A
15
82.192
−5.510
90.688
1.00
0.00
xxxx
108

ATOM
109
C
ASN
A
15
82.248
−5.882
89.215
1.00
0.00
xxxx
109

ATOM
110
O
ASN
A
15
82.458
−7.045
88.857
1.00
0.00
xxxx
110

ATOM
111
CB
ASN
A
15
80.724
−5.312
91.091
1.00
0.00
xxxx
111

ATOM
112
CG
ASN
A
15
80.549
−5.010
92.562
1.00
0.00
xxxx
112

ATOM
113
OD1
ASN
A
15
81.029
−3.996
93.054
1.00
0.00
xxxx
113

ATOM
114
ND2
ASN
A
15
79.794
−5.859
93.257
1.00
0.00
xxxx
114

ATOM
115
N
PHE
A
16
82.069
−4.897
88.348
1.00
0.00
xxxx
115

ATOM
116
CA
PHE
A
16
82.097
−5.199
86.927
1.00
0.00
xxxx
116

ATOM
117
C
PHE
A
16
83.483
−5.634
86.450
1.00
0.00
xxxx
117

ATOM
118
O
PHE
A
16
83.621
−6.611
85.693
1.00
0.00
xxxx
118

ATOM
119
CB
PHE
A
16
81.629
−3.995
86.120
1.00
0.00
xxxx
119

ATOM
120
CG
PHE
A
16
81.657
−4.235
84.646
1.00
0.00
xxxx
120

ATOM
121
CD1
PHE
A
16
80.744
−5.096
84.058
1.00
0.00
xxxx
121

ATOM
122
CD2
PHE
A
16
82.608
−3.621
83.856
1.00
0.00
xxxx
122

ATOM
123
CE1
PHE
A
16
80.778
−5.335
82.689
1.00
0.00
xxxx
123

ATOM
124
CE2
PHE
A
16
82.649
−3.859
82.482
1.00
0.00
xxxx
124

ATOM
125
CZ
PHE
A
16
81.731
−4.720
81.909
1.00
0.00
xxxx
125

ATOM
126
N
MET
A
17
84.521
−4.922
86.876
1.00
0.00
xxxx
126

ATOM
127
CA
MET
A
17
85.854
−5.278
86.404
1.00
0.00
xxxx
127

ATOM
128
C
MET
A
17
86.328
−6.622
86.968
1.00
0.00
xxxx
128

ATOM
129
O
MET
A
17
87.231
−7.255
86.403
1.00
0.00
xxxx
129

ATOM
130
CB
MET
A
17
86.862
−4.169
86.715
1.00
0.00
xxxx
130

ATOM
131
CG
MET
A
17
86.681
−2.912
85.854
1.00
0.00
xxxx
131

ATOM
132
SD
MET
A
17
86.502
−3.231
84.091
1.00
0.00
xxxx
132

ATOM
133
CE
MET
A
17
88.089
−3.976
83.741
1.00
0.00
xxxx
133

ATOM
134
N
SER
A
18
85.738
−7.047
88.082
1.00
0.00
xxxx
134

ATOM
135
CA
SER
A
18
85.999
−8.383
88.605
1.00
0.00
xxxx
135

ATOM
136
C
SER
A
18
85.524
−9.424
87.590
1.00
0.00
xxxx
136

ATOM
137
O
SER
A
18
86.219
−10.411
87.320
1.00
0.00
xxxx
137

ATOM
138
CB
SER
A
18
85.309
−8.577
89.957
1.00
0.00
xxxx
138

ATOM
139
OG
SER
A
18
85.592
−9.854
90.498
1.00
0.00
xxxx
139

ATOM
140
N
VAL
A
19
84.352
−9.186
87.004
1.00
0.00
xxxx
140

ATOM
141
CA
VAL
A
19
83.817
−10.081
85.982
1.00
0.00
xxxx
141

ATOM
142
C
VAL
A
19
84.735
−10.048
84.754
1.00
0.00
xxxx
142

ATOM
143
O
VAL
A
19
85.083
−11.091
84.201
1.00
0.00
xxxx
143

ATOM
144
CB
VAL
A
19
82.362
−9.716
85.597
1.00
0.00
xxxx
144

ATOM
145
CG1
VAL
A
19
81.813
−10.737
84.609
1.00
0.00
xxxx
145

ATOM
146
CG2
VAL
A
19
81.465
−9.656
86.832
1.00
0.00
xxxx
146

ATOM
147
N
VAL
A
20
85.130
−8.851
84.331
1.00
0.00
xxxx
147

ATOM
148
CA
VAL
A
20
85.985
−8.708
83.147
1.00
0.00
xxxx
148

ATOM
149
C
VAL
A
20
87.335
−9.385
83.329
1.00
0.00
xxxx
149

ATOM
150
O
VAL
A
20
87.796
−10.106
82.434
1.00
0.00
xxxx
150

ATOM
151
CB
VAL
A
20
86.174
−7.219
82.781
1.00
0.00
xxxx
151

ATOM
152
CG1
VAL
A
20
87.190
−7.051
81.650
1.00
0.00
xxxx
152

ATOM
153
CG2
VAL
A
20
84.842
−6.607
82.385
1.00
0.00
xxxx
153

ATOM
154
N
ARG
A
21
87.983
−9.163
84.468
1.00
0.00
xxxx
154

ATOM
155
CA
ARG
A
21
89.339
−9.679
84.636
1.00
0.00
xxxx
155

ATOM
156
C
ARG
A
21
89.329
−11.196
84.697
1.00
0.00
xxxx
156

ATOM
157
O
ARG
A
21
90.244
−11.850
84.184
1.00
0.00
xxxx
157

ATOM
158
CB
ARG
A
21
90.007
−9.074
85.875
1.00
0.00
xxxx
158

ATOM
159
CG
ARG
A
21
89.441
−9.508
87.206
1.00
0.00
xxxx
159

ATOM
160
CD
ARG
A
21
90.137
−8.696
88.303
1.00
0.00
xxxx
160

ATOM
161
NE
ARG
A
21
89.427
−8.720
89.582
1.00
0.00
xxxx
161

ATOM
162
CZ
ARG
A
21
89.481
−9.725
90.450
1.00
0.00
xxxx
162

ATOM
163
NH1
ARG
A
21
90.195
−10.809
90.165
1.00
0.00
xxxx
163

ATOM
164
NH2
ARG
A
21
88.817
−9.652
91.599
1.00
0.00
xxxx
164

ATOM
165
N
LYS
A
22
88.279
−11.766
85.280
1.00
0.00
xxxx
165

ATOM
166
CA
LYS
A
22
88.183
−13.214
85.344
1.00
0.00
xxxx
166

ATOM
167
C
LYS
A
22
87.954
−13.790
83.955
1.00
0.00
xxxx
167

ATOM
168
O
LYS
A
22
88.479
−14.857
83.616
1.00
0.00
xxxx
168

ATOM
169
CB
LYS
A
22
87.079
−13.628
86.315
1.00
0.00
xxxx
169

ATOM
170
CG
LYS
A
22
87.475
−13.286
87.751
1.00
0.00
xxxx
170

ATOM
171
CD
LYS
A
22
86.396
−13.653
88.756
1.00
0.00
xxxx
171

ATOM
172
CE
LYS
A
22
86.756
−13.110
90.132
1.00
0.00
xxxx
172

ATOM
173
NZ
LYS
A
22
85.780
−13.518
91.180
1.00
0.00
xxxx
173

ATOM
174
N
ALA
A
23
87.209
−13.060
83.132
1.00
0.00
xxxx
174

ATOM
175
CA
ALA
A
23
86.967
−13.508
81.769
1.00
0.00
xxxx
175

ATOM
176
C
ALA
A
23
88.242
−13.417
80.920
1.00
0.00
xxxx
176

ATOM
177
O
ALA
A
23
88.526
−14.326
80.142
1.00
0.00
xxxx
177

ATOM
178
CB
ALA
A
23
85.836
−12.705
81.144
1.00
0.00
xxxx
178

ATOM
179
N
ILE
A
24
89.023
−12.349
81.084
1.00
0.00
xxxx
179

ATOM
180
CA
ILE
A
24
90.285
−12.217
80.348
1.00
0.00
xxxx
180

ATOM
181
C
ILE
A
24
91.210
−13.367
80.728
1.00
0.00
xxxx
181

ATOM
182
O
ILE
A
24
91.874
−13.963
79.873
1.00
0.00
xxxx
182

ATOM
183
CB
ILE
A
24
90.973
−10.856
80.630
1.00
0.00
xxxx
183

ATOM
184
CG1
ILE
A
24
90.144
−9.700
80.088
1.00
0.00
xxxx
184

ATOM
185
CD1
ILE
A
24
90.752
−8.339
80.389
1.00
0.00
xxxx
185

ATOM
186
CG2
ILE
A
24
92.403
−10.829
80.060
1.00
0.00
xxxx
186

ATOM
187
N
GLU
A
25
91.258
−13.684
82.019
1.00
0.00
xxxx
187

ATOM
188
CA
GLU
A
25
92.092
−14.790
82.474
1.00
0.00
xxxx
188

ATOM
189
C
GLU
A
25
91.698
−16.104
81.797
1.00
0.00
xxxx
189

ATOM
190
O
GLU
A
25
92.564
−16.883
81.400
1.00
0.00
xxxx
190

ATOM
191
CB
GLU
A
25
92.012
−14.942
83.990
1.00
0.00
xxxx
191

ATOM
192
CG
GLU
A
25
93.068
−15.883
84.537
1.00
0.00
xxxx
192

ATOM
193
CD
GLU
A
25
92.979
−16.034
86.033
1.00
0.00
xxxx
193

ATOM
194
OE1
GLU
A
25
92.080
−16.763
86.503
1.00
0.00
xxxx
194

ATOM
195
OE2
GLU
A
25
93.794
−15.401
86.738
1.00
0.00
xxxx
195

ATOM
196
N
GLN
A
26
90.399
−16.344
81.657
1.00
0.00
xxxx
196

ATOM
197
CA
GLN
A
26
89.935
−17.567
81.007
1.00
0.00
xxxx
197

ATOM
198
C
GLN
A
26
90.310
−17.606
79.525
1.00
0.00
xxxx
198

ATOM
199
O
GLN
A
26
90.686
−18.654
79.004
1.00
0.00
xxxx
199

ATOM
200
CB
GLN
A
26
88.425
−17.719
81.176
1.00
0.00
xxxx
200

ATOM
201
CG
GLN
A
26
88.009
−17.965
82.613
1.00
0.00
xxxx
201

ATOM
202
CD
GLN
A
26
88.675
−19.193
83.206
1.00
0.00
xxxx
202

ATOM
203
OE1
GLN
A
26
89.258
−19.136
84.288
1.00
0.00
xxxx
203

ATOM
204
NE2
GLN
A
26
88.589
−20.314
82.497
1.00
0.00
xxxx
204

ATOM
205
N
ASP
A
27
90.225
−16.464
78.850
1.00
0.00
xxxx
205

ATOM
206
CA
ASP
A
27
90.642
−16.386
77.454
1.00
0.00
xxxx
206

ATOM
207
C
ASP
A
27
92.129
−16.674
77.293
1.00
0.00
xxxx
207

ATOM
208
O
ASP
A
27
92.544
−17.394
76.367
1.00
0.00
xxxx
208

ATOM
209
CB
ASP
A
27
90.318
−15.009
76.874
1.00
0.00
xxxx
209

ATOM
210
CG
ASP
A
27
88.819
−14.753
76.777
1.00
0.00
xxxx
210

ATOM
211
OD1
ASP
A
27
88.073
−15.718
76.515
1.00
0.00
xxxx
211

ATOM
212
OD2
ASP
A
27
88.386
−13.591
76.968
1.00
0.00
xxxx
212

ATOM
213
N
ALA
A
28
92.941
−16.146
78.202
1.00
0.00
xxxx
213

ATOM
214
CA
ALA
A
28
94.386
−16.360
78.126
1.00
0.00
xxxx
214

ATOM
215
C
ALA
A
28
94.736
−17.813
78.417
1.00
0.00
xxxx
215

ATOM
216
O
ALA
A
28
95.608
−18.381
77.762
1.00
0.00
xxxx
216

ATOM
217
CB
ALA
A
28
95.123
−15.427
79.088
1.00
0.00
xxxx
217

ATOM
218
N
LYS
A
29
94.047
−18.412
79.388
1.00
0.00
xxxx
218

ATOM
219
CA
LYS
A
29
94.249
−19.820
79.742
1.00
0.00
xxxx
219

ATOM
220
C
LYS
A
29
94.004
−20.738
78.556
1.00
0.00
xxxx
220

ATOM
221
O
LYS
A
29
94.648
−21.784
78.416
1.00
0.00
xxxx
221

ATOM
222
CB
LYS
A
29
93.326
−20.223
80.895
1.00
0.00
xxxx
222

ATOM
223
CG
LYS
A
29
93.781
−19.726
82.256
1.00
0.00
xxxx
223

ATOM
224
CD
LYS
A
29
92.810
−20.153
83.347
1.00
0.00
xxxx
224

ATOM
225
CE
LYS
A
29
93.250
−19.641
84.712
1.00
0.00
xxxx
225

ATOM
226
NZ
LYS
A
29
94.578
−20.190
85.114
1.00
0.00
xxxx
226

ATOM
227
N
ALA
A
30
93.067
−20.343
77.703
1.00
0.00
xxxx
227

ATOM
228
CA
ALA
A
30
92.696
−21.144
76.544
1.00
0.00
xxxx
228

ATOM
229
C
ALA
A
30
93.770
−21.106
75.461
1.00
0.00
xxxx
229

ATOM
230
O
ALA
A
30
93.772
−21.936
74.549
1.00
0.00
xxxx
230

ATOM
231
CB
ALA
A
30
91.364
−20.665
75.982
1.00
0.00
xxxx
231

ATOM
232
N
ALA
A
31
94.675
−20.137
75.562
1.00
0.00
xxxx
232

ATOM
233
CA
ALA
A
31
95.722
−19.923
74.566
1.00
0.00
xxxx
233

ATOM
234
C
ALA
A
31
97.061
−20.427
75.093
1.00
0.00
xxxx
234

ATOM
235
O
ALA
A
31
97.695
−19.760
75.905
1.00
0.00
xxxx
235

ATOM
236
CB
ALA
A
31
95.813
−18.437
74.210
1.00
0.00
xxxx
236

ATOM
237
N
PRO
A
32
97.511
−21.593
74.622
1.00
0.00
xxxx
237

ATOM
238
CA
PRO
A
32
98.679
−22.204
75.270
1.00
0.00
xxxx
238

ATOM
239
C
PRO
A
32
99.986
−21.419
75.097
1.00
0.00
xxxx
239

ATOM
240
O
PRO
A
32
100.924
−21.659
75.859
1.00
0.00
xxxx
240

ATOM
241
CB
PRO
A
32
98.775
−23.576
74.592
1.00
0.00
xxxx
241

ATOM
242
CG
PRO
A
32
98.064
−23.417
73.288
1.00
0.00
xxxx
242

ATOM
243
CD
PRO
A
32
96.963
−22.433
73.545
1.00
0.00
xxxx
243

ATOM
244
N
ASP
A
33
100.043
−20.498
74.139
1.00
0.00
xxxx
244

ATOM
245
CA
ASP
A
33
101.282
−19.774
73.856
1.00
0.00
xxxx
245

ATOM
246
C
ASP
A
33
101.348
−18.412
74.543
1.00
0.00
xxxx
246

ATOM
247
O
ASP
A
33
102.256
−17.629
74.271
1.00
0.00
xxxx
247

ATOM
248
CB
ASP
A
33
101.469
−19.593
72.345
1.00
0.00
xxxx
248

ATOM
249
CG
ASP
A
33
100.328
−18.833
71.692
1.00
0.00
xxxx
249

ATOM
250
OD1
ASP
A
33
99.197
−18.858
72.222
1.00
0.00
xxxx
250

ATOM
251
OD2
ASP
A
33
100.564
−18.214
70.629
1.00
0.00
xxxx
251

ATOM
252
N
VAL
A
34
100.404
−18.145
75.442
1.00
0.00
xxxx
252

ATOM
253
CA
VAL
A
34
100.292
−16.842
76.101
1.00
0.00
xxxx
253

ATOM
254
C
VAL
A
34
100.593
−16.957
77.589
1.00
0.00
xxxx
254

ATOM
255
O
VAL
A
34
100.154
−17.901
78.247
1.00
0.00
xxxx
255

ATOM
256
CB
VAL
A
34
98.890
−16.238
75.893
1.00
0.00
xxxx
256

ATOM
257
CG1
VAL
A
34
98.702
−14.981
76.733
1.00
0.00
xxxx
257

ATOM
258
CG2
VAL
A
34
98.670
−15.936
74.435
1.00
0.00
xxxx
258

ATOM
259
N
GLN
A
35
101.361
−16.004
78.108
1.00
0.00
xxxx
259

ATOM
260
CA
GLN
A
35
101.497
−15.833
79.544
1.00
0.00
xxxx
260

ATOM
261
C
GLN
A
35
100.909
−14.485
79.915
1.00
0.00
xxxx
261

ATOM
262
O
GLN
A
35
101.363
−13.445
79.433
1.00
0.00
xxxx
262

ATOM
263
CB
GLN
A
35
102.946
−15.910
79.997
1.00
0.00
xxxx
263

ATOM
264
CG
GLN
A
35
103.098
−15.718
81.497
1.00
0.00
xxxx
264

ATOM
265
CD
GLN
A
35
104.544
−15.694
81.942
1.00
0.00
xxxx
265

ATOM
266
OE1
GLN
A
35
105.461
−15.777
81.125
1.00
0.00
xxxx
266

ATOM
267
NE2
GLN
A
35
104.757
−15.577
83.249
1.00
0.00
xxxx
267

ATOM
268
N
LEU
A
36
99.886
−14.513
80.755
1.00
0.00
xxxx
268

ATOM
269
CA
LEU
A
36
99.221
−13.304
81.202
1.00
0.00
xxxx
269

ATOM
270
C
LEU
A
36
99.905
−12.748
82.449
1.00
0.00
xxxx
270

ATOM
271
O
LEU
A
36
100.128
−13.494
83.396
1.00
0.00
xxxx
271

ATOM
272
CB
LEU
A
36
97.760
−13.610
81.499
1.00
0.00
xxxx
272

ATOM
273
CG
LEU
A
36
96.815
−12.427
81.600
1.00
0.00
xxxx
273

ATOM
274
CD1
LEU
A
36
96.635
−11.829
80.218
1.00
0.00
xxxx
274

ATOM
275
CD2
LEU
A
36
95.484
−12.898
82.173
1.00
0.00
xxxx
275

ATOM
276
N
LEU
A
37
100.264
−11.462
82.445
1.00
0.00
xxxx
276

ATOM
277
CA
LEU
A
37
100.825
−10.832
83.641
1.00
0.00
xxxx
277

ATOM
278
C
LEU
A
37
99.845
−9.749
84.060
1.00
0.00
xxxx
278

ATOM
279
O
LEU
A
37
99.835
−8.664
83.473
1.00
0.00
xxxx
279

ATOM
280
CB
LEU
A
37
102.215
−10.236
83.379
1.00
0.00
xxxx
280

ATOM
281
CG
LEU
A
37
103.399
−11.161
83.055
1.00
0.00
xxxx
281

ATOM
282
CD1
LEU
A
37
103.232
−11.921
81.751
1.00
0.00
xxxx
282

ATOM
283
CD2
LEU
A
37
104.663
−10.338
82.976
1.00
0.00
xxxx
283

ATOM
284
N
MET
A
38
99.015
−10.023
85.063
1.00
0.00
xxxx
284

ATOM
285
CA
MET
A
38
97.926
−9.095
85.382
1.00
0.00
xxxx
285

ATOM
286
C
MET
A
38
98.051
−8.491
86.779
1.00
0.00
xxxx
286

ATOM
287
O
MET
A
38
98.323
−9.194
87.751
1.00
0.00
xxxx
287

ATOM
288
CB
MET
A
38
96.567
−9.791
85.224
1.00
0.00
xxxx
288

ATOM
289
CG
MET
A
38
95.373
−8.825
85.176
1.00
0.00
xxxx
289

ATOM
290
SD
MET
A
38
93.881
−9.536
84.433
1.00
0.00
xxxx
290

ATOM
291
CE
MET
A
38
93.470
−10.772
85.662
1.00
0.00
xxxx
291

ATOM
292
N
ASN
A
39
97.827
−7.180
86.849
1.00
0.00
xxxx
292

ATOM
293
CA
ASN
A
39
98.037
−6.372
88.051
1.00
0.00
xxxx
293

ATOM
294
C
ASN
A
39
96.781
−5.654
88.498
1.00
0.00
xxxx
294

ATOM
295
O
ASN
A
39
95.965
−5.230
87.672
1.00
0.00
xxxx
295

ATOM
296
CB
ASN
A
39
99.110
−5.310
87.811
1.00
0.00
xxxx
296

ATOM
297
CG
ASN
A
39
100.506
−5.893
87.698
1.00
0.00
xxxx
297

ATOM
298
OD1
ASN
A
39
101.171
−5.706
86.692
1.00
0.00
xxxx
298

ATOM
299
ND2
ASN
A
39
100.961
−6.585
88.746
1.00
0.00
xxxx
299

ATOM
300
N
ASP
A
40
96.664
−5.511
89.812
1.00
0.00
xxxx
300

ATOM
301
CA
ASP
A
40
95.672
−4.650
90.460
1.00
0.00
xxxx
301

ATOM
302
C
ASP
A
40
96.287
−3.286
90.729
1.00
0.00
xxxx
302

ATOM
303
O
ASP
A
40
97.214
−3.168
91.537
1.00
0.00
xxxx
303

ATOM
304
CB
ASP
A
40
95.198
−5.300
91.771
1.00
0.00
xxxx
304

ATOM
305
CG
ASP
A
40
94.113
−4.502
92.473
1.00
0.00
xxxx
305

ATOM
306
OD1
ASP
A
40
93.553
−3.564
91.862
1.00
0.00
xxxx
306

ATOM
307
OD2
ASP
A
40
93.809
−4.848
93.635
1.00
0.00
xxxx
307

ATOM
308
N
SER
A
41
95.772
−2.247
90.076
1.00
0.00
xxxx
308

ATOM
309
CA
SER
A
41
96.320
−0.910
90.271
1.00
0.00
xxxx
309

ATOM
310
C
SER
A
41
95.785
−0.231
91.531
1.00
0.00
xxxx
310

ATOM
311
O
SER
A
41
96.189
0.875
91.841
1.00
0.00
xxxx
311

ATOM
312
CB
SER
A
41
96.033
−0.043
89.042
1.00
0.00
xxxx
312

ATOM
313
OG
SER
A
41
96.652
−0.624
87.893
1.00
0.00
xxxx
313

ATOM
314
N
GLN
A
42
94.886
−0.899
92.258
1.00
0.00
xxxx
314

ATOM
315
CA
GLN
A
42
94.410
−0.381
93.547
1.00
0.00
xxxx
315

ATOM
316
C
GLN
A
42
93.869
1.053
93.455
1.00
0.00
xxxx
316

ATOM
317
O
GLN
A
42
94.044
1.857
94.371
1.00
0.00
xxxx
317

ATOM
318
CB
GLN
A
42
95.537
−0.455
94.588
1.00
0.00
xxxx
318

ATOM
319
CG
GLN
A
42
96.023
−1.898
94.793
1.00
0.00
xxxx
319

ATOM
320
CD
GLN
A
42
96.973
−2.066
95.967
1.00
0.00
xxxx
320

ATOM
321
OE1
GLN
A
42
97.611
−1.116
96.411
1.00
0.00
xxxx
321

ATOM
322
NE2
GLN
A
42
97.071
−3.296
96.474
1.00
0.00
xxxx
322

ATOM
323
N
ASN
A
43
93.207
1.350
92.336
1.00
0.00
xxxx
323

ATOM
324
CA
ASN
A
43
92.564
2.646
92.099
1.00
0.00
xxxx
324

ATOM
325
C
ASN
A
43
93.531
3.835
92.253
1.00
0.00
xxxx
325

ATOM
326
O
ASN
A
43
93.151
4.906
92.726
1.00
0.00
xxxx
326

ATOM
327
CB
ASN
A
43
91.361
2.817
93.037
1.00
0.00
xxxx
327

ATOM
328
CG
ASN
A
43
90.360
3.824
92.517
1.00
0.00
xxxx
328

ATOM
329
OD1
ASN
A
43
90.301
4.088
91.319
1.00
0.00
xxxx
329

ATOM
330
ND2
ASN
A
43
89.572
4.405
93.423
1.00
0.00
xxxx
330

ATOM
331
N
ASP
A
44
94.777
3.646
91.829
1.00
0.00
xxxx
331

ATOM
332
CA
ASP
A
44
95.807
4.677
91.943
1.00
0.00
xxxx
332

ATOM
333
C
ASP
A
44
96.592
4.708
90.630
1.00
0.00
xxxx
333

ATOM
334
O
ASP
A
44
97.292
3.749
90.300
1.00
0.00
xxxx
334

ATOM
335
CB
ASP
A
44
96.709
4.362
93.149
1.00
0.00
xxxx
335

ATOM
336
CG
ASP
A
44
97.743
5.434
93.437
1.00
0.00
xxxx
336

ATOM
337
OD1
ASP
A
44
98.249
6.082
92.510
1.00
0.00
xxxx
337

ATOM
338
OD2
ASP
A
44
98.075
5.609
94.631
1.00
0.00
xxxx
338

ATOM
339
N
GLN
A
45
96.493
5.798
89.872
1.00
0.00
xxxx
339

ATOM
340
CA
GLN
A
45
97.187
5.839
88.587
1.00
0.00
xxxx
340

ATOM
341
C
GLN
A
45
98.708
5.795
88.731
1.00
0.00
xxxx
341

ATOM
342
O
GLN
A
45
99.393
5.237
87.860
1.00
0.00
xxxx
342

ATOM
343
CB
GLN
A
45
96.780
7.085
87.788
1.00
0.00
xxxx
343

ATOM
344
CG
GLN
A
45
97.318
7.082
86.362
1.00
0.00
xxxx
344

ATOM
345
CD
GLN
A
45
96.752
5.928
85.546
1.00
0.00
xxxx
345

ATOM
346
OE1
GLN
A
45
95.544
5.714
85.521
1.00
0.00
xxxx
346

ATOM
347
NE2
GLN
A
45
97.630
5.174
84.884
1.00
0.00
xxxx
347

ATOM
348
N
SER
A
46
99.237
6.372
89.811
1.00
0.00
xxxx
348

ATOM
349
CA
SER
A
46
100.680
6.340
90.048
1.00
0.00
xxxx
349

ATOM
350
C
SER
A
46
101.150
4.901
90.204
1.00
0.00
xxxx
350

ATOM
351
O
SER
A
46
102.185
4.522
89.662
1.00
0.00
xxxx
351

ATOM
352
CB
SER
A
46
101.059
7.151
91.287
1.00
0.00
xxxx
352

ATOM
353
OG
SER
A
46
100.720
8.514
91.110
1.00
0.00
xxxx
353

ATOM
354
N
LYS
A
47
100.378
4.093
90.931
1.00
0.00
xxxx
354

ATOM
355
CA
LYS
A
47
100.684
2.667
91.039
1.00
0.00
xxxx
355

ATOM
356
C
LYS
A
47
100.614
1.975
89.685
1.00
0.00
xxxx
356

ATOM
357
O
LYS
A
47
101.463
1.146
89.362
1.00
0.00
xxxx
357

ATOM
358
CB
LYS
A
47
99.735
1.977
92.029
1.00
0.00
xxxx
358

ATOM
359
CG
LYS
A
47
99.961
2.404
93.475
1.00
0.00
xxxx
359

ATOM
360
CD
LYS
A
47
98.985
1.704
94.410
1.00
0.00
xxxx
360

ATOM
361
CE
LYS
A
47
99.264
2.081
95.854
1.00
0.00
xxxx
361

ATOM
362
NZ
LYS
A
47
98.268
1.501
96.800
1.00
0.00
xxxx
362

ATOM
363
N
GLN
A
48
99.592
2.305
88.898
1.00
0.00
xxxx
363

ATOM
364
CA
GLN
A
48
99.460
1.726
87.578
1.00
0.00
xxxx
364

ATOM
365
C
GLN
A
48
100.663
2.079
86.686
1.00
0.00
xxxx
365

ATOM
366
O
GLN
A
48
101.166
1.233
85.952
1.00
0.00
xxxx
366

ATOM
367
CB
GLN
A
48
98.160
2.189
86.915
1.00
0.00
xxxx
367

ATOM
368
CG
GLN
A
48
97.943
1.513
85.563
1.00
0.00
xxxx
368

ATOM
369
CD
GLN
A
48
96.543
1.713
85.022
1.00
0.00
xxxx
369

ATOM
370
OE1
GLN
A
48
96.302
2.594
84.194
1.00
0.00
xxxx
370

ATOM
371
NE2
GLN
A
48
95.611
0.888
85.477
1.00
0.00
xxxx
371

ATOM
372
N
ASN
A
49
101.128
3.320
86.760
1.00
0.00
xxxx
372

ATOM
373
CA
ASN
A
49
102.247
3.745
85.917
1.00
0.00
xxxx
373

ATOM
374
C
ASN
A
49
103.500
2.936
86.271
1.00
0.00
xxxx
374

ATOM
375
O
ASN
A
49
104.240
2.525
85.382
1.00
0.00
xxxx
375

ATOM
376
CB
ASN
A
49
102.506
5.251
86.061
1.00
0.00
xxxx
376

ATOM
377
CG
ASN
A
49
101.367
6.102
85.492
1.00
0.00
xxxx
377

ATOM
378
OD1
ASN
A
49
100.536
5.616
84.729
1.00
0.00
xxxx
378

ATOM
379
ND2
ASN
A
49
101.333
7.379
85.870
1.00
0.00
xxxx
379

ATOM
380
N
ASP
A
50
103.725
2.698
87.562
1.00
0.00
xxxx
380

ATOM
381
CA
ASP
A
50
104.856
1.874
87.982
1.00
0.00
xxxx
381

ATOM
382
C
ASP
A
50
104.697
0.440
87.487
1.00
0.00
xxxx
382

ATOM
383
O
ASP
A
50
105.672
−0.216
87.089
1.00
0.00
xxxx
383

ATOM
384
CB
ASP
A
50
104.998
1.875
89.502
1.00
0.00
xxxx
384

ATOM
385
CG
ASP
A
50
105.413
3.222
90.053
1.00
0.00
xxxx
385

ATOM
386
OD1
ASP
A
50
105.848
4.091
89.267
1.00
0.00
xxxx
386

ATOM
387
OD2
ASP
A
50
105.311
3.403
91.286
1.00
0.00
xxxx
387

ATOM
388
N
GLN
A
51
103.464
−0.055
87.526
1.00
0.00
xxxx
388

ATOM
389
CA
GLN
A
51
103.189
−1.410
87.060
1.00
0.00
xxxx
389

ATOM
390
C
GLN
A
51
103.516
−1.541
85.581
1.00
0.00
xxxx
390

ATOM
391
O
GLN
A
51
104.057
−2.559
85.142
1.00
0.00
xxxx
391

ATOM
392
CB
GLN
A
51
101.725
−1.778
87.334
1.00
0.00
xxxx
392

ATOM
393
CG
GLN
A
51
101.498
−2.034
88.824
1.00
0.00
xxxx
393

ATOM
394
CD
GLN
A
51
100.046
−2.147
89.231
1.00
0.00
xxxx
394

ATOM
395
OE1
GLN
A
51
99.148
−1.628
88.557
1.00
0.00
xxxx
395

ATOM
396
NE2
GLN
A
51
99.806
−2.809
90.361
1.00
0.00
xxxx
396

ATOM
397
N
ILE
A
52
103.182
−0.515
84.811
1.00
0.00
xxxx
397

ATOM
398
CA
ILE
A
52
103.475
−0.559
83.388
1.00
0.00
xxxx
398

ATOM
399
C
ILE
A
52
104.991
−0.557
83.135
1.00
0.00
xxxx
399

ATOM
400
O
ILE
A
52
105.474
−1.287
82.267
1.00
0.00
xxxx
400

ATOM
401
CB
ILE
A
52
102.741
0.587
82.668
1.00
0.00
xxxx
401

ATOM
402
CG1
ILE
A
52
101.226
0.330
82.728
1.00
0.00
xxxx
402

ATOM
403
CG2
ILE
A
52
103.220
0.723
81.220
1.00
0.00
xxxx
403

ATOM
404
CD1
ILE
A
52
100.376
1.551
82.399
1.00
0.00
xxxx
404

ATOM
405
N
ASP
A
53
105.747
0.215
83.917
1.00
0.00
xxxx
405

ATOM
406
CA
ASP
A
53
107.214
0.162
83.824
1.00
0.00
xxxx
406

ATOM
407
C
ASP
A
53
107.728
−1.266
84.031
1.00
0.00
xxxx
407

ATOM
408
O
ASP
A
53
108.629
−1.727
83.310
1.00
0.00
xxxx
408

ATOM
409
CB
ASP
A
53
107.869
1.079
84.857
1.00
0.00
xxxx
409

ATOM
410
CG
ASP
A
53
107.717
2.547
84.531
1.00
0.00
xxxx
410

ATOM
411
OD1
ASP
A
53
107.558
2.886
83.345
1.00
0.00
xxxx
411

ATOM
412
OD2
ASP
A
53
107.786
3.365
85.474
1.00
0.00
xxxx
412

ATOM
413
N
VAL
A
54
107.188
−1.952
85.036
1.00
0.00
xxxx
413

ATOM
414
CA
VAL
A
54
107.601
−3.323
85.328
1.00
0.00
xxxx
414

ATOM
415
C
VAL
A
54
107.221
−4.290
84.200
1.00
0.00
xxxx
415

ATOM
416
O
VAL
A
54
108.024
−5.137
83.808
1.00
0.00
xxxx
416

ATOM
417
CB
VAL
A
54
107.011
−3.791
86.677
1.00
0.00
xxxx
417

ATOM
418
CG1
VAL
A
54
107.249
−5.283
86.887
1.00
0.00
xxxx
418

ATOM
419
CG2
VAL
A
54
107.617
−2.982
87.823
1.00
0.00
xxxx
419

ATOM
420
N
LEU
A
55
106.011
−4.156
83.664
1.00
0.00
xxxx
420

ATOM
421
CA
LEU
A
55
105.582
−5.031
82.577
1.00
0.00
xxxx
421

ATOM
422
C
LEU
A
55
106.493
−4.847
81.362
1.00
0.00
xxxx
422

ATOM
423
O
LEU
A
55
106.877
−5.820
80.704
1.00
0.00
xxxx
423

ATOM
424
CB
LEU
A
55
104.120
−4.747
82.216
1.00
0.00
xxxx
424

ATOM
425
CG
LEU
A
55
103.107
−5.160
83.289
1.00
0.00
xxxx
425

ATOM
426
CD1
LEU
A
55
101.717
−4.586
83.008
1.00
0.00
xxxx
426

ATOM
427
CD2
LEU
A
55
103.031
−6.669
83.414
1.00
0.00
xxxx
427

ATOM
428
N
LEU
A
56
106.841
−3.599
81.059
1.00
0.00
xxxx
428

ATOM
429
CA
LEU
A
56
107.753
−3.336
79.949
1.00
0.00
xxxx
429

ATOM
430
C
LEU
A
56
109.150
−3.903
80.226
1.00
0.00
xxxx
430

ATOM
431
O
LEU
A
56
109.793
−4.445
79.325
1.00
0.00
xxxx
431

ATOM
432
CB
LEU
A
56
107.814
−1.839
79.657
1.00
0.00
xxxx
432

ATOM
433
CG
LEU
A
56
106.512
−1.310
79.039
1.00
0.00
xxxx
433

ATOM
434
CD1
LEU
A
56
106.505
0.221
78.986
1.00
0.00
xxxx
434

ATOM
435
CD2
LEU
A
56
106.269
−1.906
77.650
1.00
0.00
xxxx
435

ATOM
436
N
ALA
A
57
109.615
−3.812
81.472
1.00
0.00
xxxx
436

ATOM
437
CA
ALA
A
57
110.899
−4.396
81.829
1.00
0.00
xxxx
437

ATOM
438
C
ALA
A
57
110.876
−5.912
81.636
1.00
0.00
xxxx
438

ATOM
439
O
ALA
A
57
111.914
−6.521
81.319
1.00
0.00
xxxx
439

ATOM
440
CB
ALA
A
57
111.272
−4.039
83.279
1.00
0.00
xxxx
440

ATOM
441
N
LYS
A
58
109.699
−6.511
81.821
1.00
0.00
xxxx
441

ATOM
442
CA
LYS
A
58
109.515
−7.953
81.661
1.00
0.00
xxxx
442

ATOM
443
C
LYS
A
58
109.301
−8.363
80.208
1.00
0.00
xxxx
443

ATOM
444
O
LYS
A
58
109.065
−9.536
79.919
1.00
0.00
xxxx
444

ATOM
445
CB
LYS
A
58
108.349
−8.439
82.523
1.00
0.00
xxxx
445

ATOM
446
CG
LYS
A
58
108.644
−8.364
84.017
1.00
0.00
xxxx
446

ATOM
447
CD
LYS
A
58
107.485
−8.852
84.854
1.00
0.00
xxxx
447

ATOM
448
CE
LYS
A
58
107.877
−8.918
86.324
1.00
0.00
xxxx
448

ATOM
449
NZ
LYS
A
58
106.777
−9.443
87.171
1.00
0.00
xxxx
449

ATOM
450
N
GLY
A
59
109.405
−7.399
79.300
1.00
0.00
xxxx
450

ATOM
451
CA
GLY
A
59
109.364
−7.687
77.882
1.00
0.00
xxxx
451

ATOM
452
C
GLY
A
59
108.011
−8.128
77.353
1.00
0.00
xxxx
452

ATOM
453
O
GLY
A
59
107.951
−8.956
76.442
1.00
0.00
xxxx
453

ATOM
454
N
VAL
A
60
106.925
−7.596
77.908
1.00
0.00
xxxx
454

ATOM
455
CA
VAL
A
60
105.607
−7.915
77.365
1.00
0.00
xxxx
455

ATOM
456
C
VAL
A
60
105.555
−7.444
75.916
1.00
0.00
xxxx
456

ATOM
457
O
VAL
A
60
106.153
−6.430
75.552
1.00
0.00
xxxx
457

ATOM
458
CB
VAL
A
60
104.460
−7.298
78.184
1.00
0.00
xxxx
458

ATOM
459
CG1
VAL
A
60
104.425
−7.880
79.587
1.00
0.00
xxxx
459

ATOM
460
CG2
VAL
A
60
104.512
−5.757
78.190
1.00
0.00
xxxx
460

ATOM
461
N
LYS
A
61
104.831
−8.195
75.093
1.00
0.00
xxxx
461

ATOM
462
CA
LYS
A
61
104.708
−7.860
73.679
1.00
0.00
xxxx
462

ATOM
463
C
LYS
A
61
103.444
−7.049
73.388
1.00
0.00
xxxx
463

ATOM
464
O
LYS
A
61
103.270
−6.551
72.280
1.00
0.00
xxxx
464

ATOM
465
CB
LYS
A
61
104.736
−9.135
72.830
1.00
0.00
xxxx
465

ATOM
466
CG
LYS
A
61
106.049
−9.896
72.930
1.00
0.00
xxxx
466

ATOM
467
CD
LYS
A
61
106.112
−11.014
71.902
1.00
0.00
xxxx
467

ATOM
468
CE
LYS
A
61
106.273
−10.456
70.491
1.00
0.00
xxxx
468

ATOM
469
NZ
LYS
A
61
106.157
−11.505
69.426
1.00
0.00
xxxx
469

ATOM
470
N
ALA
A
62
102.561
−6.938
74.374
1.00
0.00
xxxx
470

ATOM
471
CA
ALA
A
62
101.392
−6.064
74.278
1.00
0.00
xxxx
471

ATOM
472
C
ALA
A
62
100.930
−5.706
75.670
1.00
0.00
xxxx
472

ATOM
473
O
ALA
A
62
101.185
−6.447
76.623
1.00
0.00
xxxx
473

ATOM
474
CB
ALA
A
62
100.254
−6.732
73.503
1.00
0.00
xxxx
474

ATOM
475
N
LEU
A
63
100.280
−4.553
75.787
1.00
0.00
xxxx
475

ATOM
476
CA
LEU
A
63
99.649
−4.122
77.034
1.00
0.00
xxxx
476

ATOM
477
C
LEU
A
63
98.146
−3.994
76.833
1.00
0.00
xxxx
477

ATOM
478
O
LEU
A
63
97.712
−3.472
75.820
1.00
0.00
xxxx
478

ATOM
479
CB
LEU
A
63
100.211
−2.774
77.479
1.00
0.00
xxxx
479

ATOM
480
CG
LEU
A
63
101.669
−2.776
77.919
1.00
0.00
xxxx
480

ATOM
481
CD1
LEU
A
63
102.226
−1.365
77.884
1.00
0.00
xxxx
481

ATOM
482
CD2
LEU
A
63
101.797
−3.368
79.321
1.00
0.00
xxxx
482

ATOM
483
N
ALA
A
64
97.360
−4.466
77.798
1.00
0.00
xxxx
483

ATOM
484
CA
ALA
A
64
95.934
−4.163
77.857
1.00
0.00
xxxx
484

ATOM
485
C
ALA
A
64
95.720
−3.374
79.139
1.00
0.00
xxxx
485

ATOM
486
O
ALA
A
64
95.967
−3.887
80.233
1.00
0.00
xxxx
486

ATOM
487
CB
ALA
A
64
95.091
−5.442
77.841
1.00
0.00
xxxx
487

ATOM
488
N
ILE
A
65
95.281
−2.125
79.008
1.00
0.00
xxxx
488

ATOM
489
CA
ILE
A
65
95.305
−1.184
80.125
1.00
0.00
xxxx
489

ATOM
490
C
ILE
A
65
93.900
−0.669
80.430
1.00
0.00
xxxx
490

ATOM
491
O
ILE
A
65
93.240
−0.078
79.579
1.00
0.00
xxxx
491

ATOM
492
CB
ILE
A
65
96.250
0.003
79.819
1.00
0.00
xxxx
492

ATOM
493
CG1
ILE
A
65
97.671
−0.497
79.500
1.00
0.00
xxxx
493

ATOM
494
CG2
ILE
A
65
96.178
1.044
80.964
1.00
0.00
xxxx
494

ATOM
495
CD1
ILE
A
65
98.341
−1.250
80.648
1.00
0.00
xxxx
495

ATOM
496
N
ASN
A
66
93.433
−0.941
81.640
1.00
0.00
xxxx
496

ATOM
497
CA
ASN
A
66
92.196
−0.377
82.162
1.00
0.00
xxxx
497

ATOM
498
C
ASN
A
66
92.595
0.789
83.070
1.00
0.00
xxxx
498

ATOM
499
O
ASN
A
66
92.954
0.574
84.227
1.00
0.00
xxxx
499

ATOM
500
CB
ASN
A
66
91.427
−1.500
82.892
1.00
0.00
xxxx
500

ATOM
501
CG
ASN
A
66
90.188
−1.022
83.635
1.00
0.00
xxxx
501

ATOM
502
OD1
ASN
A
66
89.988
−1.389
84.797
1.00
0.00
xxxx
502

ATOM
503
ND2
ASN
A
66
89.317
−0.271
82.953
1.00
0.00
xxxx
503

ATOM
504
N
LEU
A
67
92.595
2.007
82.519
1.00
0.00
xxxx
504

ATOM
505
CA
LEU
A
67
93.111
3.187
83.213
1.00
0.00
xxxx
505

ATOM
506
C
LEU
A
67
92.402
3.474
84.526
1.00
0.00
xxxx
506

ATOM
507
O
LEU
A
67
91.196
3.295
84.638
1.00
0.00
xxxx
507

ATOM
508
CB
LEU
A
67
92.999
4.416
82.306
1.00
0.00
xxxx
508

ATOM
509
CG
LEU
A
67
93.959
4.418
81.114
1.00
0.00
xxxx
509

ATOM
510
CD1
LEU
A
67
93.473
5.426
80.071
1.00
0.00
xxxx
510

ATOM
511
CD2
LEU
A
67
95.356
4.773
81.575
1.00
0.00
xxxx
511

ATOM
512
N
VAL
A
68
93.164
3.908
85.529
1.00
0.00
xxxx
512

ATOM
513
CA
VAL
A
68
92.548
4.499
86.712
1.00
0.00
xxxx
513

ATOM
514
C
VAL
A
68
92.007
5.883
86.328
1.00
0.00
xxxx
514

ATOM
515
O
VAL
A
68
90.839
6.205
86.572
1.00
0.00
xxxx
515

ATOM
516
CB
VAL
A
68
93.543
4.601
87.879
1.00
0.00
xxxx
516

ATOM
517
CG1
VAL
A
68
92.888
5.299
89.074
1.00
0.00
xxxx
517

ATOM
518
CG2
VAL
A
68
94.045
3.214
88.276
1.00
0.00
xxxx
518

ATOM
519
N
ASP
A
69
92.881
6.688
85.728
1.00
0.00
xxxx
519

ATOM
520
CA
ASP
A
69
92.566
8.048
85.273
1.00
0.00
xxxx
520

ATOM
521
C
ASP
A
69
92.653
8.091
83.749
1.00
0.00
xxxx
521

ATOM
522
O
ASP
A
69
93.722
7.908
83.182
1.00
0.00
xxxx
522

ATOM
523
CB
ASP
A
69
93.534
9.049
85.904
1.00
0.00
xxxx
523

ATOM
524
CG
ASP
A
69
93.300
10.481
85.454
1.00
0.00
xxxx
524

ATOM
525
OD1
ASP
A
69
92.338
10.757
84.700
1.00
0.00
xxxx
525

ATOM
526
OD2
ASP
A
69
94.096
11.344
85.886
1.00
0.00
xxxx
526

ATOM
527
N
PRO
A
70
91.519
8.324
83.081
1.00
0.00
xxxx
527

ATOM
528
CA
PRO
A
70
91.546
8.357
81.613
1.00
0.00
xxxx
528

ATOM
529
C
PRO
A
70
92.534
9.376
81.041
1.00
0.00
xxxx
529

ATOM
530
O
PRO
A
70
93.051
9.184
79.938
1.00
0.00
xxxx
530

ATOM
531
CB
PRO
A
70
90.108
8.731
81.247
1.00
0.00
xxxx
531

ATOM
532
CG
PRO
A
70
89.289
8.251
82.394
1.00
0.00
xxxx
532

ATOM
533
CD
PRO
A
70
90.154
8.424
83.624
1.00
0.00
xxxx
533

ATOM
534
N
ALA
A
71
92.813
10.440
81.784
1.00
0.00
xxxx
534

ATOM
535
CA
ALA
A
71
93.717
11.475
81.292
1.00
0.00
xxxx
535

ATOM
536
C
ALA
A
71
95.163
10.987
81.229
1.00
0.00
xxxx
536

ATOM
537
O
ALA
A
71
96.015
11.647
80.638
1.00
0.00
xxxx
537

ATOM
538
CB
ALA
A
71
93.618
12.724
82.166
1.00
0.00
xxxx
538

ATOM
539
N
ALA
A
72
95.439
9.831
81.834
1.00
0.00
xxxx
539

ATOM
540
CA
ALA
A
72
96.795
9.299
81.874
1.00
0.00
xxxx
540

ATOM
541
C
ALA
A
72
97.136
8.467
80.655
1.00
0.00
xxxx
541

ATOM
542
O
ALA
A
72
98.216
7.874
80.598
1.00
0.00
xxxx
542

ATOM
543
CB
ALA
A
72
96.998
8.462
83.127
1.00
0.00
xxxx
543

ATOM
544
N
ALA
A
73
96.221
8.396
79.693
1.00
0.00
xxxx
544

ATOM
545
CA
ALA
A
73
96.492
7.631
78.472
1.00
0.00
xxxx
545

ATOM
546
C
ALA
A
73
97.802
8.065
77.811
1.00
0.00
xxxx
546

ATOM
547
O
ALA
A
73
98.565
7.229
77.328
1.00
0.00
xxxx
547

ATOM
548
CB
ALA
A
73
95.333
7.765
77.492
1.00
0.00
xxxx
548

ATOM
549
N
GLY
A
74
98.058
9.373
77.788
1.00
0.00
xxxx
549

ATOM
550
CA
GLY
A
74
99.286
9.886
77.203
1.00
0.00
xxxx
550

ATOM
551
C
GLY
A
74
100.541
9.357
77.875
1.00
0.00
xxxx
551

ATOM
552
O
GLY
A
74
101.514
8.988
77.214
1.00
0.00
xxxx
552

ATOM
553
N
THR
A
75
100.518
9.335
79.204
1.00
0.00
xxxx
553

ATOM
554
CA
THR
A
75
101.631
8.825
79.990
1.00
0.00
xxxx
554

ATOM
555
C
THR
A
75
101.904
7.368
79.631
1.00
0.00
xxxx
555

ATOM
556
O
THR
A
75
103.051
6.971
79.433
1.00
0.00
xxxx
556

ATOM
557
CB
THR
A
75
101.340
8.945
81.489
1.00
0.00
xxxx
557

ATOM
558
OG1
THR
A
75
101.223
10.330
81.831
1.00
0.00
xxxx
558

ATOM
559
CG2
THR
A
75
102.458
8.299
82.317
1.00
0.00
xxxx
559

ATOM
560
N
VAL
A
76
100.838
6.576
79.550
1.00
0.00
xxxx
560

ATOM
561
CA
VAL
A
76
100.961
5.165
79.228
1.00
0.00
xxxx
561

ATOM
562
C
VAL
A
76
101.516
4.973
77.818
1.00
0.00
xxxx
562

ATOM
563
O
VAL
A
76
102.418
4.160
77.598
1.00
0.00
xxxx
563

ATOM
564
CB
VAL
A
76
99.600
4.457
79.384
1.00
0.00
xxxx
564

ATOM
565
CG1
VAL
A
76
99.679
3.013
78.879
1.00
0.00
xxxx
565

ATOM
566
CG2
VAL
A
76
99.164
4.498
80.850
1.00
0.00
xxxx
566

ATOM
567
N
ILE
A
77
100.990
5.747
76.874
1.00
0.00
xxxx
567

ATOM
568
CA
ILE
A
77
101.426
5.663
75.477
1.00
0.00
xxxx
568

ATOM
569
C
ILE
A
77
102.916
5.991
75.341
1.00
0.00
xxxx
569

ATOM
570
O
ILE
A
77
103.650
5.327
74.601
1.00
0.00
xxxx
570

ATOM
571
CB
ILE
A
77
100.580
6.595
74.594
1.00
0.00
xxxx
571

ATOM
572
CG1
ILE
A
77
99.207
5.972
74.345
1.00
0.00
xxxx
572

ATOM
573
CG2
ILE
A
77
101.303
6.905
73.287
1.00
0.00
xxxx
573

ATOM
574
CD1
ILE
A
77
98.218
6.927
73.704
1.00
0.00
xxxx
574

ATOM
575
N
GLU
A
78
103.371
7.007
76.065
1.00
0.00
xxxx
575

ATOM
576
C
GLU
A
78
105.680
6.225
76.455
1.00
0.00
xxxx
576

ATOM
577
0
GLU
A
78
106.718
5.967
75.839
1.00
0.00
xxxx
577

ATOM
578
CA
GLU
A
78
104.781
7.389
76.018
1.00
0.00
xxxx
578

ATOM
579
CB
GLU
A
78
105.029
8.620
76.899
1.00
0.00
xxxx
579

ATOM
580
CG
GLU
A
78
106.383
9.281
76.690
1.00
0.00
xxxx
580

ATOM
581
CD
GLU
A
78
107.463
8.732
77.605
1.00
0.00
xxxx
581

ATOM
582
OE1
GLU
A
78
107.122
8.181
78.674
1.00
0.00
xxxx
582

ATOM
583
OE2
GLU
A
78
108.658
8.853
77.255
1.00
0.00
xxxx
583

ATOM
584
N
LYS
A
79
105.275
5.512
77.504
1.00
0.00
xxxx
584

ATOM
585
CA
LYS
A
79
106.071
4.387
77.979
1.00
0.00
xxxx
585

ATOM
586
C
LYS
A
79
106.086
3.283
76.931
1.00
0.00
xxxx
586

ATOM
587
O
LYS
A
79
107.145
2.742
76.596
1.00
0.00
xxxx
587

ATOM
588
CB
LYS
A
79
105.533
3.860
79.310
1.00
0.00
xxxx
588

ATOM
589
CG
LYS
A
79
105.667
4.829
80.472
1.00
0.00
xxxx
589

ATOM
590
CD
LYS
A
79
104.967
4.284
81.707
1.00
0.00
xxxx
590

ATOM
591
CE
LYS
A
79
105.041
5.261
82.886
1.00
0.00
xxxx
591

ATOM
592
NZ
LYS
A
79
106.441
5.513
83.323
1.00
0.00
xxxx
592

ATOM
593
N
ALA
A
80
104.912
2.963
76.396
1.00
0.00
xxxx
593

ATOM
594
CA
ALA
A
80
104.798
1.881
75.427
1.00
0.00
xxxx
594

ATOM
595
C
ALA
A
80
105.539
2.204
74.126
1.00
0.00
xxxx
595

ATOM
596
O
ALA
A
80
106.202
1.333
73.546
1.00
0.00
xxxx
596

ATOM
597
CB
ALA
A
80
103.346
1.591
75.143
1.00
0.00
xxxx
597

ATOM
598
O
ARG
A
81
108.081
3.246
71.373
1.00
0.00
xxxx
598

ATOM
599
N
ARG
A
81
105.423
3.453
73.679
1.00
0.00
xxxx
599

ATOM
600
CA
ARG
A
81
106.016
3.886
72.408
1.00
0.00
xxxx
600

ATOM
601
C
ARG
A
81
107.524
3.653
72.391
1.00
0.00
xxxx
601

ATOM
602
CB
ARG
A
81
105.708
5.365
72.151
1.00
0.00
xxxx
602

ATOM
603
CG
ARG
A
81
106.197
5.872
70.789
1.00
0.00
xxxx
603

ATOM
604
CD
ARG
A
81
106.272
7.394
70.728
1.00
0.00
xxxx
604

ATOM
605
NE
ARG
A
81
105.027
8.050
71.122
1.00
0.00
xxxx
605

ATOM
606
CZ
ARG
A
81
104.003
8.258
70.302
1.00
0.00
xxxx
606

ATOM
607
NH1
ARG
A
81
102.913
8.870
70.752
1.00
0.00
xxxx
607

ATOM
608
NH2
ARG
A
81
104.058
7.843
69.041
1.00
0.00
xxxx
608

ATOM
609
N
GLY
A
82
108.172
3.884
73.530
1.00
0.00
xxxx
609

ATOM
610
CA
GLY
A
82
109.614
3.736
73.627
1.00
0.00
xxxx
610

ATOM
611
C
GLY
A
82
110.090
2.319
73.370
1.00
0.00
xxxx
611

ATOM
612
O
GLY
A
82
111.231
2.085
72.969
1.00
0.00
xxxx
612

ATOM
613
N
GLN
A
83
109.204
1.360
73.605
1.00
0.00
xxxx
613

ATOM
614
CA
GLN
A
83
109.538
−0.043
73.423
1.00
0.00
xxxx
614

ATOM
615
C
GLN
A
83
108.786
−0.651
72.231
1.00
0.00
xxxx
615

ATOM
616
O
GLN
A
83
108.819
−1.862
72.010
1.00
0.00
xxxx
616

ATOM
617
CB
GLN
A
83
109.222
−0.811
74.710
1.00
0.00
xxxx
617

ATOM
618
CG
GLN
A
83
110.081
−2.042
74.937
1.00
0.00
xxxx
618

ATOM
619
CD
GLN
A
83
110.165
−2.422
76.404
1.00
0.00
xxxx
619

ATOM
620
OE1
GLN
A
83
110.622
−1.635
77.235
1.00
0.00
xxxx
620

ATOM
621
NE2
GLN
A
83
109.712
−3.626
76.730
1.00
0.00
xxxx
621

ATOM
622
N
ASN
A
84
108.101
0.202
71.474
1.00
0.00
xxxx
622

ATOM
623
CA
ASN
A
84
107.310
−0.224
70.321
1.00
0.00
xxxx
623

ATOM
624
C
ASN
A
84
106.233
−1.246
70.709
1.00
0.00
xxxx
624

ATOM
625
O
ASN
A
84
105.928
−2.157
69.939
1.00
0.00
xxxx
625

ATOM
626
CB
ASN
A
84
108.221
−0.799
69.228
1.00
0.00
xxxx
626

ATOM
627
CG
ASN
A
84
109.380
0.118
68.896
1.00
0.00
xxxx
627

ATOM
628
OD1
ASN
A
84
109.186
1.296
68.588
1.00
0.00
xxxx
628

ATOM
629
ND2
ASN
A
84
110.600
−0.413
68.978
1.00
0.00
xxxx
629

ATOM
630
N
VAL
A
85
105.647
−1.074
71.891
1.00
0.00
xxxx
630

ATOM
631
CA
VAL
A
85
104.676
−2.033
72.419
1.00
0.00
xxxx
631

ATOM
632
C
VAL
A
85
103.254
−1.525
72.172
1.00
0.00
xxxx
632

ATOM
633
O
VAL
A
85
102.932
−0.393
72.508
1.00
0.00
xxxx
633

ATOM
634
CB
VAL
A
85
104.952
−2.294
73.920
1.00
0.00
xxxx
634

ATOM
635
CG1
VAL
A
85
103.787
−3.044
74.592
1.00
0.00
xxxx
635

ATOM
636
CG2
VAL
A
85
106.258
−3.072
74.068
1.00
0.00
xxxx
636

ATOM
637
N
PRO
A
86
102.388
−2.363
71.577
1.00
0.00
xxxx
637

ATOM
638
CA
PRO
A
86
101.004
−1.920
71.366
1.00
0.00
xxxx
638

ATOM
639
C
PRO
A
86
100.209
−1.854
72.666
1.00
0.00
xxxx
639

ATOM
640
O
PRO
A
86
100.501
−2.592
73.621
1.00
0.00
xxxx
640

ATOM
641
CB
PRO
A
86
100.421
−2.989
70.437
1.00
0.00
xxxx
641

ATOM
642
CG
PRO
A
86
101.291
−4.205
70.660
1.00
0.00
xxxx
642

ATOM
643
CD
PRO
A
86
102.672
−3.668
70.951
1.00
0.00
xxxx
643

ATOM
644
N
VAL
A
87
99.215
−0.973
72.707
1.00
0.00
xxxx
644

ATOM
645
CA
VAL
A
87
98.377
−0.792
73.891
1.00
0.00
xxxx
645

ATOM
646
C
VAL
A
87
96.907
−0.896
73.513
1.00
0.00
xxxx
646

ATOM
647
O
VAL
A
87
96.432
−0.123
72.687
1.00
0.00
xxxx
647

ATOM
648
CB
VAL
A
87
98.626
0.567
74.572
1.00
0.00
xxxx
648

ATOM
649
CG1
VAL
A
87
97.751
0.707
75.828
1.00
0.00
xxxx
649

ATOM
650
CG2
VAL
A
87
100.070
0.724
74.941
1.00
0.00
xxxx
650

ATOM
651
N
VAL
A
88
96.192
−1.820
74.149
1.00
0.00
xxxx
651

ATOM
652
CA
VAL
A
88
94.740
−1.888
74.015
1.00
0.00
xxxx
652

ATOM
653
C
VAL
A
88
94.141
−1.351
75.308
1.00
0.00
xxxx
653

ATOM
654
O
VAL
A
88
94.129
−2.049
76.335
1.00
0.00
xxxx
654

ATOM
655
CB
VAL
A
88
94.255
−3.317
73.725
1.00
0.00
xxxx
655

ATOM
656
CG1
VAL
A
88
92.734
−3.331
73.544
1.00
0.00
xxxx
656

ATOM
657
CG2
VAL
A
88
94.938
−3.884
72.472
1.00
0.00
xxxx
657

ATOM
658
N
PHE
A
89
93.667
−0.108
75.279
1.00
0.00
xxxx
658

ATOM
659
CA
PHE
A
89
92.946
0.445
76.426
1.00
0.00
xxxx
659

ATOM
660
C
PHE
A
89
91.574
−0.204
76.508
1.00
0.00
xxxx
660

ATOM
661
O
PHE
A
89
90.982
−0.520
75.470
1.00
0.00
xxxx
661

ATOM
662
CB
PHE
A
89
92.804
1.958
76.293
1.00
0.00
xxxx
662

ATOM
663
CG
PHE
A
89
94.107
2.702
76.394
1.00
0.00
xxxx
663

ATOM
664
CD1
PHE
A
89
94.708
2.899
77.625
1.00
0.00
xxxx
664

ATOM
665
CD2
PHE
A
89
94.734
3.190
75.259
1.00
0.00
xxxx
665

ATOM
666
CE1
PHE
A
89
95.910
3.578
77.726
1.00
0.00
xxxx
666

ATOM
667
CE2
PHE
A
89
95.936
3.879
75.355
1.00
0.00
xxxx
667

ATOM
668
CZ
PHE
A
89
96.523
4.077
76.594
1.00
0.00
xxxx
668

ATOM
669
N
PHE
A
90
91.048
−0.413
77.712
1.00
0.00
xxxx
669

ATOM
670
CA
PHE
A
90
89.680
−0.944
77.785
1.00
0.00
xxxx
670

ATOM
671
C
PHE
A
90
88.926
−0.412
78.992
1.00
0.00
xxxx
671

ATOM
672
O
PHE
A
90
89.525
−0.062
80.010
1.00
0.00
xxxx
672

ATOM
673
CB
PHE
A
90
89.660
−2.510
77.730
1.00
0.00
xxxx
673

ATOM
674
CG
PHE
A
90
90.339
−3.225
78.894
1.00
0.00
xxxx
674

ATOM
675
CD1
PHE
A
90
89.573
−3.927
79.824
1.00
0.00
xxxx
675

ATOM
676
CD2
PHE
A
90
91.729
−3.261
79.015
1.00
0.00
xxxx
676

ATOM
677
CE1
PHE
A
90
90.184
−4.611
80.886
1.00
0.00
xxxx
677

ATOM
678
CE2
PHE
A
90
92.349
−3.934
80.071
1.00
0.00
xxxx
678

ATOM
679
CZ
PHE
A
90
91.574
−4.614
81.001
1.00
0.00
xxxx
679

ATOM
680
N
ASN
A
91
87.616
−0.279
78.791
1.00
0.00
xxxx
680

ATOM
681
CA
ASN
A
91
86.590
0.085
79.786
1.00
0.00
xxxx
681

ATOM
682
C
ASN
A
91
86.587
1.542
80.258
1.00
0.00
xxxx
682

ATOM
683
O
ASN
A
91
85.510
2.087
80.492
1.00
0.00
xxxx
683

ATOM
684
CB
ASN
A
91
86.660
−0.854
80.998
1.00
0.00
xxxx
684

ATOM
685
CG
ASN
A
91
85.830
−0.351
82.182
1.00
0.00
xxxx
685

ATOM
686
OD1
ASN
A
91
86.370
0.228
83.120
1.00
0.00
xxxx
686

ATOM
687
ND2
ASN
A
91
84.522
−0.592
82.147
1.00
0.00
xxxx
687

ATOM
688
N
LYS
A
92
87.752
2.160
80.423
1.00
0.00
xxxx
688

ATOM
689
CA
LYS
A
92
87.828
3.594
80.690
1.00
0.00
xxxx
689

ATOM
690
C
LYS
A
92
88.391
4.250
79.446
1.00
0.00
xxxx
690

ATOM
691
O
LYS
A
92
89.554
4.061
79.097
1.00
0.00
xxxx
691

ATOM
692
CB
LYS
A
92
88.682
3.903
81.918
1.00
0.00
xxxx
692

ATOM
693
CG
LYS
A
92
88.035
3.444
83.217
1.00
0.00
xxxx
693

ATOM
694
CD
LYS
A
92
88.252
4.447
84.327
1.00
0.00
xxxx
694

ATOM
695
CE
LYS
A
92
88.017
3.795
85.679
1.00
0.00
xxxx
695

ATOM
696
NZ
LYS
A
92
88.338
4.731
86.803
1.00
0.00
xxxx
696

ATOM
697
N
GLU
A
93
87.546
5.014
78.770
1.00
0.00
xxxx
697

ATOM
698
CA
GLU
A
93
87.872
5.464
77.422
1.00
0.00
xxxx
698

ATOM
699
C
GLU
A
93
88.806
6.685
77.393
1.00
0.00
xxxx
699

ATOM
700
O
GLU
A
93
88.495
7.729
77.986
1.00
0.00
xxxx
700

ATOM
701
CB
GLU
A
93
86.585
5.765
76.656
1.00
0.00
xxxx
701

ATOM
702
CG
GLU
A
93
86.840
6.151
75.212
1.00
0.00
xxxx
702

ATOM
703
CD
GLU
A
93
85.572
6.254
74.396
1.00
0.00
xxxx
703

ATOM
704
OE1
GLU
A
93
84.503
5.840
74.887
1.00
0.00
xxxx
704

ATOM
705
OE2
GLU
A
93
85.652
6.749
73.252
1.00
0.00
xxxx
705

ATOM
706
N
PRO
A
94
89.950
6.563
76.701
1.00
0.00
xxxx
706

ATOM
707
CA
PRO
A
94
90.860
7.705
76.521
1.00
0.00
xxxx
707

ATOM
708
C
PRO
A
94
90.311
8.706
75.520
1.00
0.00
xxxx
708

ATOM
709
O
PRO
A
94
89.374
8.385
74.787
1.00
0.00
xxxx
709

ATOM
710
CB
PRO
A
94
92.143
7.073
75.966
1.00
0.00
xxxx
710

ATOM
711
CG
PRO
A
94
91.946
5.593
76.036
1.00
0.00
xxxx
711

ATOM
712
CD
PRO
A
94
90.473
5.342
76.071
1.00
0.00
xxxx
712

ATOM
713
N
SER
A
95
90.901
9.897
75.465
1.00
0.00
xxxx
713

ATOM
714
CA
SER
A
95
90.489
10.879
74.474
1.00
0.00
xxxx
714

ATOM
715
C
SER
A
95
90.814
10.412
73.058
1.00
0.00
xxxx
715

ATOM
716
O
SER
A
95
91.716
9.616
72.837
1.00
0.00
xxxx
716

ATOM
717
CB
SER
A
95
91.157
12.234
74.741
1.00
0.00
xxxx
717

ATOM
718
OG
SER
A
95
92.546
12.172
74.450
1.00
0.00
xxxx
718

ATOM
719
N
ARG
A
96
90.052
10.923
72.100
1.00
0.00
xxxx
719

ATOM
720
CA
ARG
A
96
90.334
10.701
70.685
1.00
0.00
xxxx
720

ATOM
721
C
ARG
A
96
91.758
11.141
70.337
1.00
0.00
xxxx
721

ATOM
722
O
ARG
A
96
92.484
10.443
69.625
1.00
0.00
xxxx
722

ATOM
723
CB
ARG
A
96
89.310
11.451
69.839
1.00
0.00
xxxx
723

ATOM
724
CG
ARG
A
96
89.369
11.185
68.347
1.00
0.00
xxxx
724

ATOM
725
CD
ARG
A
96
88.887
9.781
68.062
1.00
0.00
xxxx
725

ATOM
726
NE
ARG
A
96
89.980
8.914
67.664
1.00
0.00
xxxx
726

ATOM
727
CZ
ARG
A
96
89.860
7.604
67.479
1.00
0.00
xxxx
727

ATOM
728
NH1
ARG
A
96
88.689
7.005
67.678
1.00
0.00
xxxx
728

ATOM
729
NH2
ARG
A
96
90.914
6.900
67.099
1.00
0.00
xxxx
729

ATOM
730
N
LYS
A
97
92.165
12.301
70.845
1.00
0.00
xxxx
730

ATOM
731
CA
LYS
A
97
93.506
12.809
70.578
1.00
0.00
xxxx
731

ATOM
732
C
LYS
A
97
94.572
11.850
71.084
1.00
0.00
xxxx
732

ATOM
733
O
LYS
A
97
95.597
11.648
70.428
1.00
0.00
xxxx
733

ATOM
734
CB
LYS
A
97
93.707
14.180
71.227
1.00
0.00
xxxx
734

ATOM
735
CG
LYS
A
97
92.951
15.304
70.545
1.00
0.00
xxxx
735

ATOM
736
CD
LYS
A
97
93.178
16.635
71.271
1.00
0.00
xxxx
736

ATOM
737
CE
LYS
A
97
92.407
17.759
70.595
1.00
0.00
xxxx
737

ATOM
738
NZ
LYS
A
97
92.712
19.098
71.206
1.00
0.00
xxxx
738

ATOM
739
N
ALA
A
98
94.344
11.277
72.264
1.00
0.00
xxxx
739

ATOM
740
CA
ALA
A
98
95.304
10.315
72.803
1.00
0.00
xxxx
740

ATOM
741
C
ALA
A
98
95.394
9.077
71.917
1.00
0.00
xxxx
741

ATOM
742
O
ALA
A
98
96.488
8.650
71.553
1.00
0.00
xxxx
742

ATOM
743
CB
ALA
A
98
94.942
9.925
74.237
1.00
0.00
xxxx
743

ATOM
744
N
LEU
A
99
94.246
8.518
71.551
1.00
0.00
xxxx
744

ATOM
745
CA
LEU
A
99
94.233
7.346
70.673
1.00
0.00
xxxx
745

ATOM
746
C
LEU
A
99
94.941
7.608
69.346
1.00
0.00
xxxx
746

ATOM
747
O
LEU
A
99
95.665
6.754
68.837
1.00
0.00
xxxx
747

ATOM
748
CB
LEU
A
99
92.801
6.895
70.397
1.00
0.00
xxxx
748

ATOM
749
CG
LEU
A
99
92.063
6.297
71.584
1.00
0.00
xxxx
749

ATOM
750
CD1
LEU
A
99
90.656
5.872
71.158
1.00
0.00
xxxx
750

ATOM
751
CD2
LEU
A
99
92.829
5.105
72.167
1.00
0.00
xxxx
751

ATOM
752
N
ASP
A
100
94.725
8.790
68.779
1.00
0.00
xxxx
752

ATOM
753
CA
ASP
A
100
95.310
9.129
67.489
1.00
0.00
xxxx
753

ATOM
754
C
ASP
A
100
96.808
9.421
67.547
1.00
0.00
xxxx
754

ATOM
755
O
ASP
A
100
97.473
9.473
66.508
1.00
0.00
xxxx
755

ATOM
756
CB
ASP
A
100
94.579
10.332
66.887
1.00
0.00
xxxx
756

ATOM
757
CG
ASP
A
100
93.189
9.971
66.389
1.00
0.00
xxxx
757

ATOM
758
OD1
ASP
A
100
92.927
8.762
66.198
1.00
0.00
xxxx
758

ATOM
759
OD2
ASP
A
100
92.362
10.887
66.199
1.00
0.00
xxxx
759

ATOM
760
N
SER
A
101
97.343
9.599
68.750
1.00
0.00
xxxx
760

ATOM
761
CA
SER
A
101
98.739
9.990
68.902
1.00
0.00
xxxx
761

ATOM
762
C
SER
A
101
99.714
8.818
68.752
1.00
0.00
xxxx
762

ATOM
763
O
SER
A
101
100.925
9.023
68.654
1.00
0.00
xxxx
763

ATOM
764
CB
SER
A
101
98.958
10.666
70.263
1.00
0.00
xxxx
764

ATOM
765
OG
SER
A
101
98.904
9.725
71.329
1.00
0.00
xxxx
765

ATOM
766
N
TYR
A
102
99.187
7.595
68.740
1.00
0.00
xxxx
766

ATOM
767
CA
TYR
A
102
100.022
6.395
68.694
1.00
0.00
xxxx
767

ATOM
768
C
TYR
A
102
99.397
5.398
67.737
1.00
0.00
xxxx
768

ATOM
769
O
TYR
A
102
98.228
5.037
67.906
1.00
0.00
xxxx
769

ATOM
770
CB
TYR
A
102
100.154
5.792
70.096
1.00
0.00
xxxx
770

ATOM
771
CG
TYR
A
102
101.138
4.640
70.265
1.00
0.00
xxxx
771

ATOM
772
CD1
TYR
A
102
100.853
3.593
71.138
1.00
0.00
xxxx
772

ATOM
773
CD2
TYR
A
102
102.352
4.614
69.596
1.00
0.00
xxxx
773

ATOM
774
CE1
TYR
A
102
101.740
2.541
71.330
1.00
0.00
xxxx
774

ATOM
775
CE2
TYR
A
102
103.260
3.563
69.780
1.00
0.00
xxxx
775

ATOM
776
CZ
TYR
A
102
102.941
2.531
70.660
1.00
0.00
xxxx
776

ATOM
777
OH
TYR
A
102
103.832
1.493
70.862
1.00
0.00
xxxx
777

ATOM
778
N
ASP
A
103
100.165
4.951
66.739
1.00
0.00
xxxx
778

ATOM
779
CA
ASP
A
103
99.602
4.085
65.707
1.00
0.00
xxxx
779

ATOM
780
C
ASP
A
103
99.207
2.719
66.273
1.00
0.00
xxxx
780

ATOM
781
O
ASP
A
103
98.417
2.008
65.670
1.00
0.00
xxxx
781

ATOM
782
CB
ASP
A
103
100.573
3.912
64.519
1.00
0.00
xxxx
782

ATOM
783
CG
ASP
A
103
101.943
3.400
64.934
1.00
0.00
xxxx
783

ATOM
784
OD1
ASP
A
103
102.138
3.062
66.120
1.00
0.00
xxxx
784

ATOM
785
OD2
ASP
A
103
102.834
3.328
64.057
1.00
0.00
xxxx
785

ATOM
786
N
LYS
A
104
99.732
2.361
67.440
1.00
0.00
xxxx
786

ATOM
787
CA
LYS
A
104
99.415
1.066
68.020
1.00
0.00
xxxx
787

ATOM
788
C
LYS
A
104
98.553
1.168
69.286
1.00
0.00
xxxx
788

ATOM
789
O
LYS
A
104
98.544
0.235
70.094
1.00
0.00
xxxx
789

ATOM
790
CB
LYS
A
104
100.710
0.295
68.321
1.00
0.00
xxxx
790

ATOM
791
CG
LYS
A
104
101.487
−0.125
67.063
1.00
0.00
xxxx
791

ATOM
792
CD
LYS
A
104
102.803
−0.812
67.409
1.00
0.00
xxxx
792

ATOM
793
CE
LYS
A
104
103.833
0.175
67.931
1.00
0.00
xxxx
793

ATOM
794
NZ
LYS
A
104
104.492
0.950
66.837
1.00
0.00
xxxx
794

ATOM
795
N
ALA
A
105
97.822
2.277
69.446
1.00
0.00
xxxx
795

ATOM
796
CA
ALA
A
105
96.825
2.410
70.515
1.00
0.00
xxxx
796

ATOM
797
C
ALA
A
105
95.419
2.078
70.019
1.00
0.00
xxxx
797

ATOM
798
O
ALA
A
105
95.012
2.542
68.957
1.00
0.00
xxxx
798

ATOM
799
CB
ALA
A
105
96.852
3.822
71.107
1.00
0.00
xxxx
799

ATOM
800
N
TYR
A
106
94.685
1.279
70.797
1.00
0.00
xxxx
800

ATOM
801
CA
TYR
A
106
93.312
0.880
70.483
1.00
0.00
xxxx
801

ATOM
802
C
TYR
A
106
92.440
1.016
71.729
1.00
0.00
xxxx
802

ATOM
803
O
TYR
A
106
92.966
1.198
72.826
1.00
0.00
xxxx
803

ATOM
804
CB
TYR
A
106
93.268
−0.567
69.975
1.00
0.00
xxxx
804

ATOM
805
CG
TYR
A
106
93.943
−0.752
68.640
1.00
0.00
xxxx
805

ATOM
806
CD1
TYR
A
106
93.197
−0.796
67.479
1.00
0.00
xxxx
806

ATOM
807
CD2
TYR
A
106
95.323
−0.852
68.546
1.00
0.00
xxxx
807

ATOM
808
CE1
TYR
A
106
93.807
−0.942
66.245
1.00
0.00
xxxx
808

ATOM
809
CE2
TYR
A
106
95.945
−1.003
67.320
1.00
0.00
xxxx
809

ATOM
810
CZ
TYR
A
106
95.180
−1.045
66.181
1.00
0.00
xxxx
810

ATOM
811
OH
TYR
A
106
95.776
−1.190
64.941
1.00
0.00
xxxx
811

ATOM
812
N
TYR
A
107
91.120
0.911
71.562
1.00
0.00
xxxx
812

ATOM
813
CA
TYR
A
107
90.212
0.931
72.709
1.00
0.00
xxxx
813

ATOM
814
C
TYR
A
107
89.090
−0.082
72.540
1.00
0.00
xxxx
814

ATOM
815
O
TYR
A
107
88.493
−0.173
71.477
1.00
0.00
xxxx
815

ATOM
816
CB
TYR
A
107
89.587
2.325
72.927
1.00
0.00
xxxx
816

ATOM
817
CG
TYR
A
107
88.629
2.324
74.109
1.00
0.00
xxxx
817

ATOM
818
CD1
TYR
A
107
89.110
2.165
75.405
1.00
0.00
xxxx
818

ATOM
819
CD2
TYR
A
107
87.255
2.445
73.935
1.00
0.00
xxxx
819

ATOM
820
CE1
TYR
A
107
88.257
2.135
76.488
1.00
0.00
xxxx
820

ATOM
821
CE2
TYR
A
107
86.387
2.414
75.024
1.00
0.00
xxxx
821

ATOM
822
CZ
TYR
A
107
86.906
2.261
76.297
1.00
0.00
xxxx
822

ATOM
823
OH
TYR
A
107
86.058
2.217
77.383
1.00
0.00
xxxx
823

ATOM
824
N
VAL
A
108
88.797
−0.817
73.612
1.00
0.00
xxxx
824

ATOM
825
CA
VAL
A
108
87.650
−1.721
73.661
1.00
0.00
xxxx
825

ATOM
826
C
VAL
A
108
86.720
−1.294
74.784
1.00
0.00
xxxx
826

ATOM
827
O
VAL
A
108
87.141
−1.184
75.934
1.00
0.00
xxxx
827

ATOM
828
CB
VAL
A
108
88.087
−3.174
73.877
1.00
0.00
xxxx
828

ATOM
829
CG1
VAL
A
108
86.848
−4.086
74.051
1.00
0.00
xxxx
829

ATOM
830
CG2
VAL
A
108
88.972
−3.674
72.718
1.00
0.00
xxxx
830

ATOM
831
N
GLY
A
109
85.460
−1.060
74.457
1.00
0.00
xxxx
831

ATOM
832
CA
GLY
A
109
84.492
−0.672
75.470
1.00
0.00
xxxx
832

ATOM
833
C
GLY
A
109
83.079
−0.729
74.934
1.00
0.00
xxxx
833

ATOM
834
O
GLY
A
109
82.682
−1.717
74.321
1.00
0.00
xxxx
834

ATOM
835
N
THR
A
110
82.328
0.335
75.196
1.00
0.00
xxxx
835

ATOM
836
CA
THR
A
110
80.915
0.410
74.843
1.00
0.00
xxxx
836

ATOM
837
C
THR
A
110
80.556
1.792
74.323
1.00
0.00
xxxx
837

ATOM
838
O
THR
A
110
81.343
2.734
74.442
1.00
0.00
xxxx
838

ATOM
839
CB
THR
A
110
80.007
0.132
76.056
1.00
0.00
xxxx
839

ATOM
840
OG1
THR
A
110
80.096
1.231
76.975
1.00
0.00
xxxx
840

ATOM
841
CG2
THR
A
110
80.418
−1.153
76.784
1.00
0.00
xxxx
841

ATOM
842
N
ASP
A
111
79.354
1.912
73.755
1.00
0.00
xxxx
842

ATOM
843
CA
ASP
A
111
78.733
3.209
73.500
1.00
0.00
xxxx
843

ATOM
844
C
ASP
A
111
77.968
3.609
74.757
1.00
0.00
xxxx
844

ATOM
845
O
ASP
A
111
76.852
3.152
74.977
1.00
0.00
xxxx
845

ATOM
846
CB
ASP
A
111
77.803
3.122
72.295
1.00
0.00
xxxx
846

ATOM
847
CG
ASP
A
111
77.151
4.443
71.945
1.00
0.00
xxxx
847

ATOM
848
OD1
ASP
A
111
77.546
5.492
72.494
1.00
0.00
xxxx
848

ATOM
849
OD2
ASP
A
111
76.245
4.423
71.089
1.00
0.00
xxxx
849

ATOM
850
N
SER
A
112
78.584
4.441
75.593
1.00
0.00
xxxx
850

ATOM
851
CA
SER
A
112
78.050
4.710
76.928
1.00
0.00
xxxx
851

ATOM
852
C
SER
A
112
76.588
5.181
76.924
1.00
0.00
xxxx
852

ATOM
853
O
SER
A
112
75.783
4.735
77.743
1.00
0.00
xxxx
853

ATOM
854
CB
SER
A
112
78.938
5.743
77.632
1.00
0.00
xxxx
854

ATOM
855
OG
SER
A
112
80.282
5.273
77.722
1.00
0.00
xxxx
855

ATOM
856
N
LYS
A
113
76.241
6.053
75.980
1.00
0.00
xxxx
856

ATOM
857
CA
LYS
A
113
74.895
6.620
75.943
1.00
0.00
xxxx
857

ATOM
858
C
LYS
A
113
73.836
5.534
75.701
1.00
0.00
xxxx
858

ATOM
859
O
LYS
A
113
72.714
5.640
76.197
1.00
0.00
xxxx
859

ATOM
860
CB
LYS
A
113
74.799
7.711
74.877
1.00
0.00
xxxx
860

ATOM
861
CG
LYS
A
113
74.780
7.203
73.457
1.00
0.00
xxxx
861

ATOM
862
CD
LYS
A
113
74.286
8.278
72.499
1.00
0.00
xxxx
862

ATOM
863
CE
LYS
A
113
74.040
7.701
71.113
1.00
0.00
xxxx
863

ATOM
864
NZ
LYS
A
113
75.238
6.994
70.575
1.00
0.00
xxxx
864

ATOM
865
N
GLU
A
114
74.207
4.479
74.975
1.00
0.00
xxxx
865

ATOM
866
CA
GLU
A
114
73.270
3.394
74.699
1.00
0.00
xxxx
866

ATOM
867
C
GLU
A
114
72.725
2.781
75.978
1.00
0.00
xxxx
867

ATOM
868
O
GLU
A
114
71.543
2.448
76.065
1.00
0.00
xxxx
868

ATOM
869
CB
GLU
A
114
73.926
2.311
73.843
1.00
0.00
xxxx
869

ATOM
870
CG
GLU
A
114
72.990
1.167
73.486
1.00
0.00
xxxx
870

ATOM
871
CD
GLU
A
114
73.671
0.061
72.706
1.00
0.00
xxxx
871

ATOM
872
OE1
GLU
A
114
74.705
−0.457
73.172
1.00
0.00
xxxx
872

ATOM
873
OE2
GLU
A
114
73.176
−0.290
71.615
1.00
0.00
xxxx
873

ATOM
874
N
SER
A
115
73.574
2.631
76.987
1.00
0.00
xxxx
874

ATOM
875
CA
SER
A
115
73.103
2.025
78.228
1.00
0.00
xxxx
875

ATOM
876
C
SER
A
115
72.096
2.924
78.953
1.00
0.00
xxxx
876

ATOM
877
O
SER
A
115
71.162
2.423
79.559
1.00
0.00
xxxx
877

ATOM
878
CB
SER
A
115
74.274
1.673
79.162
1.00
0.00
xxxx
878

ATOM
879
OG
SER
A
115
75.073
2.791
79.501
1.00
0.00
xxxx
879

ATOM
880
N
GLY
A
116
72.282
4.240
78.880
1.00
0.00
xxxx
880

ATOM
881
CA
GLY
A
116
71.305
5.160
79.446
1.00
0.00
xxxx
881

ATOM
882
C
GLY
A
116
69.965
5.122
78.724
1.00
0.00
xxxx
882

ATOM
883
O
GLY
A
116
68.903
5.120
79.354
1.00
0.00
xxxx
883

ATOM
884
N
ILE
A
117
70.015
5.109
77.396
1.00
0.00
xxxx
884

ATOM
885
CA
ILE
A
117
68.807
5.008
76.584
1.00
0.00
xxxx
885

ATOM
886
C
ILE
A
117
68.071
3.700
76.894
1.00
0.00
xxxx
886

ATOM
887
O
ILE
A
117
66.856
3.699
77.111
1.00
0.00
xxxx
887

ATOM
888
CB
ILE
A
117
69.147
5.119
75.093
1.00
0.00
xxxx
888

ATOM
889
CG2
ILE
A
117
67.908
4.856
74.240
1.00
0.00
xxxx
889

ATOM
890
CG1
ILE
A
117
69.742
6.498
74.792
1.00
0.00
xxxx
890

ATOM
891
CD1
ILE
A
117
70.257
6.656
73.391
1.00
0.00
xxxx
891

ATOM
892
N
ILE
A
118
68.800
2.586
76.914
1.00
0.00
xxxx
892

ATOM
893
CA
ILE
A
118
68.164
1.304
77.208
1.00
0.00
xxxx
893

ATOM
894
C
ILE
A
118
67.565
1.302
78.609
1.00
0.00
xxxx
894

ATOM
895
O
ILE
A
118
66.458
0.799
78.810
1.00
0.00
xxxx
895

ATOM
896
CB
ILE
A
118
69.142
0.147
77.015
1.00
0.00
xxxx
896

ATOM
897
CG1
ILE
A
118
69.483
0.027
75.528
1.00
0.00
xxxx
897

ATOM
898
CG2
ILE
A
118
68.535
−1.155
77.531
1.00
0.00
xxxx
898

ATOM
899
CD1
ILE
A
118
70.562
−1.000
75.227
1.00
0.00
xxxx
899

ATOM
900
N
GLN
A
119
68.272
1.879
79.579
1.00
0.00
xxxx
900

ATOM
901
CA
GLN
A
119
67.736
1.916
80.932
1.00
0.00
xxxx
901

ATOM
902
C
GLN
A
119
66.448
2.738
80.996
1.00
0.00
xxxx
902

ATOM
903
O
GLN
A
119
65.490
2.339
81.649
1.00
0.00
xxxx
903

ATOM
904
CB
GLN
A
119
68.773
2.472
81.915
1.00
0.00
xxxx
904

ATOM
905
CG
GLN
A
119
68.356
2.257
83.375
1.00
0.00
xxxx
905

ATOM
906
CD
GLN
A
119
69.334
2.890
84.345
1.00
0.00
xxxx
906

ATOM
907
OE1
GLN
A
119
69.729
4.042
84.172
1.00
0.00
xxxx
907

ATOM
908
NE2
GLN
A
119
69.722
2.145
85.371
1.00
0.00
xxxx
908

ATOM
909
N
GLY
A
120
66.422
3.866
80.296
1.00
0.00
xxxx
909

ATOM
910
CA
GLY
A
120
65.229
4.697
80.240
1.00
0.00
xxxx
910

ATOM
911
C
GLY
A
120
64.057
3.986
79.584
1.00
0.00
xxxx
911

ATOM
912
O
GLY
A
120
62.917
4.111
80.032
1.00
0.00
xxxx
912

ATOM
913
N
ASP
A
121
64.323
3.268
78.502
1.00
0.00
xxxx
913

ATOM
914
CA
ASP
A
121
63.258
2.583
77.780
1.00
0.00
xxxx
914

ATOM
915
C
ASP
A
121
62.695
1.453
78.634
1.00
0.00
xxxx
915

ATOM
916
O
ASP
A
121
61.505
1.142
78.573
1.00
0.00
xxxx
916

ATOM
917
CB
ASP
A
121
63.779
2.051
76.446
1.00
0.00
xxxx
917

ATOM
918
CG
ASP
A
121
62.776
1.169
75.740
1.00
0.00
xxxx
918

ATOM
919
OD1
ASP
A
121
61.896
1.712
75.040
1.00
0.00
xxxx
919

ATOM
920
OD2
ASP
A
121
62.875
−0.068
75.881
1.00
0.00
xxxx
920

ATOM
921
N
LEU
A
122
63.564
0.857
79.444
1.00
0.00
xxxx
921

ATOM
922
CA
LEU
A
122
63.166
−0.201
80.362
1.00
0.00
xxxx
922

ATOM
923
C
LEU
A
122
62.257
0.340
81.468
1.00
0.00
xxxx
923

ATOM
924
O
LEU
A
122
61.190
−0.218
81.734
1.00
0.00
xxxx
924

ATOM
925
CB
LEU
A
122
64.407
−0.866
80.964
1.00
0.00
xxxx
925

ATOM
926
CG
LEU
A
122
64.182
−2.202
81.667
1.00
0.00
xxxx
926

ATOM
927
CD1
LEU
A
122
63.510
−3.183
80.713
1.00
0.00
xxxx
927

ATOM
928
CD2
LEU
A
122
65.509
−2.750
82.177
1.00
0.00
xxxx
928

ATOM
929
N
ILE
A
123
62.684
1.429
82.108
1.00
0.00
xxxx
929

ATOM
930
CA
ILE
A
123
61.890
2.097
83.132
1.00
0.00
xxxx
930

ATOM
931
C
ILE
A
123
60.524
2.474
82.561
1.00
0.00
xxxx
931

ATOM
932
O
ILE
A
123
59.499
2.260
83.206
1.00
0.00
xxxx
932

ATOM
933
CB
ILE
A
123
62.622
3.325
83.692
1.00
0.00
xxxx
933

ATOM
934
CG1
ILE
A
123
63.881
2.881
84.437
1.00
0.00
xxxx
934

ATOM
935
CG2
ILE
A
123
61.692
4.122
84.624
1.00
0.00
xxxx
935

ATOM
936
CD1
ILE
A
123
64.782
4.037
84.868
1.00
0.00
xxxx
936

ATOM
937
N
ALA
A
124
60.501
3.010
81.346
1.00
0.00
xxxx
937

ATOM
938
CA
ALA
A
124
59.229
3.381
80.715
1.00
0.00
xxxx
938

ATOM
939
C
ALA
A
124
58.288
2.187
80.553
1.00
0.00
xxxx
939

ATOM
940
O
ALA
A
124
57.078
2.294
80.797
1.00
0.00
xxxx
940

ATOM
941
CB
ALA
A
124
59.483
4.037
79.367
1.00
0.00
xxxx
941

ATOM
942
N
LYS
A
125
58.845
1.052
80.141
1.00
0.00
xxxx
942

ATOM
943
CA
LYS
A
125
58.071
−0.168
79.945
1.00
0.00
xxxx
943

ATOM
944
C
LYS
A
125
57.428
−0.619
81.251
1.00
0.00
xxxx
944

ATOM
945
O
LYS
A
125
56.235
−0.954
81.296
1.00
0.00
xxxx
945

ATOM
946
CB
LYS
A
125
58.978
−1.267
79.381
1.00
0.00
xxxx
946

ATOM
947
CG
LYS
A
125
58.334
−2.651
79.292
1.00
0.00
xxxx
947

ATOM
948
CD
LYS
A
125
59.409
−3.705
79.021
1.00
0.00
xxxx
948

ATOM
949
CE
LYS
A
125
58.828
−5.103
79.036
1.00
0.00
xxxx
949

ATOM
950
NZ
LYS
A
125
59.892
−6.128
78.877
1.00
0.00
xxxx
950

ATOM
951
N
HIS
A
126
58.221
−0.622
82.320
1.00
0.00
xxxx
951

ATOM
952
CA
HIS
A
126
57.741
−1.131
83.596
1.00
0.00
xxxx
952

ATOM
953
C
HIS
A
126
56.831
−0.138
84.296
1.00
0.00
xxxx
953

ATOM
954
O
HIS
A
126
55.894
−0.543
84.980
1.00
0.00
xxxx
954

ATOM
955
CB
HIS
A
126
58.922
−1.523
84.478
1.00
0.00
xxxx
955

ATOM
956
CG
HIS
A
126
59.593
−2.778
84.016
1.00
0.00
xxxx
956

ATOM
957
ND1
HIS
A
126
59.266
−4.020
84.514
1.00
0.00
xxxx
957

ATOM
958
CD2
HIS
A
126
60.510
−2.990
83.041
1.00
0.00
xxxx
958

ATOM
959
CE1
HIS
A
126
59.983
−4.943
83.893
1.00
0.00
xxxx
959

ATOM
960
NE2
HIS
A
126
60.744
−4.343
82.995
1.00
0.00
xxxx
960

ATOM
961
N
TRP
A
127
57.089
1.154
84.103
1.00
0.00
xxxx
961

ATOM
962
CA
TRP
A
127
56.206
2.213
84.605
1.00
0.00
xxxx
962

ATOM
963
C
TRP
A
127
54.805
2.083
84.022
1.00
0.00
xxxx
963

ATOM
964
O
TRP
A
127
53.803
2.171
84.746
1.00
0.00
xxxx
964

ATOM
965
CB
TRP
A
127
56.807
3.583
84.262
1.00
0.00
xxxx
965

ATOM
966
CG
TRP
A
127
56.008
4.805
84.674
1.00
0.00
xxxx
966

ATOM
967
CD1
TRP
A
127
55.272
5.607
83.854
1.00
0.00
xxxx
967

ATOM
968
CD2
TRP
A
127
55.927
5.383
85.982
1.00
0.00
xxxx
968

ATOM
969
NE1
TRP
A
127
54.716
6.642
84.574
1.00
0.00
xxxx
969

ATOM
970
CE2
TRP
A
127
55.110
6.527
85.882
1.00
0.00
xxxx
970

ATOM
971
CE3
TRP
A
127
56.469
5.045
87.229
1.00
0.00
xxxx
971

ATOM
972
CZ2
TRP
A
127
54.818
7.335
86.982
1.00
0.00
xxxx
972

ATOM
973
CZ3
TRP
A
127
56.183
5.849
88.320
1.00
0.00
xxxx
973

ATOM
974
CH2
TRP
A
127
55.360
6.980
88.191
1.00
0.00
xxxx
974

ATOM
975
N
ALA
A
128
54.730
1.869
82.713
1.00
0.00
xxxx
975

ATOM
976
CA
ALA
A
128
53.439
1.684
82.051
1.00
0.00
xxxx
976

ATOM
977
C
ALA
A
128
52.691
0.465
82.606
1.00
0.00
xxxx
977

ATOM
978
O
ALA
A
128
51.459
0.480
82.734
1.00
0.00
xxxx
978

ATOM
979
CB
ALA
A
128
53.635
1.546
80.545
1.00
0.00
xxxx
979

ATOM
980
N
ALA
A
129
53.438
−0.580
82.946
1.00
0.00
xxxx
980

ATOM
981
CA
ALA
A
129
52.859
−1.837
83.413
1.00
0.00
xxxx
981

ATOM
982
C
ALA
A
129
52.549
−1.841
84.911
1.00
0.00
xxxx
982

ATOM
983
O
ALA
A
129
51.876
−2.744
85.401
1.00
0.00
xxxx
983

ATOM
984
CB
ALA
A
129
53.793
−2.995
83.084
1.00
0.00
xxxx
984

ATOM
985
N
ASN
A
130
53.053
−0.847
85.636
1.00
0.00
xxxx
985

ATOM
986
CA
ASN
A
130
52.878
−0.801
87.086
1.00
0.00
xxxx
986

ATOM
987
C
ASN
A
130
52.454
0.593
87.527
1.00
0.00
xxxx
987

ATOM
988
O
ASN
A
130
53.226
1.290
88.173
1.00
0.00
xxxx
988

ATOM
989
CB
ASN
A
130
54.179
−1.189
87.808
1.00
0.00
xxxx
989

ATOM
990
CG
ASN
A
130
54.592
−2.629
87.551
1.00
0.00
xxxx
990

ATOM
991
OD1
ASN
A
130
54.189
−3.538
88.283
1.00
0.00
xxxx
991

ATOM
992
ND2
ASN
A
130
55.397
−2.847
86.509
1.00
0.00
xxxx
992

ATOM
993
N
GLN
A
131
51.236
1.005
87.192
1.00
0.00
xxxx
993

ATOM
994
CA
GLN
A
131
50.836
2.377
87.493
1.00
0.00
xxxx
994

ATOM
995
C
GLN
A
131
50.664
2.616
88.994
1.00
0.00
xxxx
995

ATOM
996
O
GLN
A
131
50.591
3.756
89.434
1.00
0.00
xxxx
996

ATOM
997
CB
GLN
A
131
49.558
2.737
86.743
1.00
0.00
xxxx
997

ATOM
998
CG
GLN
A
131
49.811
3.113
85.287
1.00
0.00
xxxx
998

ATOM
999
CD
GLN
A
131
50.666
4.365
85.145
1.00
0.00
xxxx
999

ATOM
1000
OE1
GLN
A
131
50.176
5.486
85.303
1.00
0.00
xxxx
1000

ATOM
1001
NE2
GLN
A
131
51.949
4.180
84.848
1.00
0.00
xxxx
1001

ATOM
1002
N
GLY
A
132
50.627
1.541
89.773
1.00
0.00
xxxx
1002

ATOM
1003
CA
GLY
A
132
50.664
1.661
91.218
1.00
0.00
xxxx
1003

ATOM
1004
C
GLY
A
132
51.957
2.255
91.741
1.00
0.00
xxxx
1004

ATOM
1005
O
GLY
A
132
52.014
2.708
92.886
1.00
0.00
xxxx
1005

ATOM
1006
N
TRP
A
133
52.999
2.266
90.906
1.00
0.00
xxxx
1006

ATOM
1007
CA
TRP
A
133
54.265
2.885
91.281
1.00
0.00
xxxx
1007

ATOM
1008
C
TRP
A
133
54.179
4.410
91.375
1.00
0.00
xxxx
1008

ATOM
1009
O
TRP
A
133
55.021
5.042
92.004
1.00
0.00
xxxx
1009

ATOM
1010
CB
TRP
A
133
55.369
2.526
90.279
1.00
0.00
xxxx
1010

ATOM
1011
CG
TRP
A
133
55.859
1.107
90.312
1.00
0.00
xxxx
1011

ATOM
1012
CD1
TRP
A
133
55.501
0.119
91.186
1.00
0.00
xxxx
1012

ATOM
1013
CD2
TRP
A
133
56.818
0.531
89.423
1.00
0.00
xxxx
1013

ATOM
1014
NE1
TRP
A
133
56.190
−1.045
90.890
1.00
0.00
xxxx
1014

ATOM
1015
CE2
TRP
A
133
56.997
−0.816
89.806
1.00
0.00
xxxx
1015

ATOM
1016
CE3
TRP
A
133
57.544
1.025
88.334
1.00
0.00
xxxx
1016

ATOM
1017
CZ2
TRP
A
133
57.877
−1.674
89.140
1.00
0.00
xxxx
1017

ATOM
1018
CZ3
TRP
A
133
58.422
0.170
87.671
1.00
0.00
xxxx
1018

ATOM
1019
CH2
TRP
A
133
58.574
−1.165
88.079
1.00
0.00
xxxx
1019

ATOM
1020
N
ASP
A
134
53.175
4.985
90.714
1.00
0.00
xxxx
1020

ATOM
1021
CA
ASP
A
134
52.940
6.423
90.756
1.00
0.00
xxxx
1021

ATOM
1022
C
ASP
A
134
52.241
6.760
92.070
1.00
0.00
xxxx
1022

ATOM
1023
O
ASP
A
134
51.022
6.966
92.112
1.00
0.00
xxxx
1023

ATOM
1024
CB
ASP
A
134
52.105
6.842
89.543
1.00
0.00
xxxx
1024

ATOM
1025
CG
ASP
A
134
51.978
8.350
89.382
1.00
0.00
xxxx
1025

ATOM
1026
OD1
ASP
A
134
52.701
9.111
90.044
1.00
0.00
xxxx
1026

ATOM
1027
OD2
ASP
A
134
51.151
8.771
88.554
1.00
0.00
xxxx
1027

ATOM
1028
N
LEU
A
135
53.031
6.800
93.141
1.00
0.00
xxxx
1028

ATOM
1029
CA
LEU
A
135
52.494
6.901
94.496
1.00
0.00
xxxx
1029

ATOM
1030
C
LEU
A
135
51.661
8.154
94.723
1.00
0.00
xxxx
1030

ATOM
1031
O
LEU
A
135
50.639
8.093
95.418
1.00
0.00
xxxx
1031

ATOM
1032
CB
LEU
A
135
53.636
6.852
95.513
1.00
0.00
xxxx
1032

ATOM
1033
CG
LEU
A
135
54.522
5.609
95.486
1.00
0.00
xxxx
1033

ATOM
1034
CD1
LEU
A
135
55.586
5.739
96.573
1.00
0.00
xxxx
1034

ATOM
1035
CD2
LEU
A
135
53.695
4.365
95.696
1.00
0.00
xxxx
1035

ATOM
1036
N
ASN
A
136
52.068
9.286
94.148
1.00
0.00
xxxx
1036

ATOM
1037
CA
ASN
A
136
51.282
10.512
94.345
1.00
0.00
xxxx
1037

ATOM
1038
C
ASN
A
136
50.294
10.778
93.208
1.00
0.00
xxxx
1038

ATOM
1039
O
ASN
A
136
49.642
11.827
93.183
1.00
0.00
xxxx
1039

ATOM
1040
CB
ASN
A
136
52.193
11.729
94.543
1.00
0.00
xxxx
1040

ATOM
1041
CG
ASN
A
136
52.931
12.144
93.274
1.00
0.00
xxxx
1041

ATOM
1042
OD1
ASN
A
136
53.028
11.386
92.307
1.00
0.00
xxxx
1042

ATOM
1043
ND2
ASN
A
136
53.477
13.362
93.289
1.00
0.00
xxxx
1043

ATOM
1044
N
LYS
A
137
50.197
9.830
92.280
1.00
0.00
xxxx
1044

ATOM
1045
CA
LYS
A
137
49.160
9.856
91.239
1.00
0.00
xxxx
1045

ATOM
1046
C
LYS
A
137
49.225
11.109
90.363
1.00
0.00
xxxx
1046

ATOM
1047
O
LYS
A
137
48.189
11.578
89.883
1.00
0.00
xxxx
1047

ATOM
1048
CB
LYS
A
137
47.770
9.736
91.883
1.00
0.00
xxxx
1048

ATOM
1049
CG
LYS
A
137
47.571
8.425
92.630
1.00
0.00
xxxx
1049

ATOM
1050
CD
LYS
A
137
46.208
8.359
93.303
1.00
0.00
xxxx
1050

ATOM
1051
CE
LYS
A
137
45.091
8.308
92.284
1.00
0.00
xxxx
1051

ATOM
1052
NZ
LYS
A
137
45.115
7.052
91.487
1.00
0.00
xxxx
1052

ATOM
1053
N
ASP
A
138
50.428
11.638
90.135
1.00
0.00
xxxx
1053

ATOM
1054
CA
ASP
A
138
50.554
12.869
89.344
1.00
0.00
xxxx
1054

ATOM
1055
C
ASP
A
138
51.009
12.636
87.901
1.00
0.00
xxxx
1055

ATOM
1056
O
ASP
A
138
51.184
13.594
87.148
1.00
0.00
xxxx
1056

ATOM
1057
CB
ASP
A
138
51.499
13.865
90.041
1.00
0.00
xxxx
1057

ATOM
1058
CG
ASP
A
138
52.965
13.447
90.011
1.00
0.00
xxxx
1058

ATOM
1059
OD1
ASP
A
138
53.285
12.308
89.603
1.00
0.00
xxxx
1059

ATOM
1060
OD2
ASP
A
138
53.812
14.281
90.431
1.00
0.00
xxxx
1060

ATOM
1061
N
GLY
A
139
51.196
11.374
87.519
1.00
0.00
xxxx
1061

ATOM
1062
CA
GLY
A
139
51.634
11.030
86.174
1.00
0.00
xxxx
1062

ATOM
1063
C
GLY
A
139
53.104
11.282
85.887
1.00
0.00
xxxx
1063

ATOM
1064
O
GLY
A
139
53.555
11.086
84.757
1.00
0.00
xxxx
1064

ATOM
1065
N
GLN
A
140
53.859
11.686
86.910
1.00
0.00
xxxx
1065

ATOM
1066
CA
GLN
A
140
55.299
11.929
86.779
1.00
0.00
xxxx
1066

ATOM
1067
C
GLN
A
140
56.057
10.927
87.603
1.00
0.00
xxxx
1067

ATOM
1068
O
GLN
A
140
55.531
10.447
88.612
1.00
0.00
xxxx
1068

ATOM
1069
CB
GLN
A
140
55.682
13.327
87.258
1.00
0.00
xxxx
1069

ATOM
1070
CG
GLN
A
140
54.808
14.436
86.722
1.00
0.00
xxxx
1070

ATOM
1071
CD
GLN
A
140
55.319
15.804
87.115
1.00
0.00
xxxx
1071

ATOM
1072
OE1
GLN
A
140
56.517
15.992
87.328
1.00
0.00
xxxx
1072

ATOM
1073
NE2
GLN
A
140
54.412
16.769
87.222
1.00
0.00
xxxx
1073

ATOM
1074
N
ILE
A
141
57.275
10.594
87.178
1.00
0.00
xxxx
1074

ATOM
1075
CA
ILE
A
141
58.146
9.760
88.003
1.00
0.00
xxxx
1075

ATOM
1076
C
ILE
A
141
58.978
10.606
88.964
1.00
0.00
xxxx
1076

ATOM
1077
O
ILE
A
141
59.856
11.347
88.538
1.00
0.00
xxxx
1077

ATOM
1078
CB
ILE
A
141
59.090
8.899
87.162
1.00
0.00
xxxx
1078

ATOM
1079
CG1
ILE
A
141
58.300
7.990
86.223
1.00
0.00
xxxx
1079

ATOM
1080
CD1
ILE
A
141
59.209
7.121
85.330
1.00
0.00
xxxx
1080

ATOM
1081
CG2
ILE
A
141
60.002
8.087
88.090
1.00
0.00
xxxx
1081

ATOM
1082
N
GLN
A
142
58.695
10.497
90.256
1.00
0.00
xxxx
1082

ATOM
1083
CA
GLN
A
142
59.568
11.073
91.274
1.00
0.00
xxxx
1083

ATOM
1084
C
GLN
A
142
60.693
10.084
91.528
1.00
0.00
xxxx
1084

ATOM
1085
O
GLN
A
142
60.453
8.970
92.000
1.00
0.00
xxxx
1085

ATOM
1086
CB
GLN
A
142
58.795
11.361
92.562
1.00
0.00
xxxx
1086

ATOM
1087
CG
GLN
A
142
58.111
12.718
92.607
1.00
0.00
xxxx
1087

ATOM
1088
CD
GLN
A
142
56.877
12.796
91.716
1.00
0.00
xxxx
1088

ATOM
1089
OE1
GLN
A
142
56.161
11.805
91.524
1.00
0.00
xxxx
1089

ATOM
1090
NE2
GLN
A
142
56.611
13.987
91.190
1.00
0.00
xxxx
1090

ATOM
1091
N
PHE
A
143
61.916
10.468
91.183
1.00
0.00
xxxx
1091

ATOM
1092
CA
PHE
A
143
63.031
9.530
91.267
1.00
0.00
xxxx
1092

ATOM
1093
C
PHE
A
143
64.180
10.041
92.119
1.00
0.00
xxxx
1093

ATOM
1094
O
PHE
A
143
64.290
11.237
92.391
1.00
0.00
xxxx
1094

ATOM
1095
CB
PHE
A
143
63.561
9.192
89.856
1.00
0.00
xxxx
1095

ATOM
1096
CG
PHE
A
143
64.318
10.321
89.187
1.00
0.00
xxxx
1096

ATOM
1097
CD1
PHE
A
143
65.710
10.383
89.259
1.00
0.00
xxxx
1097

ATOM
1098
CD2
PHE
A
143
63.647
11.301
88.475
1.00
0.00
xxxx
1098

ATOM
1099
CE1
PHE
A
143
66.415
11.409
88.638
1.00
0.00
xxxx
1099

ATOM
1100
CE2
PHE
A
143
64.342
12.332
87.852
1.00
0.00
xxxx
1100

ATOM
1101
CZ
PHE
A
143
65.730
12.386
87.936
1.00
0.00
xxxx
1101

ATOM
1102
N
VAL
A
144
65.038
9.116
92.534
1.00
0.00
xxxx
1102

ATOM
1103
CA
VAL
A
144
66.359
9.480
93.032
1.00
0.00
xxxx
1103

ATOM
1104
C
VAL
A
144
67.364
8.774
92.143
1.00
0.00
xxxx
1104

ATOM
1105
O
VAL
A
144
67.052
7.737
91.557
1.00
0.00
xxxx
1105

ATOM
1106
CB
VAL
A
144
66.579
9.111
94.511
1.00
0.00
xxxx
1106

ATOM
1107
CG1
VAL
A
144
65.579
9.852
95.400
1.00
0.00
xxxx
1107

ATOM
1108
CG2
VAL
A
144
66.496
7.598
94.726
1.00
0.00
xxxx
1108

ATOM
1109
N
LEU
A
145
68.556
9.348
92.009
1.00
0.00
xxxx
1109

ATOM
1110
CA
LEU
A
145
69.551
8.754
91.118
1.00
0.00
xxxx
1110

ATOM
1111
C
LEU
A
145
70.913
8.649
91.792
1.00
0.00
xxxx
1111

ATOM
1112
O
LEU
A
145
71.429
9.620
92.348
1.00
0.00
xxxx
1112

ATOM
1113
CB
LEU
A
145
69.651
9.557
89.808
1.00
0.00
xxxx
1113

ATOM
1114
CG
LEU
A
145
70.503
8.946
88.676
1.00
0.00
xxxx
1114

ATOM
1115
CD1
LEU
A
145
69.943
9.329
87.311
1.00
0.00
xxxx
1115

ATOM
1116
CD2
LEU
A
145
71.965
9.353
88.771
1.00
0.00
xxxx
1116

ATOM
1117
N
LEU
A
146
71.462
7.438
91.735
1.00
0.00
xxxx
1117

ATOM
1118
CA
LEU
A
146
72.791
7.139
92.259
1.00
0.00
xxxx
1118

ATOM
1119
C
LEU
A
146
73.792
7.143
91.107
1.00
0.00
xxxx
1119

ATOM
1120
O
LEU
A
146
73.759
6.267
90.238
1.00
0.00
xxxx
1120

ATOM
1121
CB
LEU
A
146
72.770
5.788
92.984
1.00
0.00
xxxx
1121

ATOM
1122
CG
LEU
A
146
71.969
5.854
94.285
1.00
0.00
xxxx
1122

ATOM
1123
CD1
LEU
A
146
71.464
4.479
94.688
1.00
0.00
xxxx
1123

ATOM
1124
CD2
LEU
A
146
72.845
6.450
95.380
1.00
0.00
xxxx
1124

ATOM
1125
N
LYS
A
147
74.662
8.147
91.095
1.00
0.00
xxxx
1125

ATOM
1126
CA
LYS
A
147
75.583
8.361
89.986
1.00
0.00
xxxx
1126

ATOM
1127
C
LYS
A
147
76.939
7.700
90.244
1.00
0.00
xxxx
1127

ATOM
1128
O
LYS
A
147
77.365
7.565
91.391
1.00
0.00
xxxx
1128

ATOM
1129
CB
LYS
A
147
75.739
9.871
89.754
1.00
0.00
xxxx
1129

ATOM
1130
CG
LYS
A
147
76.634
10.249
88.595
1.00
0.00
xxxx
1130

ATOM
1131
CD
LYS
A
147
76.509
11.741
88.309
1.00
0.00
xxxx
1131

ATOM
1132
CE
LYS
A
147
77.457
12.148
87.189
1.00
0.00
xxxx
1132

ATOM
1133
NZ
LYS
A
147
77.409
13.623
86.951
1.00
0.00
xxxx
1133

ATOM
1134
N
GLY
A
148
77.607
7.268
89.174
1.00
0.00
xxxx
1134

ATOM
1135
CA
GLY
A
148
78.973
6.778
89.262
1.00
0.00
xxxx
1135

ATOM
1136
C
GLY
A
148
80.004
7.876
89.465
1.00
0.00
xxxx
1136

ATOM
1137
O
GLY
A
148
79.660
8.999
89.844
1.00
0.00
xxxx
1137

ATOM
1138
N
GLU
A
149
81.269
7.544
89.201
1.00
0.00
xxxx
1138

ATOM
1139
CA
GLU
A
149
82.387
8.479
89.315
1.00
0.00
xxxx
1139

ATOM
1140
C
GLU
A
149
82.189
9.673
88.390
1.00
0.00
xxxx
1140

ATOM
1141
O
GLU
A
149
82.058
9.502
87.185
1.00
0.00
xxxx
1141

ATOM
1142
CB
GLU
A
149
83.705
7.763
88.982
1.00
0.00
xxxx
1142

ATOM
1143
CG
GLU
A
149
84.892
8.693
88.733
1.00
0.00
xxxx
1143

ATOM
1144
CD
GLU
A
149
86.163
7.955
88.312
1.00
0.00
xxxx
1144

ATOM
1145
OE1
GLU
A
149
86.160
7.257
87.269
1.00
0.00
xxxx
1145

ATOM
1146
OE2
GLU
A
149
87.176
8.078
89.032
1.00
0.00
xxxx
1146

ATOM
1147
N
PRO
A
150
82.139
10.889
88.954
1.00
0.00
xxxx
1147

ATOM
1148
CA
PRO
A
150
81.976
12.064
88.090
1.00
0.00
xxxx
1148

ATOM
1149
C
PRO
A
150
83.076
12.150
87.034
1.00
0.00
xxxx
1149

ATOM
1150
O
PRO
A
150
84.254
11.966
87.346
1.00
0.00
xxxx
1150

ATOM
1151
CB
PRO
A
150
82.054
13.233
89.076
1.00
0.00
xxxx
1151

ATOM
1152
CG
PRO
A
150
81.585
12.636
90.382
1.00
0.00
xxxx
1152

ATOM
1153
CD
PRO
A
150
82.150
11.246
90.383
1.00
0.00
xxxx
1153

ATOM
1154
N
GLY
A
151
82.686
12.388
85.786
1.00
0.00
xxxx
1154

ATOM
1155
CA
GLY
A
151
83.636
12.431
84.690
1.00
0.00
xxxx
1155

ATOM
1156
C
GLY
A
151
83.867
11.116
83.969
1.00
0.00
xxxx
1156

ATOM
1157
O
GLY
A
151
84.386
11.100
82.852
1.00
0.00
xxxx
1157

ATOM
1158
N
HIS
A
152
83.505
10.008
84.610
1.00
0.00
xxxx
1158

ATOM
1159
CA
HIS
A
152
83.556
8.702
83.962
1.00
0.00
xxxx
1159

ATOM
1160
C
HIS
A
152
82.542
8.753
82.832
1.00
0.00
xxxx
1160

ATOM
1161
O
HIS
A
152
81.397
9.117
83.066
1.00
0.00
xxxx
1161

ATOM
1162
CB
HIS
A
152
83.232
7.586
84.968
1.00
0.00
xxxx
1162

ATOM
1163
CG
HIS
A
152
83.493
6.196
84.467
1.00
0.00
xxxx
1163

ATOM
1164
ND1
HIS
A
152
82.885
5.674
83.345
1.00
0.00
xxxx
1164

ATOM
1165
CD2
HIS
A
152
84.270
5.205
84.966
1.00
0.00
xxxx
1165

ATOM
1166
CE1
HIS
A
152
83.290
4.429
83.162
1.00
0.00
xxxx
1166

ATOM
1167
NE2
HIS
A
152
84.131
4.120
84.134
1.00
0.00
xxxx
1167

ATOM
1168
N
PRO
A
153
82.963
8.418
81.602
1.00
0.00
xxxx
1168

ATOM
1169
CA
PRO
A
153
82.057
8.494
80.450
1.00
0.00
xxxx
1169

ATOM
1170
C
PRO
A
153
80.746
7.753
80.671
1.00
0.00
xxxx
1170

ATOM
1171
O
PRO
A
153
79.684
8.243
80.282
1.00
0.00
xxxx
1171

ATOM
1172
CB
PRO
A
153
82.867
7.831
79.330
1.00
0.00
xxxx
1172

ATOM
1173
CG
PRO
A
153
84.262
8.052
79.713
1.00
0.00
xxxx
1173

ATOM
1174
CD
PRO
A
153
84.313
7.980
81.209
1.00
0.00
xxxx
1174

ATOM
1175
N
ASP
A
154
80.818
6.585
81.300
1.00
0.00
xxxx
1175

ATOM
1176
CA
ASP
A
154
79.619
5.782
81.532
1.00
0.00
xxxx
1176

ATOM
1177
C
ASP
A
154
78.681
6.459
82.518
1.00
0.00
xxxx
1177

ATOM
1178
O
ASP
A
154
77.477
6.478
82.308
1.00
0.00
xxxx
1178

ATOM
1179
CB
ASP
A
154
79.969
4.382
82.041
1.00
0.00
xxxx
1179

ATOM
1180
CG
ASP
A
154
80.549
3.488
80.956
1.00
0.00
xxxx
1180

ATOM
1181
OD1
ASP
A
154
80.282
3.727
79.756
1.00
0.00
xxxx
1181

ATOM
1182
OD2
ASP
A
154
81.262
2.524
81.298
1.00
0.00
xxxx
1182

ATOM
1183
N
ALA
A
155
79.225
6.992
83.609
1.00
0.00
xxxx
1183

ATOM
1184
CA
ALA
A
155
78.391
7.677
84.597
1.00
0.00
xxxx
1184

ATOM
1185
C
ALA
A
155
77.702
8.915
84.010
1.00
0.00
xxxx
1185

ATOM
1186
O
ALA
A
155
76.509
9.131
84.215
1.00
0.00
xxxx
1186

ATOM
1187
CB
ALA
A
155
79.220
8.067
85.819
1.00
0.00
xxxx
1187

ATOM
1188
N
GLU
A
156
78.455
9.731
83.276
1.00
0.00
xxxx
1188

ATOM
1189
CA
GLU
A
156
77.885
10.946
82.720
1.00
0.00
xxxx
1189

ATOM
1190
C
GLU
A
156
76.799
10.637
81.693
1.00
0.00
xxxx
1190

ATOM
1191
O
GLU
A
156
75.725
11.221
81.738
1.00
0.00
xxxx
1191

ATOM
1192
CB
GLU
A
156
78.973
11.814
82.091
1.00
0.00
xxxx
1192

ATOM
1193
CG
GLU
A
156
80.011
12.322
83.083
1.00
0.00
xxxx
1193

ATOM
1194
CD
GLU
A
156
79.422
13.168
84.199
1.00
0.00
xxxx
1194

ATOM
1195
OE1
GLU
A
156
78.347
13.780
84.009
1.00
0.00
xxxx
1195

ATOM
1196
OE2
GLU
A
156
80.049
13.224
85.276
1.00
0.00
xxxx
1196

ATOM
1197
N
ALA
A
157
77.090
9.729
80.765
1.00
0.00
xxxx
1197

ATOM
1198
CA
ALA
A
157
76.121
9.347
79.745
1.00
0.00
xxxx
1198

ATOM
1199
C
ALA
A
157
74.890
8.658
80.332
1.00
0.00
xxxx
1199

ATOM
1200
O
ALA
A
157
73.761
8.943
79.922
1.00
0.00
xxxx
1200

ATOM
1201
CB
ALA
A
157
76.775
8.439
78.713
1.00
0.00
xxxx
1201

ATOM
1202
N
ARG
A
158
75.085
7.754
81.286
1.00
0.00
xxxx
1202

ATOM
1203
CA
ARG
A
158
73.946
7.028
81.836
1.00
0.00
xxxx
1203

ATOM
1204
C
ARG
A
158
73.015
7.947
82.600
1.00
0.00
xxxx
1204

ATOM
1205
O
ARG
A
158
71.795
7.761
82.596
1.00
0.00
xxxx
1205

ATOM
1206
CB
ARG
A
158
74.413
5.885
82.742
1.00
0.00
xxxx
1206

ATOM
1207
CG
ARG
A
158
74.963
4.718
81.960
1.00
0.00
xxxx
1207

ATOM
1208
CD
ARG
A
158
75.686
3.742
82.866
1.00
0.00
xxxx
1208

ATOM
1209
NE
ARG
A
158
76.454
2.793
82.073
1.00
0.00
xxxx
1209

ATOM
1210
CZ
ARG
A
158
77.307
1.917
82.597
1.00
0.00
xxxx
1210

ATOM
1211
NH1
ARG
A
158
77.475
1.869
83.918
1.00
0.00
xxxx
1211

ATOM
1212
NH2
ARG
A
158
78.000
1.104
81.808
1.00
0.00
xxxx
1212

ATOM
1213
N
THR
A
159
73.595
8.932
83.272
1.00
0.00
xxxx
1213

ATOM
1214
CA
THR
A
159
72.812
9.858
84.072
1.00
0.00
xxxx
1214

ATOM
1215
C
THR
A
159
72.016
10.787
83.165
1.00
0.00
xxxx
1215

ATOM
1216
O
THR
A
159
70.829
11.022
83.385
1.00
0.00
xxxx
1216

ATOM
1217
CB
THR
A
159
73.723
10.662
85.000
1.00
0.00
xxxx
1217

ATOM
1218
OG1
THR
A
159
74.339
9.767
85.931
1.00
0.00
xxxx
1218

ATOM
1219
CG2
THR
A
159
72.930
11.692
85.772
1.00
0.00
xxxx
1219

ATOM
1220
N
THR
A
160
72.673
11.288
82.128
1.00
0.00
xxxx
1220

ATOM
1221
CA
THR
A
160
72.007
12.189
81.196
1.00
0.00
xxxx
1221

ATOM
1222
C
THR
A
160
70.915
11.488
80.402
1.00
0.00
xxxx
1222

ATOM
1223
O
THR
A
160
69.805
12.009
80.263
1.00
0.00
xxxx
1223

ATOM
1224
CB
THR
A
160
73.011
12.800
80.207
1.00
0.00
xxxx
1224

ATOM
1225
OG1
THR
A
160
73.939
13.636
80.914
1.00
0.00
xxxx
1225

ATOM
1226
CG2
THR
A
160
72.289
13.625
79.137
1.00
0.00
xxxx
1226

ATOM
1227
N
TYR
A
161
71.237
10.319
79.853
1.00
0.00
xxxx
1227

ATOM
1228
CA
TYR
A
161
70.351
9.740
78.847
1.00
0.00
xxxx
1228

ATOM
1229
C
TYR
A
161
69.210
8.901
79.417
1.00
0.00
xxxx
1229

ATOM
1230
O
TYR
A
161
68.202
8.711
78.729
1.00
0.00
xxxx
1230

ATOM
1231
CB
TYR
A
161
71.169
8.925
77.829
1.00
0.00
xxxx
1231

ATOM
1232
CG
TYR
A
161
71.871
9.815
76.830
1.00
0.00
xxxx
1232

ATOM
1233
CD1
TYR
A
161
71.230
10.222
75.669
1.00
0.00
xxxx
1233

ATOM
1234
CD2
TYR
A
161
73.154
10.285
77.067
1.00
0.00
xxxx
1234

ATOM
1235
CE1
TYR
A
161
71.864
11.051
74.756
1.00
0.00
xxxx
1235

ATOM
1236
CE2
TYR
A
161
73.792
11.120
76.162
1.00
0.00
xxxx
1236

ATOM
1237
CZ
TYR
A
161
73.142
11.496
75.012
1.00
0.00
xxxx
1237

ATOM
1238
OH
TYR
A
161
73.775
12.323
74.112
1.00
0.00
xxxx
1238

ATOM
1239
N
VAL
A
162
69.310
8.434
80.659
1.00
0.00
xxxx
1239

ATOM
1240
CA
VAL
A
162
68.155
7.734
81.220
1.00
0.00
xxxx
1240

ATOM
1241
C
VAL
A
162
66.970
8.708
81.360
1.00
0.00
xxxx
1241

ATOM
1242
O
VAL
A
162
65.832
8.370
81.008
1.00
0.00
xxxx
1242

ATOM
1243
CB
VAL
A
162
68.495
7.027
82.564
1.00
0.00
xxxx
1243

ATOM
1244
CG1
VAL
A
162
68.829
8.019
83.681
1.00
0.00
xxxx
1244

ATOM
1245
CG2
VAL
A
162
67.339
6.120
82.992
1.00
0.00
xxxx
1245

ATOM
1246
N
ILE
A
163
67.249
9.926
81.817
1.00
0.00
xxxx
1246

ATOM
1247
CA
ILE
A
163
66.213
10.941
81.981
1.00
0.00
xxxx
1247

ATOM
1248
C
ILE
A
163
65.767
11.478
80.631
1.00
0.00
xxxx
1248

ATOM
1249
O
ILE
A
163
64.561
11.682
80.412
1.00
0.00
xxxx
1249

ATOM
1250
CB
ILE
A
163
66.702
12.073
82.917
1.00
0.00
xxxx
1250

ATOM
1251
CG1
ILE
A
163
67.019
11.507
84.306
1.00
0.00
xxxx
1251

ATOM
1252
CG2
ILE
A
163
65.663
13.205
83.013
1.00
0.00
xxxx
1252

ATOM
1253
CD1
ILE
A
163
65.913
10.626
84.897
1.00
0.00
xxxx
1253

ATOM
1254
N
LYS
A
164
66.717
11.716
79.730
1.00
0.00
xxxx
1254

ATOM
1255
CA
LYS
A
164
66.365
12.228
78.410
1.00
0.00
xxxx
1255

ATOM
1256
C
LYS
A
164
65.417
11.261
77.700
1.00
0.00
xxxx
1256

ATOM
1257
O
LYS
A
164
64.434
11.676
77.079
1.00
0.00
xxxx
1257

ATOM
1258
CB
LYS
A
164
67.619
12.461
77.565
1.00
0.00
xxxx
1258

ATOM
1259
CG
LYS
A
164
67.313
13.048
76.199
1.00
0.00
xxxx
1259

ATOM
1260
CD
LYS
A
164
68.579
13.299
75.394
1.00
0.00
xxxx
1260

ATOM
1261
CE
LYS
A
164
68.249
13.646
73.948
1.00
0.00
xxxx
1261

ATOM
1262
NZ
LYS
A
164
67.187
14.687
73.850
1.00
0.00
xxxx
1262

ATOM
1263
N
GLU
A
165
65.703
9.969
77.807
1.00
0.00
xxxx
1263

ATOM
1264
CA
GLU
A
165
64.888
8.978
77.119
1.00
0.00
xxxx
1264

ATOM
1265
C
GLU
A
165
63.478
8.925
77.699
1.00
0.00
xxxx
1265

ATOM
1266
O
GLU
A
165
62.496
8.895
76.948
1.00
0.00
xxxx
1266

ATOM
1267
CB
GLU
A
165
65.548
7.595
77.174
1.00
0.00
xxxx
1267

ATOM
1268
CG
GLU
A
165
64.772
6.517
76.425
1.00
0.00
xxxx
1268

ATOM
1269
CD
GLU
A
165
64.739
6.725
74.909
1.00
0.00
xxxx
1269

ATOM
1270
OE1
GLU
A
165
65.462
7.595
74.383
1.00
0.00
xxxx
1270

ATOM
1271
OE2
GLU
A
165
63.978
6.001
74.234
1.00
0.00
xxxx
1271

ATOM
1272
N
LEU
A
166
63.367
8.943
79.024
1.00
0.00
xxxx
1272

ATOM
1273
CA
LEU
A
166
62.055
8.996
79.661
1.00
0.00
xxxx
1273

ATOM
1274
C
LEU
A
166
61.286
10.233
79.214
1.00
0.00
xxxx
1274

ATOM
1275
O
LEU
A
166
60.137
10.134
78.784
1.00
0.00
xxxx
1275

ATOM
1276
CB
LEU
A
166
62.196
8.980
81.185
1.00
0.00
xxxx
1276

ATOM
1277
CG
LEU
A
166
62.545
7.614
81.779
1.00
0.00
xxxx
1277

ATOM
1278
CD1
LEU
A
166
63.027
7.747
83.220
1.00
0.00
xxxx
1278

ATOM
1279
CD2
LEU
A
166
61.331
6.689
81.686
1.00
0.00
xxxx
1279

ATOM
1280
N
ASN
A
167
61.932
11.393
79.281
1.00
0.00
xxxx
1280

ATOM
1281
CA
ASN
A
167
61.263
12.636
78.902
1.00
0.00
xxxx
1281

ATOM
1282
C
ASN
A
167
60.872
12.639
77.421
1.00
0.00
xxxx
1282

ATOM
1283
O
ASN
A
167
59.788
13.121
77.071
1.00
0.00
xxxx
1283

ATOM
1284
CB
ASN
A
167
62.147
13.848
79.210
1.00
0.00
xxxx
1284

ATOM
1285
CG
ASN
A
167
62.376
14.059
80.705
1.00
0.00
xxxx
1285

ATOM
1286
OD1
ASN
A
167
61.698
13.473
81.558
1.00
0.00
xxxx
1286

ATOM
1287
ND2
ASN
A
167
63.329
14.915
81.024
1.00
0.00
xxxx
1287

ATOM
1288
N
ASP
A
168
61.730
12.088
76.558
1.00
0.00
xxxx
1288

ATOM
1289
CA
ASP
A
168
61.453
12.062
75.119
1.00
0.00
xxxx
1289

ATOM
1290
C
ASP
A
168
60.230
11.206
74.833
1.00
0.00
xxxx
1290

ATOM
1291
O
ASP
A
168
59.505
11.452
73.863
1.00
0.00
xxxx
1291

ATOM
1292
CB
ASP
A
168
62.658
11.534
74.332
1.00
0.00
xxxx
1292

ATOM
1293
CG
ASP
A
168
63.759
12.576
74.153
1.00
0.00
xxxx
1293

ATOM
1294
OD1
ASP
A
168
63.522
13.772
74.436
1.00
0.00
xxxx
1294

ATOM
1295
OD2
ASP
A
168
64.861
12.194
73.707