Water-soluble transmembrane proteins and methods for preparation and use thereof

Abstract

To provide a method of designing, selecting and / or producing water-soluble membrane proteins and peptides.SOLUTION: Provided is a computer-implemented method for executing a procedure to select a water-soluble variant of a G Protein-Coupled Receptor (GPCR). The method comprises: entering a sequence of the GPCR for analysis; obtaining a variant of the GPCR, in which a plurality of hydrophobic amino acids in transmembrane (TM) domain α-helical segments of the GPCR are substituted; subsequently, obtaining a α-helical secondary structure result for the variant to verify maintenance of α-helical secondary structures in the variant; and obtaining a transmembrane region result for the variant to verify water solubility of the variant. Thereby the method selects the water-soluble variant of the GPCR.SELECTED DRAWING: None

Description

[Technical field]

[0001] (Related Applications) This application claims priority to U.S. application Ser. No. 14 / 669,753, both filed March 26, 2015, and International Application No. PCT / US2015 / 022780, both of which claim the benefit under 35 U.S.C. 119(e) of the filing dates of U.S. Provisional Application No. 62 / 117,550, filed February 18, 2015, U.S. Provisional Application No. 61 / 993,783, filed May 15, 2014, and U.S. Provisional Application No. 61 / 971,388, filed March 27, 2014.

[0002] This application also claims the benefit under 35 U.S.C. 119(e) of the filing dates of U.S. Provisional Application No. 62 / 117,550, filed February 18, 2015, U.S. Provisional Application No. 61 / 993,783, filed May 15, 2014, and U.S. Provisional Application No. 61 / 971,388, filed March 27, 2014.

[0003] The entire contents of each of the above-referenced applications, including all figures and sequence listings, are hereby incorporated by reference. [Background technology]

[0004] Membrane proteins play an important role in all biological systems. Approximately 30% of all genes in almost all sequenced genomes code for membrane proteins. However, our detailed understanding of their structure and function lags far behind that of soluble proteins. As of March 2015, the Protein Structure Data Bank contains over 100,000 structures. However, only 945 membrane protein structures are registered, including 28 G protein-coupled receptors and 530 unique structures that do not include tetraspanin membrane proteins.

[0005] Although membrane receptors are very important, there are several obstacles to elucidating the structure and function of membrane receptors and their recognition and ligand binding properties. The most significant and challenging challenge is that it is extremely difficult to produce milligram quantities of soluble and stable receptors. Inexpensive large-scale production methods are in high demand and have therefore been the focus of extensive research. Only when these preceding obstacles are overcome can detailed structural studies be performed.

[0006] Zhang et al. (U.S. Patent No. 8,637,452) (hereby incorporated by reference) describe an improved method for water-soluble GPCRs in which certain hydrophobic amino acids located in the transmembrane region are replaced by polar amino acids. However, this method is labor-intensive. Furthermore, although the modified transmembrane region meets the water-solubility criteria, improvements in water solubility and ligand binding are desired. Thus, there is a need in the art for improved methods for studying G protein-coupled receptors. Summary of the Invention

[0007] The present invention relates to a method for designing, selecting and / or producing water-soluble membrane proteins and peptides, peptides (and transmembrane domains) designed, selected or produced therefrom, compositions comprising said peptides and methods of using the same. In particular, the method relates to a method for designing libraries of water-soluble membrane peptides, such as GPCR mutants and tetraspanin membrane proteins, using the "QTY principle" of changing water-insoluble amino acids (leucine, isoleucine, valine and phenylalanine or simple letter codes L, I, V, F) to non-ionic water-soluble amino acids (glutamine, threonine and tyrosine or simple letter codes Q, T, Y). In addition, two additional non-ionic amino acids, asparagine (N) and serine (S), may be used for the substitution of L, I and V other than F. It should be understood that in the embodiments discussed below, asparagine (N) and serine (S) are envisaged as possible substitutes for Q and T (described as mutants) or L, I or V (described as native proteins). However, for the sake of brevity, the details of these other embodiments will not be described in further detail in this application, as they will be known to those of ordinary skill in the art from the teachings herein.

[0008] The present invention encompasses modified, synthetic and / or non-naturally occurring one or more α-helical domains and water-soluble polypeptides (e.g., "sGPCRs") comprising such modified one or more α-helical domains, where the modified one or more α-helical domains comprise an amino acid sequence in which a plurality of hydrophobic amino acid residues (L, I, V, F) in the α-helical domain of a native membrane protein are replaced with non-ionizable hydrophilic amino acid residues (Q, T, T, Y or "Q, T, Y", respectively) and / or N and S. The present invention also encompasses a method of preparing a water-soluble polypeptide comprising replacing a plurality of hydrophobic amino acid residues (L, I, V, F) in the α-helical domain of a native membrane protein with non-ionizable hydrophilic amino acid residues (Q / N / S, T / N / S, Y, respectively). The present invention further encompasses polypeptides prepared by replacing a plurality of hydrophobic amino acid residues (L, I, V, F) in the α-helical domain of a native membrane protein with non-ionizable hydrophilic amino acid residues (Q / N / S, T / N / S, Y, respectively). The variants may be characterized by their name, which is the parent or native protein (eg, CXCR4) followed by the abbreviation "QTY" (eg, CXCR4-QTY).

[0009] Accordingly, one aspect of the invention includes the steps of: (1) inputting a sequence of a membrane protein (e.g., a GPCR) for analysis; and (2) obtaining mutants of the membrane protein (e.g., a GPCR) in which multiple hydrophobic amino acids within a transmembrane (TM) domain α-helical segment ("TM region") of the membrane protein (e.g., a GPCR) have been substituted, wherein (a) the hydrophobic amino acids are selected from the group consisting of leucine (L), isoleucine (I), valine (V) and phenylalanine (F), (b) the leucines (L) are each independently substituted with glutamine (Q), asparagine (N) or serine (S), and (c) the isoleucines (I) and the valines (V) are each independently substituted with The present invention provides a method of operating a computer program to execute a scripted procedure for designing water soluble mutants of membrane proteins (e.g., G protein-coupled receptors (GPCRs)), comprising the steps of: (a) replacing each of said amino acids with a phenylalanine (P), threonine (T), asparagine (N), or serine (S); and (b) replacing each of said phenylalanines with a tyrosine (Y); and thereafter, (3) obtaining an alpha-helical secondary structure result for said mutants to confirm maintenance of an alpha-helical secondary structure in said mutants; and (4) obtaining a transmembrane domain result for said mutants to confirm water solubility of said mutants, thereby designing a water soluble mutant of a membrane protein (e.g., a GPCR).

[0010] In certain embodiments, step (3) is performed before, simultaneously with or after step (4). Additional steps described herein can be incorporated into the procedure. The process preferably uses a calculation step preformed by a data processing system. The system utilizes an automated calculation system and a method for selecting protein variants.

[0011] In certain embodiments, in step (2), a subset of the plurality of hydrophobic amino acids in the same TM region of the GPCR is substituted to generate one member of a candidate mutant library, and one or more different subsets of the plurality of hydrophobic amino acids are substituted to generate additional members of the library. In certain embodiments, the method may further comprise ranking all members of the library based on a combined score, the combined score being a weighted combination of the alpha-helix secondary structure prediction result and the transmembrane domain prediction result. In certain embodiments, the method may further comprise ranking the mutants using a ranking function. In certain embodiments, the ranking function may comprise secondary structure components and water-soluble components. For example, the ranking function may comprise weighted values ​​of secondary structure components and / or water-soluble components. In certain embodiments, the method may further comprise performing the method by a data processor, the data processor may further comprise a memory connected thereto.

[0012] In certain embodiments, the method may further comprise selecting the N members with the highest combination scores to form a first library of mutant candidates for the TM region, where N is a predetermined integer (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more). In certain embodiments, the method may further comprise creating one library of mutant candidates for one, two, three, four, five or all six other TM regions of the GPCR. In certain embodiments, the method may further comprise replacing two or more TM regions of the GPCR with corresponding TM regions in the mutant candidate library to create a combinatorial mutant library. In certain embodiments, the method further comprises producing / expressing the combinatorial mutants. In certain embodiments, the method further comprises testing the combinatorial mutants for ligand binding (e.g., in a yeast two-hybrid assay), where those with substantially the same ligand binding compared to the GPCR are selected. In certain embodiments, the method further comprises testing the combination mutants for the biological function of the GPCR, where those having substantially the same biological function as the GPCR are selected.

[0013] Certain water-soluble polypeptides of the invention have the ability to bind to a ligand that normally binds to a wild-type or native membrane protein (e.g., a GPCR). In certain embodiments, amino acids within the putative ligand-binding site of a native membrane protein (e.g., a GPCR) are not substituted and / or the sequences of the extracellular and / or intracellular domains of the native membrane protein (e.g., a GPCR) are identical.

[0014] The (non-ionic) hydrophilic residues, which replace one or more hydrophobic residues in the α-helical domain of the native membrane protein, are selected from the group consisting of glutamine (Q), threonine (T), tyrosine (Y), asparagine (N) and serine (S), and any combination thereof. In a further aspect, hydrophobic residues selected from leucine (L), isoleucine (I), valine (V) and phenylalanine (F) are substituted. In a particular embodiment, a phenylalanine residue in the α-helical domain of the protein is substituted with a tyrosine, an isoleucine and / or a valine residue in the α-helical domain of the protein is each independently substituted with a threonine (or S or N), and / or a leucine residue in the α-helical domain of the protein is each independently substituted with a glutamine (or S or N).

[0015] In certain embodiments, substantially all (e.g., 96%, 97%, 98%, 99% or 100%) of the leucines are replaced with glutamines, or 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%. In certain embodiments, substantially all (e.g., 96%, 97%, 98%, 99% or 100%) of the isoleucines are replaced with threonines, or 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%. In certain embodiments, substantially all (e.g., 96%, 97%, 98%, 99% or 100%) of the valines are replaced with threonines, or 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%. In certain embodiments, substantially all (e.g., 96%, 97%, 98%, 99% or 100%) or 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% of the phenylalanines are substituted with tyrosine. In certain embodiments, one or more (e.g., one, two or three) of the leucines are not substituted. In certain embodiments, one or more (e.g., one, two or three) of the isoleucines are not substituted. In certain embodiments, one or more (e.g., one, two or three) of the valines are not substituted. In certain embodiments, one or more (e.g., one, two or three) of the phenylalanines are not substituted.

[0016] In certain embodiments, the combinatorial mutant library comprises less than about 2 million members. In certain embodiments, the sequence of the GPCR comprises information about the TM region of the GPCR. In certain embodiments, the sequence of the GPCR is obtained from a protein structure database (e.g., PDB, UniProt). In certain embodiments, the TM region of the GPCR is predicted based on the sequence of the GPCR. For example, the TMHMM2.0 (transmembrane prediction using hidden Markov models) software module / package can be used to predict the TM region of the GPCR. In certain embodiments, the TMHMM2.0 software module / package utilizes dynamic baseline for peak search.

[0017] In certain embodiments, the method further comprises providing a polynucleotide sequence for each mutant of said GPCR, which may be codon optimized for expression in a host (e.g., a bacterium such as E. coli, a yeast such as Saccharomyces cerevisiae or Schizosaccharomyces pombe, an insect cell such as an Sf9 cell, a non-human mammalian cell, or a human cell).

[0018] In certain embodiments, the scripted procedures may include VBA scripts. In certain embodiments, the scripted procedures may be operable on a Linux system (e.g., Ubuntu 12.04 LTS), a Unix system, a Microsoft Windows operating system, an Android operating system, or an Apple iOS operating system. Different programming languages ​​may be used with the implementation of the present invention, including C++, JavaScript, MATLAB, and the like. The coded instructions may be stored in a memory device, such as a non-transitory computer-readable medium that may be used with a computer system known to those skilled in the art.

[0019] In certain embodiments, the α-helical domain is one of seven transmembrane α-helical domains in a native membrane protein that is a G protein-coupled receptor (GPCR). In some embodiments, the GPCR is a purinergic receptor (P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 ), M 1 and M. 3 Muscarinic acetylcholine receptors, thrombin receptors (protease-activated receptor (PAR)-1, PAR-2), thromboxane (TXA 2 ), sphingosine 1-phosphate (S1P 2 , S1P 3 , S1P 4 and S1P 5 ), lysophosphatidic acid (LPA 1 , L.P.A.2 , L.P.A. 3 ), angiotensin II (AT 1 ), serotonin (5-HT 2c and 5-HT 4 ), somatostatin (sst 5 ), endothelin (ET A and E.T. B ), cholecystokinin (CCK 1 ), V 1a Vasopressin receptor, D 5 Dopamine receptor, fMLP formyl peptide receptor, GAL 2 Galanin receptor, EP 3 Prostanoid receptor, A 1 Adenosine receptor, alpha 1 Adrenergic receptor, BB 2 Bombesin receptor, B 2 Bradykinin receptor, calcium-sensing receptor, chemokine receptor, KSHV-ORF74 chemokine receptor, NK 1 The receptor is selected from the group consisting of tachykinin receptors, thyroid stimulating hormone (TSH) receptors, protease activated receptors, neuropeptide receptors, adenosine A2B receptors, P2Y purinergic receptors, metabotropic glutamate receptors, GRK5, GPCR-30 and CXCR4.

[0020] In other embodiments, the native membrane protein or membrane protein is an essential membrane protein. In a further aspect, the native membrane protein is a mammalian protein. The protein of the present invention is preferably a human protein. In certain embodiments, reference to a specific GPCR protein (e.g., CXCR4) refers to a mammalian GPCR, such as a non-human mammalian GPCR, or a human GPCR.

[0021] In some embodiments, the α-helical domain is one of seven transmembrane α-helical domains in a mutant G protein-coupled receptor (GPCR) that has been modified, e.g., in an extracellular or intracellular loop, to improve or alter ligand binding, as described elsewhere in the literature. For purposes of the present invention, the term "native" or "wild-type" refers to the protein (or α-helical domain) prior to aqueous solubilization according to the methods described herein.

[0022] In certain embodiments, the membrane protein may be a tetraspanin membrane protein characterized by four transmembrane alpha helices. Approximately 54 human tetraspanin membrane proteins have been reviewed and annotated. Many are known to mediate cell-cell signaling events that play important roles in controlling cell development, activation, proliferation and motility. For example, the CD81 receptor plays an important role as a receptor for Hepatitis C virus entry and malaria parasite infection. The CD81 gene is localized in a tumor suppressor gene region and may be a candidate for mediating cancer malignancy. CD151 is involved in increasing cell motility, invasion and metastasis of cancer cells. Expression of CD63 correlates with ovarian cancer invasiveness. A hallmark of tetraspanin membrane proteins is a cysteine-cysteine-glycine motif in the second or large extracellular loop.

[0023] Another aspect of the invention provides water soluble mutants of G protein-coupled receptors (GPCRs) characterized in that (1) multiple hydrophobic amino acids within a transmembrane (TM) domain α-helical segment ("TM region") of the GPCR have been substituted, wherein (a) the hydrophobic amino acids are selected from the group consisting of leucine (L), isoleucine (I), valine (V) and phenylalanine (F); (b) the leucines (L) are each independently substituted with glutamine (Q), asparagine (N) or serine (S); (c) the isoleucine (I) and the valine (V) are each independently substituted with threonine (T), asparagine (N) or serine (S); and (d) the phenylalanines are each independently substituted with tyrosine (Y); thereafter, (2) an α-helical secondary structure is maintained by all seven TM regions of the mutant; and (3) no predicted transmembrane region is present.

[0024] In certain embodiments, the water-soluble variant comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 4-11, 13-20, 22-29, 31-38, 40-47, 49-56, and 58-64, which may further comprise one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 3, 12, 21, 30, 39, 48, and 57. In certain embodiments, the water-soluble variant binds to a CXCR4 ligand.

[0025] In certain embodiments, the water-soluble variant comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 69-76, 78-85, 87, 89-96, 98-105, 107-114, and 116-123, which may further comprise one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 68, 77, 86, 88, 97, 106, 115, and 124. In certain embodiments, the water-soluble variant binds to a CX3CR1 ligand.

[0026] In certain embodiments, the water-soluble variants are selected from the group consisting of SEQ ID NOs: 128-135, 137-144, 146-153, 155-162, 164-171, 173 and 175-176. 182. It may further comprise one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 127, 136, 145, 154, 163, 172, 174 and 183. In certain embodiments, the water-soluble variant binds to a CCR3 ligand.

[0027] In certain embodiments, the water-soluble variant comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 187-194, 196-203, 205-206, 208, 210-217, 219-225, 227-234, which may further comprise one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 186, 195, 204, 207, 209, 218, 226, and 235. In certain embodiments, the water-soluble variant binds to a CCR5 ligand.

[0028] In certain embodiments, the water-soluble variant comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 236-243, 245-252, 254-261, 263-270, 272, 274-281 and 283-290, which may further comprise one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 235, 244, 253, 262, 271, 273, 282 and 291. In certain embodiments, the water-soluble variant binds to a CXCR3 ligand.

[0029] In certain embodiments, the water soluble variants comprise one or more transmembrane domains set forth in any one of SEQ ID NOs: 2, 67, 126, 185, 327, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, or 325. In certain embodiments, the variants are water soluble and bind to a ligand of the homologous native transmembrane protein.

[0030] Another aspect of the invention provides a method for producing a protein in a bacterium (e.g., E. coli), comprising: (a) culturing the bacterium in a growth medium under conditions suitable for protein production; (b) fractionating a lysate of the bacteria to produce a soluble fraction and an insoluble pellet fraction; and (c) isolating the protein of interest from the soluble fraction, wherein (1) the protein is a mutant of a G protein-coupled receptor (GPCR) according to any one of claims 29-46; and (2) the yield of the protein is at least 20 mg / L (e.g., 30 mg / L, 40 mg / L, 50 mg / L or more) of growth medium.

[0031] In certain embodiments, the bacterium is E. coli BL21 and the growth medium is LB medium. In certain embodiments, the protein is encoded by a plasmid in the bacterium. In certain embodiments, expression of the protein is under the control of an inducible promoter, such as an inducible promoter inducible by IPTG. In certain embodiments, the lysate is generated by sonication. In certain embodiments, the lysate is centrifuged at 14,500 x g or greater to generate a soluble fraction.

[0032] Another aspect of the invention provides a method for treating a disorder or disease mediated by membrane protein activity in a subject in need thereof, comprising administering to the subject an effective amount of a water soluble polypeptide described herein.

[0033] In certain embodiments, the water-soluble polypeptide retains the ligand binding activity of the membrane protein. Examples of disorders and diseases that can be treated by administering the water-soluble peptides of the present invention include, but are not limited to, cancer (e.g., small cell lung cancer, melanoma, triple-negative breast cancer), Parkinson's disease, cardiovascular disease, hypertension, and bronchial asthma.

[0034] Another aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a water-soluble polypeptide of the present invention and a pharma- ceutically acceptable carrier or diluent.

[0035] In yet another aspect, the invention provides cells transfected with a subject water-soluble peptide comprising a modified α-helical domain, hi certain embodiments, the cell is an animal cell (e.g., a human, non-human mammalian, insect, avian, fish, reptile, amphibian or other cell), yeast or bacterial cell.

[0036] The present invention also includes a computer-implemented method executed on a computer system, comprising one or more of the methods (or steps thereof) described herein. The computer system includes a non-transitory computer-readable medium having computer-executable instructions stored thereon, which, when executed by the computer system, cause the computer system to perform the method, and which, when executed by the computer system, cause the computer system to perform the method envisioned herein. Further, a computer system is envisioned that includes at least one memory for storing sequence data and quantitative results described herein, and at least one processor connected to the memory, configured to perform the method described herein. A user interface, such as a graphical user interface (GUI) in conjunction with an electronic display device, can be used to select process parameters that operate to control the selection methods, including the computational methods described herein.

[0037] Another aspect of the invention provides a non-transitory computer readable medium having stored thereon a set of instructions for performing any of the methods of the invention.

[0038] A further aspect of the present invention provides a data processing system comprising a data processor operative to carry out amino acid substitutions as in any of the methods of the present invention, and operative to rank the protein variants according to a ranking function and to select a water soluble variant of a G protein coupled receptor.

[0039] It should be understood that all embodiments of the present invention, including those described in only one aspect of the invention (e.g., screening methods), should be construed as applicable to all aspects of the invention (e.g., water-soluble proteins or methods of use) and should be construed as combinable with any one or more additional embodiments of the invention, unless expressly disclaimed or otherwise inappropriate, as should be readily understood by one of skill in the art.

[0040] The above and other objects, features and advantages of the present invention will become apparent from the following more detailed description of representative embodiments of the invention, as illustrated in the accompanying drawings, in which like numerals refer to the same parts throughout the different views, and in which the drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention. [Brief description of the drawings]

[0041] [Figure 1A] Figures 1A-1D are general illustrations of the QTY code, which systematically substitutes the hydrophobic amino acids L, I, V, and F with Q, T, T, and Y, respectively. (Figure 1A) The molecular shapes of the amino acids leucine and glutamine are similar, and similarly, the molecular shapes of isoleucine and valine are similar to threonine, and the molecular shapes of phenylalanine and tyrosine are similar. Leucine, isoleucine, valine, and phenylalanine are hydrophobic and cannot bind water molecules. In contrast, glutamine can bind four water molecules, i.e., two hydrogen donors and two hydrogen acceptors, and the -OH groups on threonine and tyrosine can bind three water molecules, i.e., one hydrogen donor and two acceptors. [Figure 1B] Figure 1B shows a side view of the α-helix. After applying the QTY code of systematic amino acid changes, the α-helix becomes water soluble. [Figure 1C]Figure 1C shows a top view of the α-helix before and after the QTY code substitution. The helix on the left is the native membrane helix with mostly hydrophobic amino acids, and the helix on the right is the same helix after applying the QTY code substitution. This helix now has the most hydrophilic amino acids. [Figure 1D] (Figure 1D) Before the QTY code, the GPCR membrane proteins are surrounded by hydrophobic lipid molecules so that they are embedded in the lipid membrane (left part of Figure 1D). After applying the QTY code, the GPCR membrane proteins become water-soluble and no longer require the surfactant surrounding them for stabilization (right part of Figure 1D). [Diagram 2] Figure 1 shows a TMHMM prediction of the transmembrane domain region of CXCR4. The prediction shows seven distinct hydrophobic transmembrane segments. In contrast, the TMHMM prediction of a mutant of CXCR4 subjected to the QTY replacement method of the present invention (CXCR4-QTY) shows that there are no longer seven distinct hydrophobic transmembrane segments visible. [Diagram 3] 1 shows the predicted α-helical wheel structure of the fully QTY-encoded TM1 domain of CXCR4. [Figure 4] Illustrative of such potential mutations in each of the seven TM regions of the GPCR CXCR4. [Diagram 5] Sequence alignment of the wild type protein and the respective QTY variants of CXCR4, CXCR3, CCR3 and CCR5. The QTY code applies only to the seven hydrophobic transmembrane segments and not to the extracellular and intracellular segments. [Figure 6] Sequence alignment of the wild type protein and the respective QTY variants of CXCR4, CXCR3, CCR3 and CCR5. The QTY code applies only to the seven hydrophobic transmembrane segments and not to the extracellular and intracellular segments. [Figure 7]Sequence alignment of the wild type protein and the respective QTY variants of CXCR4, CXCR3, CCR3 and CCR5. The QTY code applies only to the seven hydrophobic transmembrane segments and not to the extracellular and intracellular segments. [Figure 8] Sequence alignment of the wild type protein and the respective QTY variants of CXCR4, CXCR3, CCR3 and CCR5. The QTY code applies only to the seven hydrophobic transmembrane segments and not to the extracellular and intracellular segments. [Figure 9A] 1 is a flow chart of an exemplary embodiment of the method. [Figure 9B] 4 is another flow chart of an exemplary embodiment of the present method. [Figure 10] 1 is an example of a computer system of the present invention. [Figure 11A] FIG. 1 is a schematic diagram of a flow chart describing the process steps of certain preferred embodiments of the present invention. [Figure 11B] FIG. 1 is a schematic diagram of a flow chart describing the process steps of certain preferred embodiments of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] A description of the preferred embodiments of the present invention follows: The words "a" or "an" are intended to include one or more unless otherwise specified.

[0043] In some aspects, the invention relates to the use of the QTY (Glutamine, Threonine and Tyrosine) substitution (i.e., "QTY Code") method (or "principle") to change the hydrophobic residues leucine (L), isoleucine (I), valine (V) and phenylalanine (F) of the seven transmembrane alpha-helices of native proteins to the hydrophilic residues glutamine (Q), threonine (T) and tyrosine (Y). In certain embodiments, as described above, asparagine (N) and serine (S) can also be used as replacement residues for L, I and / or V other than F. The invention can convert water-insoluble native membrane proteins into more water-soluble counterparts that still maintain some or substantially all of the functions of the native proteins.

[0044] The present invention includes a method for designing water-soluble peptides. The method is illustrated primarily with respect to GPCR proteins, with human CCR3, CCR5, CXCR4 and CX3CR1 as specific examples, although the general principles of the invention also apply to other proteins with transmembrane (α-helical) regions.

[0045] GPCRs typically have seven transmembrane α-helices (seven TMs) and eight loops (eight NTMs) connected by seven TM regions. These transmembrane segments may be referred to as TM1, TM2, TM3, TM4, TM5, TM6 and TM7. The eight non-transmembrane loops are divided into four extracellular loops EL1, EL2, EL3 and EL4 and four intracellular loops IL1, IL2, IL3 and IL4, thus making a total of eight loops (including N- and C-terminal loops, each with a free end, each connected to only one TM region). Thus, the 7TM-GPCR protein can be divided into 15 fragments based on transmembrane and non-transmembrane features.

[0046] One aspect of the invention is a method of operating a computer program to execute scripted procedures to select or prepare water soluble mutants of a membrane protein (e.g., a G protein-coupled receptor (GPCR)), comprising: (1) inputting the sequence of a membrane protein of interest (e.g., a GPCR) for analysis; (2) obtaining a mutant of a membrane protein (e.g., a GPCR) having multiple hydrophobic amino acid substitutions within a transmembrane (TM) domain α-helical segment ("TM region") of the membrane protein (e.g., a GPCR), (a) the hydrophobic amino acid is selected from the group consisting of leucine (L), isoleucine (I), valine (V) and phenylalanine (F); (b) each said leucine (L) is independently replaced with glutamine (Q), asparagine (N), or serine (S); (c) said isoleucine (I) and said valine (V) are each independently replaced with threonine (T), asparagine (N) or serine (S); and (d) each phenylalanine is replaced with a tyrosine (Y); and then (3) obtaining alpha helix secondary structure results for the variants to confirm maintenance of alpha helix secondary structure in the variants; (4) obtaining a transmembrane domain profile for the variant and confirming the water solubility of the variant; thereby selecting a water-soluble mutant of the membrane protein (e.g., GPCR).

[0047] As used herein, the terms "water-soluble mutant of a (spanning) membrane protein" or "water-soluble (spanning) membrane mutant" can be used interchangeably.

[0048] The exact order in which the steps of the present invention are performed may vary. For example, in certain embodiments, step (3) is performed before step (4). In certain embodiments, step (3) is performed simultaneously with step (4). In certain embodiments, step (3) is performed after step (4).

[0049] In certain embodiments, the plurality of hydrophobic amino acids is randomly selected from all hydrophobic amino acid candidates L, I, V, and F located in all TM regions of the protein. In certain embodiments, the plurality of hydrophobic amino acids is selected from about 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%, 100%, 102%, 104%, 106%, 108%, 109%, 109%, 109%, 102%, 105%, 106%, 107%, 108%, 109%, 109%, 11 8%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In certain embodiments, the plurality of hydrophobic amino acids is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% of all potential hydrophobic amino acids L, I, V and F located in all TM regions of the protein. In certain embodiments, the plurality of hydrophobic amino acids is less than or equal to about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60% or 50% of all potential hydrophobic amino acids L, I, V and F located in all TM regions of the protein. In certain embodiments, the randomly selected hydrophobic amino acids L, I, V and F may be approximately evenly distributed among all TM regions, or may be preferentially or exclusively distributed among 1, 2, 3, 4, 5 or 6 TM regions.

[0050] In certain embodiments, all potential hydrophobic amino acids L, I, V and F on all TM regions of the protein are substituted. For example, all L's are independently substituted with Q (or S or N), and / or all I's and V's are independently substituted with T (or S or N), and / or all F's are substituted with Y. In certain embodiments, all L's are substituted with Q, all I's and V's are substituted with T, and all F's are substituted with Y.

[0051] In certain embodiments, instead of randomly substituting selected hydrophobic amino acids L, I, V and F in all TM regions, all substitutions can be initially limited to any one of the TM regions (e.g., the most N- or C-terminal TM region), and only the desired substitution mutants are selected as members of the mutant candidate library. All members of the library differ in the substitutions in the selected TM region, either by the position at which the substitution is made (e.g., the 3rd residue is substituted for the 10th residue in the TM region), or by the identity of the substituted residue (e.g., S to T for an I or V substitution), or both. The desired substitution mutants are selected based on a predetermined criterion, such as a scoring system that considers α-helical secondary structure prediction results and / or transmembrane domain prediction results.

[0052] This method can be repeated for 1, 2, 3, 4, 5, 6 additional TM regions of the protein, or for all remaining TM regions of the protein, with each iteration generating a library of candidate mutants that can be stored in electronic memory or a database. Within the same library, all mutants differ in substitutions within the selected TM region (see above), but are otherwise identical in the remaining TM and non-TM regions.

[0053] Domain swapping, or domain shuffling, using sequences in two or more such libraries produces combinatorial variants with hydrophobic amino acid L, I, V, F substitutions in two or more TM regions. Depending on the number of members in each library, the total number of possible combinations of combinatorial variants can approach millions with only a few members in each library. For example, for a GPCR with seven TM regions, if there are eight members in each of the seven libraries, the total number of combinatorial variants based on the libraries would be 8. 7 That is, about 2.1 million. In certain embodiments, the combinatorial variant library contains less than about 5 million, 4 million, 3 million, 2 million, 1 million, or 500,000 members.

[0054] Thus, in certain embodiments, in step (2), one subset of the plurality of hydrophobic amino acids within the same TM region of a protein of interest (e.g., a GPCR) is substituted to generate one member of a mutant candidate library, and one or more different subsets of the plurality of hydrophobic amino acids are substituted to generate further members of the library.

[0055] In certain embodiments, the method further comprises ranking all members of said library based on a combined score, the combined score being a weighted combination of the alpha helix secondary structure prediction results and the transmembrane domain prediction results.

[0056] As will be appreciated by those skilled in the art, domains with different sequences will likely predict different water solubility and propensity for α-helix formation. A "score" is assigned to a particular predicted water solubility or range of water solubility, propensity or range of propensity to form α-helix structures. The score may be qualitative (0, 1), where 0 may represent, for example, a domain with unacceptable predicted water solubility and 1 may represent, for example, a domain with acceptable predicted water solubility. The score may be based on, for example, a threshold. Alternatively, the score may be assessed, for example, on a scale of 1-10 established to characterize increasing degrees of water solubility. Alternatively, the score may be quantitative, such as describing predicted solubility in mg / mL. Once the scores have been assessed for each domain, the domain variants can be easily compared (or ranked) by one or, preferably, both of the scores to select domain variants that are both water soluble and form α-helices. Thus, preferred embodiments may utilize a ranking function that can be used to calculate ranking data. It should also be noted that water-soluble proteins prepared based on the presently described system can be analyzed and characterized, and those substitution combinations that are not effective in achieving a given biological function can be used to constrain the computational model, thereby providing input to the system that allows for more efficient information processing.

[0057] For example, one or more mutants can be designed and produced in vitro and / or in vivo using the methods of the present invention, and one or more biological functions of the mutants can be determined based on any of many art-recognized methods. For GPCRs, for example, the ligand binding and / or downstream signaling by the mutants can be compared to that of wild-type GPCRs, and the QTY substitution pattern used to produce a particular mutant can be associated with increased, maintained, or decreased biological activity. Such structure / function relationship information obtained based on one or more mutants can be used for machine learning, or additional constraints can be imposed on the computational model of the present invention to more efficiently rank the mutants produced by the methods of the present invention. In this way, a new candidate mutant with a substitution pattern that more closely matches the substitution pattern of a known successful mutant can be ranked higher than another candidate mutant with a substitution pattern that is less closely matched to the substitution pattern of a known successful mutant or that is more closely matched to the substitution pattern of a known unsuccessful mutant.

[0058] The TMHMM program, when run as a standalone software module / package (e.g., for Linux systems), generates a score between 0 and 1 that can be used to predict the propensity to form transmembrane domains / proteins. This score can be used as a quantitative predictor of water solubility in the methods of the invention.

[0059] Thus, in certain embodiments, the α-helical secondary structure component of the ranking function may be a quantitative score, such as 0.5 or 1 for no predicted α-helical secondary structure, and 0 for maintaining predicted α-helical secondary structure. In certain embodiments, transmembrane region results can be obtained by a TM region prediction program such as TMHMM2.0, which provides a numerical value between 0 and 1, where 0 is no predicted TM region and 1 has the strongest tendency to form one or more TM regions. Thus, the two scores can be combined directly or with a weight, such that the combined score represents an overall assessment of maintained secondary structure as well as predicted water solubility (measured by tendency to form TM regions). For example, a combined score of 0 indicates that the variant does not have a predicted TM region but maintains a predicted α-helical secondary structure, and is therefore a desirable variant. On the other hand, variants that have a strong tendency to form TM regions (e.g., due to the presence of many hydrophobic residues) tend to have a larger combined score and are therefore less desirable under this scoring scheme.

[0060] In certain embodiments, the method includes removing variants with alpha helix secondary structure predictions that tend to indicate that the alpha helix secondary structure is broken or disrupted. In certain embodiments, the method includes removing variants with transmembrane domain predictions that tend to indicate a strong tendency to form TM domains. Thus, the system can include a BEAMing module that can filter out variants through further selection processes.

[0061] In certain embodiments, the ranking function can be selected to include a weighting scheme that assigns 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% weight to the alpha helix secondary structure prediction results and the remainder to the transmembrane domain prediction results. Depending on the desired properties, such as biological function, the weighting features can be selected manually by the user or automatically by the software.

[0062] In certain embodiments, the method further comprises selecting the N members having the highest combination scores to form a first library of mutant candidates for said TM region, where N is a predetermined integer (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more).

[0063] In certain embodiments, the method further comprises generating a library of mutant candidates for one, two, three, four, five, six or all remaining TM regions of the protein (e.g., a GPCR). Each entry in the library can include fields used to define attributes of the entry, including ranking data generated by one or more ranking functions.

[0064] In certain embodiments, the method further comprises replacing two or more (e.g., all) TM regions of the protein (e.g., GPCR) with corresponding TM regions in the mutant candidate library to generate a combinatorial mutant library. As used herein, "corresponding TM regions" refers to TM regions in the mutant candidate library that are the same as or homologous to the TM regions of the protein (e.g., GPCR) being combined. For example, if the second and third TM regions from the N-terminus of a GPCR are replaced, the TM region sequence in the library that has replacements only in the second TM region and the TM region sequence in the library that has replacements only in the third TM region are imported / pasted / transferred to the second and third TM regions of the GPCR to generate combinatorial mutants.

[0065] In certain embodiments, substantially all (e.g., 96%, 97%, 98%, 99% or 100%) of the leucines or 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% are replaced with glutamine. In certain embodiments, substantially all (e.g., 96%, 97%, 98%, 99% or 100%) of the isoleucines or 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% are replaced with threonine. In certain embodiments, substantially all (e.g., 96%, 97%, 98%, 99% or 100%) of the valines or 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% are replaced with threonine. In certain embodiments, substantially all (e.g., 96%, 97%, 98%, 99% or 100%) or 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% of the phenylalanines are substituted with tyrosine. In certain embodiments, one or more (e.g., 1, 2 or 3) of the leucines are not substituted. In certain embodiments, one or more (e.g., 1, 2 or 3) of the isoleucines are not substituted. In certain embodiments, one or more (e.g., 1, 2 or 3) of the valines are not substituted. In certain embodiments, one or more (e.g., 1, 2 or 3) of the phenylalanines are not substituted.

[0066] In certain embodiments, the method further comprises producing / expressing the combination mutants. In certain embodiments, the method further comprises testing the combination mutants for ligand binding (e.g., in vitro or in a biological system such as yeast two-hybrid), whereby those with substantially the same ligand binding are selected compared to the ligand binding of the GPCR. In certain embodiments, the method further comprises testing the combination mutants for the biological function of the GPCR, whereby those with substantially the same biological function are selected compared to the biological function of the GPCR.

[0067] In certain embodiments, the sequence of a TM protein (e.g., a GPCR) includes information about the TM regions of the protein, e.g., the location of one or more transmembrane regions of the TM protein, such as the location of all TM regions. Such sequences may belong to proteins that have a solved crystal structure with defined TM regions. Also, such sequences may belong to proteins that have TM region information annotated based on prior studies, such information being readily available from public or proprietary databases such as PDB, UniProt, Genbank, EMBL, DBJ, etc.

[0068] The Protein Data Bank (PDB) is a weekly updated repository for three-dimensional structural data of large biomolecules such as proteins and nucleic acids. Data, typically obtained by X-ray crystallography or NMR spectroscopy, and submitted by biologists and biochemists worldwide, are freely accessible on the Internet via the websites of its member organizations (PDBe, PDBj and RCSB). The PDB is overseen by the World Protein Data Bank, or wwPDB. The PDB is an important resource in the area of ​​structural biology, such as structural genomics, and most major scientific journals and several funding agencies are currently calling for scientists to submit their structural data to the PDB.

[0069] Considering the contents of the PDB as primary data, there are hundreds of derived (i.e., secondary) databases that classify the data differently. For example, SCOP and CATH both classify structures according to structural type and presumed evolutionary relationships, GO classifies structures based on genes, while crystallographic databases store information about the 3D structure of proteins. All such publicly available databases may be used to obtain input sequence information, including information about the presence and location of transmembrane regions.

[0070] Another publicly available database that can provide sequence information for use in the methods of the invention is UniProt. UniProt is a comprehensive, high-quality, freely accessible database of protein sequence and function information, with many entries derived from genome sequencing projects. It contains a lot of information on the biological functions of proteins derived from the research literature. UniProt provides four core databases: UniProtKB (with subdivisions Swiss-Prot and TrEMBL), UniParc, UniRef and UniMes. Of these, UniProtKB / Swiss-Prot is a manually annotated, non-redundant protein sequence database that combines information extracted from the scientific literature and computational analysis evaluated by biocurators. The aim of UniProtKB / Swiss-Prot is to provide all known relevant information about a particular protein. The annotation is regularly reviewed to keep up with current scientific findings. The manual annotation of the entries includes a detailed analysis of the protein sequence and the scientific literature. Sequences from the same gene and the same organism species are merged into the same database entry. Differences between sequences are identified and their causes are documented (e.g., alternative splicing, natural mutations, etc.). Computer predictions are manually evaluated and relevant results are selected for inclusion in the entry. These predictions include post-translational modifications, transmembrane domains and topology, signal peptides, domain identification and protein family classification, all of which may be used to obtain useful sequence information related to the TM regions used in the methods of the invention.

[0071] In certain embodiments, the sequence of a TM protein (e.g., a GPCR) does not include information regarding the location of one or more (e.g., any) transmembrane regions, although the one or more TM regions can be predicted based on sequence homology to related proteins with known TM regions. For example, the related proteins may be homologous proteins in different species.

[0072] In certain embodiments, the sequence of the TM protein (e.g., GPCR) does not contain information regarding the location of one or more (e.g., any) transmembrane regions, and such information is not readily available based on publicly available information. In this embodiment, the present invention uses art-recognized methods to calculate the TM regions, such as the TMHMM2.0 (Transmembrane Prediction Using Hidden Markov Models) program developed by the Center for Biological Sequence Analysis. See further details on this below.

[0073] In certain embodiments, the method further comprises providing a polynucleotide sequence for each variant of the protein (e.g., GPCR). Such polynucleotide sequences can be readily generated based on the protein sequence of the protein (e.g., GPCR) and the known genetic code. In certain embodiments, the polynucleotide sequences are codon optimized for expression in a host. The host may be a bacterium, such as E. coli, a yeast, such as Saccharomyces cerevisiae or Schizosaccharomyces pombe, an insect cell, such as an Sf9 cell, a non-human mammalian cell, or a human cell.

[0074] In certain embodiments, the protein is a GPCR, e.g., a purinergic receptor (P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 ), M 1 and M. 3 Muscarinic acetylcholine receptors, thrombin receptors (protease-activated receptor (PAR)-1, PAR-2), thromboxane (TXA 2 ), sphingosine 1-phosphate (S1P 2 , S1P 3 , S1P 4 and S1P 5 ), lysophosphatidic acid (LPA 1 , L.P.A. 2 , L.P.A. 3 ), angiotensin II (AT 1 ), serotonin (5-HT 2cand 5-HT 4 ), somatostatin (sst 5 ), endothelin (ET A and E.T. B ), cholecystokinin (CCK 1 ), V 1a Vasopressin receptor, D 5 Dopamine receptor, fMLP formyl peptide receptor, GAL 2 Galanin receptor, EP 3 Prostanoid receptor, A 1 Adenosine receptor, alpha 1 Adrenergic receptor, BB 2 Bombesin receptor, B 2 Bradykinin receptor, calcium-sensing receptor, chemokine receptor, KSHV-ORF74 chemokine receptor, NK 1 The GPCR is selected from the group consisting of tachykinin receptors, thyroid stimulating hormone (TSH) receptors, protease-activated receptors, neuropeptide receptors, adenosine A2B receptors, P2Y purinergic receptors, metabotropic glutamate receptors, GRK5, GPCR-30, and CXCR4.

[0075] In certain embodiments, the scripted steps of the method include a VBA script.

[0076] In certain embodiments, the scripted procedures are operable on a Linux system (eg, Ubuntu 12.04 LTS), a Microsoft Windows operating system, or an Apple iOS operating system.

[0077] In certain embodiments, the method comprises: (1) identifying the first transmembrane domain of a membrane protein, if necessary by predicting the alpha-helical structure of the protein (e.g., a GPCR); (2) modifying a plurality of hydrophobic amino acids according to the QTY codes defined herein to obtain a modified first transmembrane sequence; (3) scoring the propensity for alpha-helical structure of the first modified transmembrane sequence of (2) (e.g., in the context of a modified membrane protein having the first modified transmembrane sequence) to obtain a structure score; (4) scoring the solubility prediction of the first modified transmembrane sequence of (2) (e.g., in the context of a modified membrane protein having the first modified transmembrane sequence) to obtain a solubility score; (5) repeating steps (2) to (4) to obtain a first library of putatively water-soluble modified transmembrane mutants; (6) comparing the structural and solubility scores of each of the first modified transmembrane variants that are putatively water-soluble in the first library, and preferably ranking the first modified transmembrane variants that are putatively water-soluble using said structural and solubility scores; (7) selecting a plurality of the putatively water-soluble first modified transmembrane variants, where the plurality is an integer H or preferably less than 10, 9, 8, 7, 6, 5 or 4, to obtain a second library of putatively water-soluble first modified transmembrane variants; (8) repeating steps (1)-(7) for the second, third, fourth, fifth, sixth, seventh or preferably all transmembrane domains of the protein, where the total number of transmembrane domains modified by this method is an integer n; (9) identifying an amino acid sequence of the protein that is not contained in any of the transmembrane regions modified in steps (1) to (8) and that includes any extracellular or intracellular domain of the protein; (10) Producing a combinatorial mutant of a modified transmembrane protein that is putatively water-soluble (see above), and (11) Optionally, identifying the nucleic acid sequence of each of the modified transmembrane mutants that are putatively water soluble. The present invention includes all or substantially all of the above.

[0078] Using the nucleic acid sequences identified in the above method, nucleic acid sequences for each of the putatively water-soluble modified transmembrane variants and each of the non-transmembrane domains (including extracellular and intracellular domains) are generated and combinatorially expressed to produce a maximum H n A library of 10 million putatively water-soluble transmembrane protein variants can be generated. For example, if H is 8 and n is 7, a library of approximately 2 million water-soluble protein variants can be designed.

[0079] Another aspect of the present invention relates to the expression of soluble mutant proteins (e.g., GPCRs) designed according to the methods of the present invention. This aspect of the present invention is based in part on the surprising discovery that high levels of expression can be achieved with soluble mutant proteins (e.g., GPCRs) designed according to the methods of the present invention, both in cell-free expression systems in vitro and in commonly used cellular expression systems such as E. coli. In addition, the expressed proteins are highly soluble and can be easily purified from the soluble fraction of the expression system, such as the soluble fraction of the lysate of an E. coli culture, as opposed to the insoluble aggregates or pellets that most membrane proteins typically reside in.

[0080] Thus, one aspect of the present invention is (a) culturing the bacteria in a growth medium under conditions suitable for protein production; (b) fractionating the bacterial lysate to produce a soluble fraction and an insoluble pellet fraction; (c) isolating the protein from the soluble fraction, (1) the protein is a mutant protein of the subject invention (e.g., a G protein-coupled receptor (GPCR)); and (2) the protein yield is at least 20 mg / L (e.g., 30 mg / L, 40 mg / L, 50 mg / L or more) of growth medium; and The present invention provides a method for producing a protein in bacteria (e.g., E. coli), comprising:

[0081] In certain embodiments, the bacterium is E. coli BL21 and the growth medium is LB medium. In certain embodiments, the protein is encoded by a plasmid in the bacterium. In certain embodiments, expression of the protein is under the control of an inducible promoter. For example, the inducible promoter may be inducible by IPTG. In certain embodiments, the lysate is generated by sonication. In certain embodiments, the lysate is centrifuged at 14,500 x g or greater to generate a soluble fraction.

[0082] With the general aspects of the invention described above, specific features or specific embodiments of the invention are further described below.

[0083] Transmembrane domain prediction Certain methods of the present invention include predicting transmembrane regions of proteins such as GPCRs. There are many programs and software known in the art for TM regions, any of which may be used individually or in combination in the methods of the present invention that require a TM region prediction step. These programs usually have a very simple user interface that requires the user to provide an input sequence in a typically specified format (such as FASTA or text format), and provide prediction results using text or graphics or both. Some programs also provide more advanced features, such as allowing the user to specify certain parameters to fine-tune the prediction results. All such programs can be used in the methods of the present invention.

[0084] One exemplary TM region prediction program is TMHMM (provided by the Center for Biological Sequences at the Technical University of Denmark), which accurately predicts 97-98% of TM region helices. It uses a hidden Markov model to predict transmembrane helices in proteins. The input protein sequence may be in FASTA format, and the output can be presented as an html page containing images of the predicted locations of TM regions. In a study by Moller et al. entitled "Evaluation of Methods for the Prediction of Membrane Spanning Regions" (Bioinformatics 17(7):646-653, 2001), TMHMM was determined to be the best transmembrane prediction program at the time of evaluation.

[0085] Its specifications include the following:TMH MM1.0, 2.0, and 2.0 of 2.0 stirrups (Sonnhammer et al., Conf. Mol. AAAI Press, pp. 176-182, 1998; 1994, Eisenberg (Eisenberg et al., Nature 299:371-374, 1982), Kyte / Doolittle (Kyte and Doolittle, J. Mol. Biol. 157:105-132, 1982), TMAP (Persson and Argos, J. Protein Chem. 16:453-457, 1997), DAS (Cserzo et al., Protein Eng. 10:673-676, 1997), HMMTOP (Tusnady and Simon, J. Mol. Biol. 283:489-506, 1998), SOSUI (Hirokawa et al., Bioinformatics). 14:378-379, 1998)、PHD(Rost et al., Int. Conf. Intell. Syst. Mol. Biol. AAAI Press, St. Louis, USA, pp.192-200, 1996)、TMpred(Hofmann and Stoffel, Biol. Chem. Horse-Seyler 374:166, 1996). 1993), KKD (Klein et al., Biochim. Biophys. Acta. 815:468-476, 1985), ALOM2 (Nakai and Kanehisa, Genomics 14:489-911, 1992), and Topred2 (Claros and Heijne, Comput. Appl. Biosci.10:685-686, 1994), all of which can be used to predict TM regions in the methods of the present invention. All cited references are incorporated herein by reference.

[0086] The principles of TMHMM are described in Krogh et al., Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes. Journal of Molecular Biology, 305(3):567-580, January 2001 (incorporated by reference) and Sonnhammer et al., A hidden Markov model for predicting transmembrane helices in protein sequences In J. Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C. Sensen, editors, Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology, pages 175-182, Menlo Park, Calif., 1998, AAAI Press (incorporated by reference).

[0087] DAS (Dense Alignment Surface, Cserzo et al., “Prediction of transmembrane alpha-helices in procariotic membrane proteins: the Dense Alignment Surface method,” Prot. Eng. 10(6): 673-676, 1997, Stockholm University, Sweden) predicts transmembrane regions using the dense alignment surface method. DAS is based on a low stringency dot plot of a query sequence against a set of library sequences (non-homologous membrane proteins) using a special previously obtained score matrix. The method provides a highly accurate hydrophobicity profile of the query, from which the positions of potential transmembrane segments can be obtained. The novelty of the DAS-TMfilter algorithm is the second prediction cycle to predict TM segments in sequences of the TM library. To use the DAS server, users input a protein sequence at www.sbc.su.se / ~miklos / DAS / and the DA server predicts the TM regions of the input sequence.

[0088] HMMTOP (Hungarian Academy of Sciences, Budapest) is an automated server for predicting the topology of transmembrane helices and proteins using hidden Markov models developed by GE Tusnady at the Institute of Enzymology. The methods used by this prediction server are described in GE Tusnady and I. Simon (1998) "Principles Governing Amino Acid Composition of Integral Membrane Proteins: Applications to Topology Prediction," J. Mol. Biol. 283: 489-506 (incorporated by reference). New features of the HMMTOP 2.0 version are described in GE Tusnady and I. Simon (2001) "The HMMTOP transmembrane topology prediction server," Bioinformatics 17: 849-850 (incorporated by reference).

[0089] The MEMSAT2 transmembrane prediction page (www.sacs.ucsf.edu / cgi-bin / memsat.py) predicts transmembrane segments in proteins using FASTA or text format as input. The associated program, MEMSAT(1.5) software, is copyrighted by Dr. David Jones (Jones et al., Biochemistry 33:3038-3049, 1994). The latest version of MEMSTAT, MEMSAT V3, is a widely used all-helical membrane protein prediction method, MEMSAT. The method was benchmarked on a test set of transmembrane proteins of known topology. MEMSAT was estimated to be over 78% accurate in predicting the structure of all-helical transmembrane proteins and the location of their constituent helical elements in the membrane from sequence data. MEMSAT-SVM is a highly accurate predictor of transmembrane helix topology. It can distinguish signal peptides and identify cytoplasmic and extracellular loops. Both MEMSAT3 and MEMSAT-SVM are part of the PSIPRED protein sequence analysis workbench, which brings together several structure prediction methods at University College London.

[0090] The Phobius server (phobius.sbc.su.se) is for predicting transmembrane topology and signal peptides from FASTA formatted protein amino acid sequences. Phobius is described in Lukas et al., "A Combined Transmembrane Topology and Signal Peptide Prediction Method," Journal of Molecular Biology 338(5):1027-1036, 2004). PoyPhobius is described in Lukas et al., "An HMM posterior decoder for sequence feature prediction that includes homology information," Bioinformatics, 21 (Suppl 1):i251-i257, 2005. The Phobius web server is described in Lukas et al., "Advantages of combined transmembrane topology and signal peptide prediction--the Phobius web server," Nucleic Acids Res. 35:W429-32, 2007 (all technical content cited is incorporated by reference).

[0091] SOSUI is for distinguishing between membrane and soluble proteins along with the prediction of transmembrane helices. SOSUI predicts transmembrane regions using Hydrophobicity Analysis for Topology and the Probe Helix Method for Tertial Structure. The accuracy of protein classification is said to be as high as 99%, with the corresponding value for transmembrane helix prediction being about 97%. The SOSUI system is available by internet access at www.tuat.ac.jp / mitaku / sosui.

[0092] TMPred (European Molecular Biology Network, Swiss Node) predicts transmembrane regions and protein orientation in a query sequence. Specifically, the TMPred algorithm is based on a statistical analysis of TMbase (a database of naturally occurring transmembrane proteins). The prediction is made using a combination of several weight matrices for scoring. See Hofmann & Stoffel (1993) "TMbase - A database of membrane spanning proteins segments," Biol. Chem. Hoppe-Seyler, 374:166.

[0093] The SPLIT4.0 server is a membrane protein secondary structure prediction server (split.pmfst.hr / split / 4) that predicts transmembrane (TM) secondary structures of membrane proteins in the SWISS-PROT format using a preference function method. See Juretic et al., "Basic charge clusters and predictions of membrane protein topology," J. Chem. Inf. Comput. Sci., 42:620-632, 2002 (incorporated by reference).

[0094] PRED-TMR predicts transmembrane domains in proteins simply using the protein sequence itself. The algorithm refines standard hydrophobicity analysis by detecting potential ends ("ends", beginning and end) of transmembrane regions. This allows discarding highly hydrophobic regions that are not bounded by a clear beginning and end configuration, and validating putative transmembrane segments that are not distinguishable by their hydrophobic composition. The accuracy obtained on a test set of 101 non-homologous transmembrane proteins with reliable topology compares well with that of other popular existing methods. Only a slight decrease in prediction accuracy was observed when the algorithm was applied to all transmembrane proteins in the SwissProt database (release 35). See Pasquier et al., "A novel method for predicting transmembrane segments in proteins based on a statistical analysis of the SwissProt database: the PRED-TMR algorithm," Protein Eng., 12(5):381-385, 1999 (incorporated by reference).

[0095] In the related PRED-TMR2, its application is extended by a preprocessing step represented by an artificial neural network that is able to distinguish transmembrane proteins from soluble or fibrous proteins with high accuracy. When applied to several test sets of transmembrane proteins, the system gives a perfect prediction score of 100% by classifying all sequences within the transmembrane class. When applied to 995 non-transmembrane proteins extracted from the PDBselect database, the neural network erroneously predicts 23 of them as transmembrane (97.7% correct assignment). See Pasquier and Hamodrakas, "An hierarchical artificial neural network system for the classification of transmembrane proteins," Protein Eng., 12(8):631-634, 1999 (incorporated by reference).

[0096] Protein α-helix secondary structure prediction Certain methods of the invention include predicting the alpha-helix secondary structure of proteins, such as GPCRs. Many such programs and software are known in the art, any of which may be used individually or in combination in the methods of the invention that require an alpha-helix secondary structure prediction step. All such programs may be used in the methods of the invention.

[0097] Early methods of secondary structure prediction were restricted to predicting three predominant states: helix, sheet or random coil. These methods were based on the helix- or sheet-forming tendencies of individual amino acids sometimes coupled with rules to estimate the free energy of forming secondary structure elements. Such methods were typically about 60% accurate in predicting which of the three states (helix / sheet / coil) a residue would adopt. The first widely used technique for predicting protein secondary structure from amino acid sequences was the Chou-Fasman method.

[0098] A significant increase in accuracy (almost to about 80%) is made by utilizing the information provided by multiple sequence alignments; knowing the complete distribution of amino acids occurring at a position (and nearby, typically about 7 residues on either side) throughout evolution gives a very good picture of the structural trends near that position. For example, a given protein may have a glycine at a given position, which in itself may suggest a random coil. However, multiple sequence alignments may reveal that a helix-preferring amino acid occurs at that position (and nearby positions) in 95% of homologous proteins throughout evolution. Furthermore, by examining the average hydrophobicity at that position and nearby positions, the same alignment may also suggest a residue solvent accessibility pattern consistent with an alpha helix. Taken together, these factors may suggest that the glycine in the original protein adopts an alpha helix structure rather than a random coil. Thus, in the methods of the present invention, alpha helix secondary structure prediction programs, including neural networks, hidden Markov models, and support vector machines, may combine all available data to make predictions of the three states. Such prediction methods also provide confidence scores for their predictions at all positions.

[0099] Benchmarking is continuously performed on secondary structure prediction methods (e.g., EVA (Benchmark)). EVA runs a continuous benchmarking project to evaluate the quality of protein structure prediction and secondary structure prediction methods. Methods that predict both secondary and tertiary structure, such as homology modeling, protein threading and contact order prediction, are compared with results from newly solved protein structures every week that are deposited in the Protein Data Bank (PDB). The project aims to determine the prediction accuracy that can be expected for non-expert users of a common publicly available prediction web server.

[0100] According to these tests, the most accurate methods at present are Psipred, SAM (Karplus, "SAM-T08, HMM-based protein structure prediction," Nucleic Acids Res. (2009) 37 (Web Server issue): W492-497. doi:10.1093 / nar / gkp403), PORTER (Pollastri & McLysaght, "Porter: a new, accurate server for protein secondary structure prediction," Bioinformatics 21 (8):1719-1720, 2005), and PROF (Yachdav et al. (2014). "PredictProtein--an open resource for online prediction of protein structural and functional features," Nucleic Acids Res. 42 (Web Server issue): W337-343. doi:10.1093 / nar / gku366) and SABLE (Adamczak et al. (2005) "Combining prediction of secondary structure and solvent accessibility in proteins," Proteins 59 (3): 467-475. doi:10.1002 / prot.20441).Also, a standard method for assigning secondary structure classes (helix / strand / coil) to PDB structures is DSSP (Kabsch W and Sander (1983) "Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features," Biopolymers 22 (12): 2577-2637. doi:10.1002 / bip.360221211), against which predictions are benchmarked. All are incorporated by reference and all may be used in the methods of the invention.

[0101] The DSSP algorithm is a standard method for assigning secondary structure to the amino acids of a protein given the atomic resolution coordinates of the protein. DSSP starts by identifying the intrabackbone hydrogen bonds of the protein using a simple electrostatic definition, assuming partial charges of -0.42e and +0.20e for the carbonyl oxygen and amide hydrogen, respectively, and assigning their inverses to the carbonyl carbon and amide nitrogen. Hydrogen bonds are identified if E in the following equation is less than -0.5 kcal / mol:

number

[0102] Based on this, eight different secondary structures can be assigned. 3 10 Helices, α-helices and π-helices, are identified by their repeated sequences of hydrogen bonds separated by 3, 4 or 5 residues, respectively, with the symbols G, H and I. Two types of β-sheet structures exist, with β-bridges having the symbol B and longer sets of hydrogen bonds and β-bulges having the symbol E. T is used for turns characterized by hydrogen bonds typical of helices, and S for regions of high curvature. (where:

number

number

[0103] PSIPRED (Secondary Structure Prediction by PSI-BLAST) is a technique used to investigate protein structures. It uses neural networks, i.e. machine learning methods, in its algorithm. It is a server-side program that features a website serving as a front-end interface, and is able to predict the secondary structure of proteins (beta-sheets, alpha-helices and coils) from the primary sequence. See bioinf.cs.ucl.ac.uk / psipred. The idea of ​​this method is a machine learning method that uses information on evolutionarily related proteins to predict the secondary structure of a new amino acid sequence. Specifically, PSI-BLAST is used to find related sequences and build a position-specific score matrix. This matrix is ​​processed by a neural network that has been built and trained to predict the secondary structure of the input sequence. This prediction method or algorithm is divided into three stages: creating a sequence profile, predicting the initial secondary structure and filtering the predicted structure. PSIPRED works to normalize the sequence profile created by PSI-BLAST. It then predicts the initial secondary structure by using neural networking. For each amino acid in the sequence, the neural network is given a window of 15 acids. Further information is attached indicating whether these windows span the N- or C-terminus of the chain. This results in a final input layer of 315 input units divided into 15 groups of 21 units. The network has a single hidden layer of 75 units and three output nodes (one for each secondary structure element, i.e., helix, sheet, coil). A second neural network is used to filter the predicted structures of the first network. A window of 15 positions is given to this network. An indication of the possible positions of this window at the chain end is also transferred. This results in 60 input units divided into 15 groups of 4. The network has a single hidden layer of 60 units and results in three output nodes (one for each secondary structure element, i.e., helix, sheet, coil).Three final output nodes deliver the score of each secondary structure element for the center position of this window. The secondary structure with the highest score is used by PSIPRED to generate the protein prediction. The Q3 value is the percentage of correctly predicted residues in the secondary structure states, helices, strands and coils.

[0104] Step-by-step description of an exemplary embodiment With the invention generally described above, certain non-limiting, exemplary embodiments are described below with reference to representative flow charts in the figures.

[0105] Figure 9A illustrates one non-limiting embodiment of the present invention, which generally depicts a method 200 of the present invention in which selected hydrophobic amino acids L, I, V and F within the TM region of a protein of interest (e.g., a GPCR) are substituted according to the "QTY Code" of the present invention, without limiting the substitution to any particular TM region / domain.

[0106] In a specific embodiment thereof, the method starts by obtaining or reading (204) an input of a protein sequence, which may or may not be a transmembrane protein (202). The protein sequence can then be subjected to TM region prediction (206) (if such information is not already available from the input protein sequence) and alpha helix secondary structure prediction based on any of the art-recognized methods. For example, a program such as the TMHMM program can be used to perform the TM region prediction (240). If this prediction does not yield any TM regions in 242, one or more different TM region prediction programs, such as SOSUI, may be used (250) to allow prediction of the presence / absence of TM regions. If no TM regions are predicted based on such programs in 252, then there may be no TM regions in the protein (254) and the method ends (260).

[0107] On the other hand, if one or more TM regions are predicted by any of the suitable programs in 242, a TM region protein sequence is obtained (244), and the QTY codes of the present invention can be applied to the hydrophobic amino acids L, I, V, and F in such one or more TM regions. More specifically, according to the QTY codes, leucines in the TM region can be independently substituted with glutamine (Q), serine (S), or asparagine (N) (212) or left unsubstituted, isoleucines and valines in the TM region can be independently substituted with threonine (T), serine (S), or asparagine (N) or left unsubstituted, and phenylalanines in the TM region can be independently substituted with tyrosine (Y) or left unsubstituted. Such QTY substitutions result in the production of one or more putatively water-soluble variants of the original transmembrane protein. Note that the number of substitutions made for each amino acid in a region can be selected as a parameter.

[0108] The α-helical secondary structure of each of the putatively water-soluble variants can then be predicted using any art-recognized program, such as PORTER (210). The results can then be compared with those of the original protein, which is preferably predicted using the same program (e.g., PORTER) (208). Note that the α-helical secondary structure of the original protein can be predicted using any art-recognized program before, simultaneously with, or after the TM region prediction step of the original protein.

[0109] If the α-helical secondary structure prediction results indicate that the candidate water-soluble variant maintains or largely maintains the same α-helical secondary structure as the original protein at 214, it suggests that the particular pattern of QTY substitutions in the variant does not affect or does not significantly affect the α-helical secondary structure in the original protein. A prediction of this TM region can then be made (220), confirmed (222), and variant sequences generated (224). Optionally, if the results indicate that one or more of the α-helical secondary structures in the original protein are disrupted at 214, the variant can be discarded as undesirable at this step, thus terminating the method.

[0110] On the other hand, the method of the present invention also requires that the predicted QTY variants show a lower or no tendency to form TM regions compared to the original protein. Thus, the putatively water-soluble variants can be subjected to TM region prediction (if necessary), such as using the same TM region prediction program used for the initial TM region prediction in the original protein. If the results show that significant TM regions are still present, the variants may be discarded. On the other hand, if the results show that there are no TM regions or that there is a lower tendency to form TM regions, the variants can be selected as desirable variants that have a higher water solubility than the original protein but maintain an α-helical secondary structure, and thus likely maintain the function of the original protein.

[0111] If desired, further steps can be taken to further characterize the resulting water-soluble variants. Such further characterization may include calculating the pI of the variant (226) and comparing this to the pI of the original protein. This pI should not change or should only change very slightly (i.e., less than 30%, or preferably less than 20%, or more preferably less than 10%). Another further characterization may include creating a helical wheel model (246), such as that shown in FIG. 3, to show the location and any clustering of QTY substitutions in any particular TM region.

[0112] Another exemplary embodiment of the invention for designing transmembrane regions of proteins (e.g., GPCRs) according to the QTY codes of the invention can be performed on a computer system using the representative method 10 depicted in Figure 9B, some of the detailed steps of which are further described below, many of which are optional or can be combined according to the methods of the invention.

[0113] 1: In step 1, a computer interface of a computer system receives a protein sequence, selects for analysis, and uploads or inputs data describing the protein (e.g., its sequence) via the computer interface of the computer system (12). The input data may be a protein name, a database reference, or a protein sequence. For example, the protein sequence may be uploaded via the computer interface.

[0114] 2: In step 2, further data regarding the protein, including its name or sequence, can be identified, determined, obtained and / or entered, and can be entered via a computer interface. One source for obtaining protein data (20) is a database named UniProt (www.uniprot.org). Alternatively, the method of the invention can store data regarding the protein or sequences associated with the protein for later retrieval by the user in this step. In an embodiment, the program can prompt the user to select a database or file to retrieve further data (e.g., sequence data) regarding the protein selected for analysis.

[0115] 3: In step 3, the user can input, upload, or obtain data identifying transmembrane regions. For example, the user can be prompted to obtain data from a public source such as UniProt. That information can be verified (30) and gleaned from that database for use in step 5.

[0116] 4: Alternatively or additionally, transmembrane regions can be established by any art-recognized method even if TM region information is not readily available from the input protein sequence (40). Transmembrane regions are generally characterized by an α-helical conformation. For example, a software module / package named TMHMM2.0 (Transmembrane Prediction Using Hidden Markov Models) developed by the Center for Biological Sequences (www.cbs.dtu.dk / services / TMHMM) can be used to perform transmembrane helix prediction. This software version may have problems with peak searching and may fail to find the seven TM regions for GPCRs. Therefore, a modified version of this program in which a dynamic baseline is introduced into the peak searching method performed by the computer system may be used as needed. Here, for example, for GPCRs, if all seven TM regions are not found using the initial baseline value, the baseline can be changed to a lower value. For example, the default baseline may be set to 0.2. To identify the missing seventh transmembrane region, the baseline value can be set to 0.1. If more than eight TM regions are found, the baseline can be changed to a higher value, such as 0.15, to filter out off-point TM predictions. For example, when the amino acid sequence of CCR-2 was subjected to the TMHMM2.0 software, only six transmembrane regions were initially identified. However, when the baseline value of TMHMM2.0 was set to 0.07, a total of seven correct transmembrane regions were identified. The results of the TM region predictions are then fed into step 5.

[0117] 5: In step 5, after identifying TM data, either by de novo prediction or by obtaining such information from the initial sequence input, the sequence of the GPCR is divided into a total of 15 fragments according to the TM region information, i.e., seven transmembrane segments (seven TMs) (52) and eight non-transmembrane segments (eight NTMs) (54) (50). That is, there should be seven TM and eight NTM fragments for each typical GPCR.

[0118] Of course, the system may perform one or more, such as all, of the above steps using a computer interface for input by a user, and of course, the system may omit one or more of the above steps or combine two or more of the steps.

[0119] 6: In step 6, partial QTY substitutions are made for a selected subset of hydrophobic amino acids L, I, V, and F in a given TM region of the protein (60). Specifically, a first transmembrane region (typically, but not necessarily, the transmembrane region most proximal to the N-terminus of the protein) is first selected for mutation. Some or all of the hydrophobic amino acids (L, I, V, and F) in the first transmembrane region are then replaced with corresponding non-ionic hydrophilic amino acids (Q / S / N, T / S / N, T / S / N, or Y). Of course, in this case, the amino acids are not actually replaced in the protein. Rather, the amino acid designations are replaced in the sequence for modeling. Thus, the term "sequence" is intended to include "sequence data." Typically, most or all of the hydrophobic amino acids are selected for substitution. If less than all amino acids are selected, it may be desirable to select internal hydrophobic amino acids while leaving one or more N- and / or C-terminal amino acids of the transmembrane region hydrophobic. Additionally or alternatively, it may be desirable to select to replace all of the leucines (L) in the transmembrane region. Additionally or alternatively, it may be desirable to select to replace all of the isoleucines (I) in the transmembrane region. Additionally or alternatively, it may be desirable to select to replace all of the valines (V) in the transmembrane region. Additionally or alternatively, it may be desirable to select to replace all of the phenylalanines (F) in the transmembrane region. Additionally or alternatively, it may be advantageous to retain one or more phenylalanines in the transmembrane region. Additionally or alternatively, it may be advantageous to retain one or more valines in the transmembrane region. Additionally or alternatively, it may be advantageous to retain one or more leucines in the transmembrane region. Additionally or alternatively, it may be advantageous to retain one or more isoleucines in the transmembrane region. Additionally or alternatively, it may be advantageous to retain one or more hydrophobic amino acids in a transmembrane region whose wild type sequence is characterized by three or more consecutive hydrophobic amino acids.

[0120] 7: In step 7, the so designed transmembrane region is placed back into the sequence context of the original protein: since each set of substitutions generates one specific putative mutant for that TM region, the mutated or redesigned TM region with QTY substitutions (62) is swapped with the corresponding TM region of the original protein to generate transmembrane mutants or "putative mutants" (70). Together, these related putative mutants form a first library of putative mutants.

[0121] 8: Then, in steps 82 and 84, each putative variant is subjected to the transmembrane domain prediction method (84) described herein (e.g., loss of predicted TM domains). The variants are also evaluated for a score of the tendency of the sequence to form an alpha helix (82). The variants are also subjected to the water solubility prediction method described herein. For example, the variants are evaluated for a score of the tendency of the sequence to be water soluble. Such a score may be based on the predicted tendency to form TM domains, where a strong tendency to form TM domains is associated with low water solubility, and low or no tendency to form TM domains is associated with high water solubility. Of course, complete water solubility at all concentrations is not necessary for most commercial purposes. Water solubility is preferably determined to be that required for functionality in the predicted conditions of use (e.g., ligand binding assays).

[0122] 9: In step 9, putative variants that predict loss of α-helical structure and / or "water insolubility" (predicted under expected conditions of use) are discarded. For example, a combined score or rank (90), which is a weighted combination based on a ranking function of the α-helical secondary structure prediction results and the TM region / water solubility prediction results, can be used to select putative variants that predict α-helical structure and water solubility. For example, if a prediction can be made that the α-helical structure may be compromised, transmembrane variants that are highly water soluble or feature 0, 1, 2 or 3 hydrophobic amino acids (e.g., higher weight for water solubility prediction results) can be selected. Alternatively or additionally, highly α-helical structures that feature 3, 4, 5 or 6 hydrophobic amino acids (e.g., higher weight for α-helical secondary structure prediction results) can be selected.

[0123] 10: In step 10, the putative variants in the same library can be sorted or ranked (100) based on the score calculation scheme (94) outlined above. A predetermined number of putative variants can then be selected as the final members of the first putative variant library. For example, in the above combination score, a score of 0 means no tendency to form TM regions and perfect maintenance of the original α-helical secondary structure, and thus the most desirable putative variant. A slightly higher score may indicate a slight tendency to form TM regions (or a low tendency to be water-soluble). Thus, this putative variant is less desirable, but can still be selected based on its superior combination score compared to other putative variants in the library.

[0124] In certain embodiments, a predetermined number of desired putative mutants may be selected, such as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1.

[0125] These steps (e.g., steps 6-10) can be repeated for the second, third, fourth, fifth, sixth and / or seventh (or more) transmembrane regions or domains to generate a library of putative mutants, one for each such TM region or domain.

[0126] 11: In step 11, combinations of TM regions or domains with putative mutants and non-substituted non-TM regions can be selected (110). For example, one, two, three or four domains containing putative mutants with high α-helical structure scores can be combined with one, two, three, four, five or six domains containing putative mutants with high water solubility scores. In another example, in the selection of multiple mutants, a domain / TM region characterized by replacing all hydrophobic amino acids with hydrophilic amino acids, thus maximizing its water solubility score, can be combined with a second domain / TM region that retains three, four or five hydrophobic amino acids. As known in the art, such selected putative mutants can be "recombined" with extracellular and intracellular domains to generate an initial combinatorial library of putatively water soluble protein mutants.

[0127] In certain embodiments, all or a fragment of the putatively water-soluble protein variants of the initial combinatorial library designed as described herein can be prepared (produced or expressed in vitro or in a host cell) and screened for water solubility and / or ligand binding, preferably in a high-throughput screen. For example, the library can be amplified to express less than 100% of the putatively water-soluble protein variants. Reporter systems can be used to screen for ligand binding, as is well known in the art. The methods of the present invention can be used to rapidly identify libraries of putatively water-soluble modified transmembrane combinatorial variants that contain functionally combined extracellular and intracellular domains to produce water-soluble protein variants that have the proper three-dimensional structure of the wild-type protein and retain ligand binding function (including binding affinity) or other functions. The software can include a learning module that uses the confirmed functionality of the protein variants to exclude certain variants or rank them differently.

[0128] In certain embodiments, to be experimentally practical, the initial combinatorial library has about 2 million potentially water-soluble GPCR or CXCR4 variants. Of course, libraries with more or less variants can also be designed. In certain embodiments, smaller libraries may be preferred, since they can be optimized based on the analysis of the research results described herein. The analysis of the research results will likely establish the trend of optimizing the number of domain variants to be recombined and the prediction for selecting domain variants.

[0129] In certain embodiments, specific hydrophobic amino acids within the TM regions of transmembrane proteins are selected for modification based on their helix-forming propensity, also known as the "helix prediction score" (see www.proteopedia.org / wiki / index.php / Main_Page). Various fragments are randomly combined to generate approximately 2 million (8 7 ) mutants. The number of mutants expected is generally calculated using the formula H n where n=the number of transmembrane regions modified and / or altered by the method (in the example of a GPCR, n=7) and H=the number of putative mutants in each transmembrane region available to generate combinatorial mutants.

[0130] Once the initial combinatorial library, or group of domain variants to be recombined, is selected, nucleic acid molecules, or DNA or cDNA molecules, encoding the proteins in the initial combinatorial library can be designed. These nucleic acid molecules are preferably designed to be codon optimized and intron deleted for the expression system selected to generate the library of coding sequences. For example, if the expression system is E. coli, codons optimized for E. coli expression can be selected. See www.dna20.com / resources / genedesigner. Also, a promoter region, such as a promoter suitable for expression in the expression system (e.g., E. coli), is selected and operably linked to the coding sequences in the library of coding sequences.

[0131] The initial library of coding sequences, or portions thereof, are then expressed to generate a library of putatively water-soluble GPCRs. This library is then subjected to a ligand binding assay in which the putatively water-soluble GPCRs are contacted with a ligand, preferably in an aqueous medium, and ligand binding is detected.

[0132] The present invention includes transmembrane domain variants and nucleic acid molecules encoding same which are obtained or obtainable from the methods described herein.

[0133] The present invention also contemplates water-soluble GPCR mutants ("sGPCRs") characterized by multiple transmembrane domains that are independently characterized by at least 50%, preferably at least about 60%, more preferably at least about 70% or 80%, e.g., at least about 90% of the hydrophobic amino acid residues (L, I, V, and F) of the native transmembrane protein (e.g., GPCR) being replaced by Q, T, T, or Y, respectively. The sGPCRs of the present invention are characterized by water solubility and ligand binding. In particular, the sGPCRs bind the same native ligands as the corresponding native GPCRs.

[0134] The present invention further encompasses methods of treating disorders and diseases mediated by the activity of a membrane protein comprising the use of a water-soluble polypeptide to treat said disorders or diseases, wherein the water-soluble polypeptide comprises a modified α-helical domain, and wherein the water-soluble polypeptide retains the ligand binding activity of its native membrane protein. Examples of such disorders and diseases include, but are not limited to, cancer, small cell lung cancer, melanoma, breast cancer, Parkinson's disease, cardiovascular disease, hypertension, and asthma.

[0135] As described herein, the water-soluble peptides described herein can be used to treat illnesses or diseases mediated by the activity of membrane proteins. In certain embodiments, the water-soluble peptides can act as "decoys" for membrane receptors and bind to ligands that would otherwise activate the membrane receptors. Thus, the water-soluble peptides described herein can be used to reduce the activity of membrane proteins. These water-soluble peptides can remain in the circulation and competitively bind to specific ligands, thereby reducing the activity of membrane-bound receptors. For example, the GPCR CXCR4 is overexpressed in small cell lung cancer and promotes metastasis of tumor cells. Binding to this ligand by water-soluble peptides as described herein can significantly reduce metastasis.

[0136] The chemokine receptor CXCR4 is known in virology as the major co-receptor for the entry of T cell line-tropic HIV (Feng et al. (1996) Science 272: 872-877; Davis et al. (1997) J Exp Med 186: 1793-1798; Zaitseva et al. (1997) Nat Med 3: 1369-1375; Sanchez et al. (1997) J Biol Chem 272: 27529-27531). Stromal cell-derived factor 1 (SDF-1) is a chemokine that specifically interacts with CXCR4. When SDF-1 binds to CXCR4, CXCR4 activates downstream kinase pathways such as Ras / MAP kinase and phosphatidylinositol 3-kinase (PI3K) / Akt in lymphocytes, megakaryocytic and hematopoietic stem cells (Bleul et al. (1996) Nature 382: 829-833; Deng et al. (1997) Nature 388: 296-300; Kijowski et al. (2001) Stem Cells 19: 453-466; Majka et al. (2001) Folia. Histochem. Cytobiol. 39: 235-244; Sotsios et al. (1999) J. Immunol. 163: 5954-5963; Vlahakis et al. (2002) J. Immunol. 169: 5964-5966). SDF-1 activates Gαi protein-mediated signaling (pertussis toxin-sensitive) including Gαi protein-mediated signaling (Gαi protein-mediated signaling, including Gαi protein-mediated signaling, ...

[0137] Recent studies have shown that CXCR4 interaction can regulate metastatic cell migration. Hypoxia, or reduced oxygen tension, is a microenvironmental change that occurs in most solid tumors and is a major inducer of tumor angiogenesis and therapeutic resistance. Hypoxia increases CXCR4 levels (Staller et al. (2003) Nature 425: 307-311). Microarray analysis of a subpopulation of cells from a bone metastasis model with increased metastatic activity showed that CXCR4 was one of the genes increased in the metastatic phenotype. Furthermore, overexpression of CXCR4 in isolated cells significantly increased metastatic activity (Kang et al. (2003) Cancer Cell 3: 537-549). In samples from various breast cancer patients, Muller et al. (Muller et al. (2001) Nature 410: 50-56) found that CXCR4 expression levels were higher in primary tumors versus normal mammary gland or epithelial cells. Furthermore, CXCR4 antibody treatment was found to inhibit metastasis to regional lymph nodes compared to control isotypes, all of which metastasized to lymph nodes and lungs (Muller et al. (2001)). Thus, the decoy therapy model is suitable for treating CXCR4-mediated diseases and disorders.

[0138] In another embodiment, the invention relates to the treatment of a disease or disorder associated with CXCR4-dependent chemotaxis involving abnormal leukocyte recruitment or activation, the disease being selected from the group consisting of arthritis, psoriasis, multiple sclerosis, ulcerative colitis, Crohn's disease, allergy, asthma, AIDS-associated encephalitis, AIDS-associated maculopapular rash, AIDS-associated interstitial pneumonia, AIDS-associated enteropathy, AIDS-associated periportal pneumonia and AIDS-associated glomerulonephritis.

[0139] In another aspect, the invention relates to the treatment of a disease or disorder selected from arthritis, lymphoma, non-small cell lung cancer, lung cancer, breast cancer, prostate cancer, multiple sclerosis, central nervous system developmental disorders, dementia, Parkinson's disease, Alzheimer's disease, tumors, fibromas, astrocytoma, myeloma, glioblastoma, inflammatory diseases, organ transplant rejection, AIDS, HIV infection or angiogenesis.

[0140] The present invention also includes a pharmaceutical composition comprising the water-soluble polypeptide and a pharma- ceutically acceptable carrier or diluent.

[0141] The composition may also include pharma- ceutically acceptable non-toxic carriers or diluents, defined as excipients commonly used to formulate pharmaceutical compositions for administration to animals or humans, depending on the desired formulation. The diluents are selected so as not to affect the biological activity of the drug or pharmacological composition. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solution, dextrose solution, and Hank's solution. The pharmaceutical composition or formulation may also include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, and the like. The pharmaceutical composition may also include large, slowly metabolized macromolecules, such as proteins, polysaccharides such as chitosan, polylactic acid, polyglycolic acid, and copolymers (e.g., latex functionalized SEPHAROSE™, agarose, cellulose, and the like), polymerized amino acids, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes).

[0142] The composition can be administered parenterally, for example, by intravenous, intramuscular, intrathecal or subcutaneous injection. Parenteral administration can be achieved by incorporating the composition into a solution or suspension. Such a solution or suspension may include a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents. Parenteral formulations may also include antibacterial agents, for example, benzyl alcohol or methyl parabens, antioxidants, for example, ascorbic acid or sodium bisulfite, and chelating agents, for example, EDTA. Buffers, for example, acetates, citrates or phosphates, and tonicity adjusters, for example, sodium chloride or dextrose, may also be added. The parenteral formulations can be enclosed in glass or plastic ampoules, disposable syringes or multiple dose vials.

[0143] In addition, auxiliary substances such as wetting agents, emulsifiers, surfactants, pH buffer substances, etc. may be present in the composition.Other components of pharmaceutical compositions are oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil and mineral oil.In general, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, especially for injection solutions.

[0144] Injectable preparations can be prepared either as liquid solutions or suspensions, or as solids suitable for dissolving in liquid excipients before injection, i.e., solution or suspension.These preparations can also be emulsified or encapsulated in liposomes or microparticles such as polylactides, polyglycolides or copolymers to enhance adjuvant effects as described above (Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997).The compositions and medicaments described herein can be administered in the form of depot injections or implant preparations, which can be formulated to allow sustained or pulsed release of active ingredients.

[0145] Transdermal administration includes the transdermal absorption of the composition through the skin. Transdermal formulations include patches, ointments, creams, gels, salves, etc. Transdermal delivery can be achieved using skin patches or transferosomes. See Paul et al., Eur. J. Immunol. 25: 3521-24, 1995 and Cevc et al., Biochem. Biophys. Acta 1368: 201-15, 1998.

[0146] "Treating" or "treatment" includes preventing or delaying the onset of symptoms, complications or biochemical manifestations of a disease, alleviating or ameliorating the symptoms, or arresting or inhibiting further progression of a disease, condition or disorder. A "patient" is a human subject in need of treatment.

[0147] An "effective amount" refers to an amount of a therapeutic agent sufficient to ameliorate one or more symptoms of a disease and / or prevent the progression of a disease, cause remission of a disease, and / or achieve a desired effect.

[0148] Computer Systems Various aspects and functions described herein may be implemented as dedicated hardware or software components executing on one or more computer systems. There are many examples of computer systems in use today. These examples include, among others, net appliances, personal computers, workstations, mainframes, networked clients, servers, media servers, application servers, database servers and web servers. Other examples of computer systems may include mobile computing devices such as mobile phones and personal digital assistants, network equipment such as load balancers, routers and switches. Furthermore, aspects may be located on a single computer system or may be distributed among multiple computer systems connected by one or more communication networks.

[0149] For example, various aspects, functions, and methods may be distributed among one or more computer systems configured to provide services to one or more client computers or perform tasks overall as part of a distributed system. Additionally, aspects may be performed on a client-server or multi-tier system with components distributed among one or more server systems performing various functions. Thus, the embodiments are not limited to execution on any particular system or group of systems. Furthermore, the aspects, functions, and methods may be implemented in software, hardware, or firmware, or any combination thereof. Thus, the aspects, functions, and methods may be implemented in methods, operations, systems, system elements, and components using a variety of hardware and software configurations, and these examples are not limited to any particular distributed architecture, network, or communication protocol.

[0150] Referring to FIG. 10, a block diagram of a distributed computer system 300 in which various aspects and functions are implemented is shown. As shown, the distributed computer system 300 comprises one or more computer systems that exchange information. More specifically, the distributed computer system 300 comprises computer systems 302, 304, and 306. As shown, the computer systems 302, 304, and 306 are interconnected via a communication network 308 and may exchange data via the communication network 308. The network 308 may comprise any communication network through which the computer systems may exchange data. To exchange data using the network 308, the computer systems 302, 304, and 306 and the network 308 may use various methods, protocols, and standards. Examples of these protocols and standards include NAS, Web, storage, and other data movement protocols suitable for use in big data environments. To ensure that data transfer is secure, the computer systems 302, 304, and 306 may transmit data over the network 308 using various security measures, such as, for example, SSL or VPN technology. Although distributed computer system 300 illustrates three networked computer systems, distributed computer system 300 is not so limited and may include any number of computer systems and computing devices networked using any medium and communication protocol.

[0151] As shown in FIG. 10, computer system 302 includes a processor 310, a memory 312, interconnection elements 314, an interface 316, and a data storage element 318. To implement at least some of the aspects, functions, and methods disclosed herein, processor 310 executes a sequence of instructions that result in processed data. Processor 310 may be any type of processor, multiprocessor, or control device. Examples of processors include commercially available processors such as Intel Xeon, Itanium, Core, Celeron, or Pentium processors, AMD Opteron processors, Apple A4 or A5 processors, Sun UltraSPARC processors, IBM Power5+ processors, IBM mainframe chips, or quantum computers. Processor 310 is connected to other system components, including one or more memory devices 312, by interconnection elements 314.

[0152] Memory 312 stores programs (e.g., sequences of instructions coded to be executable by processor 310) and data during operation of computer system 302. Memory 312 may thus be a relatively high performance volatile RAM, such as dynamic RAM ("DRAM") or static memory ("SRAM"). However, memory 312 may comprise any device for storing data, such as a disk drive or other non-volatile storage device. In accordance with various examples, memory 312 may be organized into individualized and possibly unique structures to perform the functions disclosed herein. These data structures may be sized and organized to store particular data values ​​and data types.

[0153] The components of computer system 302 are connected by interconnection elements, such as interconnection element 314. Interconnection element 314 may include any communication coupling between system components, such as one or more physical buses according to proprietary or standard computing bus technologies, such as IDE, SCSI, PCI, and InfiniBand. Interconnection element 314 allows communications, including instructions and data, to be exchanged between the system components of computer system 302.

[0154] The computer system 302 also includes one or more interface devices 316, such as input devices, output devices, and combination input / output devices. An interface device may receive input or provide output. More specifically, an output device may provide information for external presentation. An input device may receive information from an external source. Examples of interface devices include a keyboard, a mouse device, a trackball, a microphone, a touch screen, a printing device, a display screen, speakers, a network interface card, and the like. The interface devices enable the computer system 302 to exchange information and communicate with external entities, such as users and other systems.

[0155] The data storage element 318 comprises a computer readable and writable non-volatile or non-transitory data storage medium on which are stored instructions defining a program or other object executed by the processor 310. The data storage element 318 may also contain information recorded on or in the medium and processed by the processor 310 during execution of the program. More specifically, the information may be stored in one or more data structures specifically configured to conserve storage space or enhance data exchange performance. The instructions may be permanently stored as coded signals, and the instructions may cause the processor 310 to perform the functions described herein. The medium may be, for example, an optical disk, a magnetic disk, or a flash memory, among others. During operation, the processor 310 or some other controller causes data to be read from the non-volatile recording medium into another memory, such as the memory 312, which allows faster access to the information by the processor 310 than the storage medium included in the data storage element 318. However, the memory may be located in data storage element 318 or memory 312, with processor 310 processing the data in the memory and then copying the data to a storage medium associated with data storage element 318 after processing is complete. Various components may manage data movement between the storage medium and other memory elements, and these examples are not limited to any particular data management component. Additionally, these examples are not limited to any particular memory or data storage system.

[0156] Although computer system 302 is shown as one type of computer system that may implement various aspects and functions, for example, the aspects and functions are not limited to execution on computer system 302 as shown in FIG. 10. Various aspects and functions may be implemented on one or more computers having different architectures or components than those shown in FIG. 10. For example, computer system 302 may include specially programmed, special purpose hardware such as application specific integrated circuits ("ASICs") made to perform the specific operations disclosed herein. Meanwhile, another example may perform the same functions using a grid of several general purpose computing devices running MAC OS System X with Motorola PowerPC processors and several special purpose computing devices running proprietary hardware and operating systems.

[0157] Computer system 302 may be a computer system that includes an operating system that manages at least some of the hardware elements included in computer system 302. In some examples, a processor or controller, such as processor 310, executes an operating system. Examples of specific operating systems that may be executed include Windows-based operating systems, such as Windows NT, Windows 2000 (Windows ME), Windows XP, Windows Vista, or Windows 7 operating systems available from Microsoft Corporation, the MAC OS System X operating system or the iOS operating system available from Apple Computer, one of many Linux-based operating system distributions, such as the Enterprise Linux operating system available from Red Hat, Inc., the Solaris operating system available from Oracle, or a UNIX operating system available from a variety of sources. Many other operating systems may be used, and these examples are not limited to any particular operating system.

[0158] The processor 310 and the operating system together define a computer platform on which application programs are written in high-level programming languages. These component applications may be executable intermediate bytecodes, i.e. interpreted and executed code, that communicate over a communications network, e.g., the Internet, using a communications protocol, e.g., TCP / IP. Similarly, embodiments may be implemented using an object-oriented programming language, such as .Net, SmallTalk, Java, C++, Ada, C# (C Sharp), Python, or JavaScript. Other object-oriented programming languages ​​may also be used. Alternatively, functional, scripting, or logic programming languages ​​may be used.

[0159] Additionally, various aspects and functions may be implemented in non-programmed environments. For example, a document created in HTML, XML, or other format may provide the appearance of a graphical user interface or perform other functions when viewed in a browser program window. Additionally, various examples may be implemented as programmed or non-programmed elements or any combination thereof. For example, a web page may be implemented using HTML, but data objects required within the web page may be written in C++. Thus, the examples are not limited to a particular programming language, and any suitable programming language may be used. Thus, the functional components disclosed herein may include a wide variety of elements (e.g., dedicated hardware, executable code, data structures, or objects) configured to perform the functions described herein.

[0160] In some examples, components disclosed herein may read parameters that affect the functions performed by the components. These parameters may be physically stored in any form of suitable memory, including volatile memory (such as RAM) or non-volatile memory (such as a magnetic hard drive). The parameters may also be logically stored in proprietary data structures (such as a database or file defined by a user space application) or in commonly shared data structures (such as an application registry defined by an operating system). Some examples also provide both system and user interfaces that allow external entities to modify the parameters and thereby configure the operation of the components.

[0161] Software for performing the computational method is generally shown in FIG. 11A, where a user selects (402) operating parameters for running the procedure on a computer, as described herein above, where one or more sequences are input (404) and substitutions are made (408). The system is operable to confirm the secondary structure (408) and to confirm the water solubility of one or more variants. As shown in FIG. 11B, the program can include further processing options in addition to those described above, where one or more ranking functions can be stored (442) and the user can select the ranking function to use or the system can automatically select it (444). The system then generates a rank (446) as described herein, and the user can then produce (448) and measure the function (448) of the selected variants, and then input the functional data and modify the processing procedure based thereon (450).

[0162] The present invention will be better understood in connection with the following examples, which are intended as illustrations only and are not intended to limit the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art, and such changes can be made without departing from the spirit of the invention and the scope of the appended claims. EXAMPLES

[0163] Example 1: CXC chemokine receptor type 4 isoform a (CXCR4) CXCR4 is a chemokine receptor 356 amino acids in length. It has a pI of approximately 8.61 and a molecular weight of 40221.19 Da. The sequence of CXCR4 published in the literature is: [ka] It is.

[0164] This sequence is subjected to TMHMM to identify the transmembrane domains shown in FIG.

[0165] All or substantially all of the hydrophobic amino acids L, I, V and F are replaced with Q, T and Y (respectively) to give the following sequences: [ka]

[0166] The protein has a predicted pI of 8.54 and a molecular weight of 40551.64 Da. Each predicted transmembrane region is underlined, and a fully modified domain of the invention is illustrated. Thus, for example, the invention includes a transmembrane domain (TM1) comprising amino acids 47-70 of SEQ ID NO:2, and proteins comprising same. As an example, FIG. 3 depicts an alpha helix prediction of the TM1 sequence. Preferably, proteins comprising TM1 herein comprise one or more (e.g., all) of the extracellular and intracellular loop sequences (non-underlined sequences) of SEQ ID NO:2. Additionally or alternatively, proteins comprising TM1 herein comprise one or more further transmembrane regions (underlined sequences) within SEQ ID NO:2 or a homologous sequence that retains one, two, three or possibly four or more of the native L, I, V and F amino acids set forth in SEQ ID NO:1.

[0167] The native protein sequence of CXCR4 (which differs in the N-terminal amino acid) was again subjected to the method. The program output divided the native sequence into extracellular and intracellular regions and selected eight transmembrane domain mutants for each of the transmembrane domains. The results are shown in Figure 4 and in the table below. MEGISIYTSDNYTEEMGSGDYDSMKEPCFREENANFNK (SEQ ID NO:3; EC1) TM1 mutant: IFLPTTYSTTFQTGTTGNGQVTQVM (SEQ ID NO:4) IFQPTTYSTTFQTGTTGNGQVTQVM (SEQ ID NO:5) IFQPTTYSTTFQTGTTGNGQVTQTM (SEQ ID NO:6) IFQPTTYSTTYQTGTTGNGQVTQTM (SEQ ID NO:7) IFQPTTYSTTYQTGTTGNGQTTQVM (SEQ ID NO:8) IFQPTTYSTTYQTGTTGNGQTIQTM (SEQ ID NO: 9) IFQPTTYSTTYQTGTTGNGQTTQTM (SEQ ID NO: 10) TYQPTTYSTTYQTGTTGNGQTTQTM (SEQ ID NO:11) GYQKKLRSMTDKYR (SEQ ID NO: 12; IC1) TM2 mutant: LHLSTADQQFTTTQPFWAVDAV (SEQ ID NO:13) LHLSVADQQYTTTQPFWATDAV (SEQ ID NO:14) LHQSVADQQYVTTQPFWATDAT (SEQ ID NO:15) QHQSVADQQFTTTQPFWATDAT (SEQ ID NO:16) LHQSVADQQYTITQPYWATDAT (SEQ ID NO:17) QHLSVADQQYTITQPYWATDAT (SEQ ID NO:18) QHLSTADQQYVTTQPYWATDAT (SEQ ID NO:19) QHQSTADQQYTTTQPYWATDAT (SEQ ID NO:20) ANWYFGNFLCK (SEQ ID NO:21; EC2) TM3 Mutant: AVHVTYTVNQYSSVQIQAFT (SEQ ID NO:22) AVHTTYTVNQYSSVQIQAFT (SEQ ID NO:23) AVHTTYTVNQYSSVQTQAFT (SEQ ID NO:24) ATHTTYTVNQYSSVQTQAFT (SEQ ID NO:25) ATHTIYTTNQYSSVQTQAFT (SEQ ID NO:26) AVHTTYTTNQYSSVQTQAFT (SEQ ID NO:27) ATHTTYTTNQYSSVQTQAFT (SEQ ID NO:28) ATHTTYTTNQYSSTQTQAYT (SEQ ID NO:29) SLDRYLAIVHATNSQRPRKLLAEK (SEQ ID NO:30; IC2) TM4 mutant: VTYTGVWTPAQQQTIPDFIF (SEQ ID NO:31) TTYTGTWIPAQQQTIPDFIF (SEQ ID NO:32) TTYTGTWTPAQQQTIPDFIF (SEQ ID NO:33) TTYTGTWTPAQQQTIPDFIY (SEQ ID NO:34) TTYVGTWTPAQQQTTPDYIF (SEQ ID NO:35) TTYVGTWTPAQQQTTPDFIY (SEQ ID NO:36) TTYTGVWTPAQQQTTPDYTF (SEQ ID NO:37) TTYTGTWTPAQQQTTPDYTY (SEQ ID NO:38) ANVSEADDRYICDRFYPNDLW (SEQ ID NO:39; EC3) TM5 mutant: VVVFQFQHTMVGQTQPGTTTQ (SEQ ID NO:40) VVVFQFQHTMTGQTQPGTTTQ (SEQ ID NO:41) VVVFQYQHTMTGQTQPGTTTQ (SEQ ID NO:42) VVVYQYQHTMTGQTQPGTTTQ (SEQ ID NO:43) TVVFQYQHTMTGQTQPGTTTQ (SEQ ID NO:44) VVTFQYQHTMTGQTQPGTTTQ (SEQ ID NO:45) TVVYQYQHTMTGQTQPGTTTQ (SEQ ID NO:46) TTTYQYQHTMTGQTQPGTTTQ (SEQ ID NO:47) SCYCIIISKLSHSKGHQKRKALKTT (SEQ ID NO:48; IC3) TM6 mutant: VTQIQAFFACWQPYYTGTST (SEQ ID NO:49) VIQIQAYFACWQPYYTGTST (SEQ ID NO:50) VIQIQAYYACWQPYYTGTST (SEQ ID NO:51) VIQTQAFYACWQPYYTGTST (SEQ ID NO:52) VIQTQAYFACWQPYYTGTST (SEQ ID NO:53) VTQIQAFYACWQPYYTGTST (SEQ ID NO:54) VIQTQAYYACWQPYYTGTST (SEQ ID NO:55) TTQTQAYYACWQPYYTGTST (SEQ ID NO:56) DSFILLEIIKQGCEFENTVHK (SEQ ID NO:57; EC4) TM7 mutant WISITEAQAFFHCCLNPIQY (SEQ ID NO:58) WISITEAQAFYHCCLNPIQY (SEQ ID NO:59) WISITEAQAYFHCCQNPTLY (SEQ ID NO:60) WISTTEALAFYHCCQNPTQY (SEQ ID NO:61) WISTTEALAYFHCCQNPTQY (SEQ ID NO:62) WISITEALAYYHCCQNPTQY (SEQ ID NO:63) WISTTEALAYYHCCQNPTQY (SEQ ID NO:64) WTSTTEAQAYYHCCQNPTQY AFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS (SEQ ID NO: 65; IC4)

[0168] It will be apparent from the above that the sequences before, between and after each list of transmembrane domain mutants (SEQ ID NOs: 3, 12, 21, 30, 39, 48, 57 and 65) are the N', middle and C' extracellular and intracellular regions, respectively.

[0169] The sequences were then used to generate coding sequences suitable for expression in an expression system, in this case yeast, as known in the art, which were then recombinantly expressed to generate a library containing multiple proteins having SEQ ID NOs: 3, 12, 21, 30, 39, 48, 57, and 65, each of which contains one transmembrane domain mutant from each mutant list between its respective intracellular and extracellular domains.

[0170] The library so constructed was then assayed for the CXCR4 cognate ligand, i.e., SDF1a (or CCL12), on a plasmid expressed in yeast that binds in living yeast cells. Ligand binding was detected by gene activation with the yeast two-hybrid system, and the samples were then sequenced. Nineteen CXCR4 mutants were sequenced. The results are shown in Figure 5.

[0171] Example 2: CXC chemokine receptor type 3 isoform b (CX3CR1) CX3CR1 is a chemokine receptor 355 amino acids in length. It has a pI of approximately 6.74 and a molecular weight of 40396.4 Da. The sequence is subjected to TMHMM to identify its transmembrane domain. All or substantially all of the hydrophobic amino acids L, I, V and F in the transmembrane domain are substituted with Q, T and Y (respectively) to obtain the following sequence (lower line) aligned to the wild type (upper line): [ka]

[0172] The predicted pI of this protein variant is 6.74 and molecular weight is 41027.17 Da. Each predicted transmembrane region is underlined, and a fully modified domain of the invention is illustrated. Thus, for example, the invention includes a transmembrane domain that includes the underlined amino acids of SEQ ID NO:67. Preferably, a protein comprising TM1 herein includes one or more (e.g., all) of the extracellular and intracellular loop sequences (non-underlined sequences) of SEQ ID NO:66. Additionally or alternatively, a protein comprising TM1 herein includes one or more additional transmembrane regions (underlined sequences) within SEQ ID NO:67 or a homologous sequence that retains one, two, three, or possibly four or more of the native V, L, I, and F amino acids set forth in SEQ ID NO:66.

[0173] The native protein sequence of CX3CR1 was again subjected to the method. The program output divided the native sequence into extracellular and intracellular regions and selected eight transmembrane domain mutants for each of the transmembrane domains. The results are shown in the table below. MDQFPESVTENFEYDDLAEACYIGDIVVFGT (SEQ ID NO:68) TM1 mutant: TYQSTYYSTTFATGQVGNQQVVFALTNS (SEQ ID NO:69) TYQSTYYSTTYATGQVGNQQVVFALTNS (SEQ ID NO:70) TYQSTYYSTTYATGQVGNQQVVFAQTNS (SEQ ID NO:71) TYQSTYYSTTYATGQTGNLQVTFAQTNS (SEQ ID NO:72) TYQSTYYSTTYATGQTGNQLVTFAQTNS (SEQ ID NO:73) TYQSTYYSTTYATGQTGNQQVVFAQTNS (SEQ ID NO:74) TYQSTYYSTTYATGQTGNLQVTYAQTNS (SEQ ID NO:75) TYQSTYYSTTYATGQTGNQQTTYAQTNS (SEQ ID NO:76) KKPKSVTDIY (SEQ ID NO:77) TM2 mutant LLNQAQSDQLFVATQPFWTHY (SEQ ID NO:78) LLNQAQSDQQFVATQPFWTHY (SEQ ID NO:79) QQNLAQSDQQFVATQPFWTHY (SEQ ID NO:80) LQNLAQSDQQYTATQPFWTHY (SEQ ID NO:81) QLNLAQSDQQYTATQPFWTHY (SEQ ID NO:82) LLNQAQSDQQFTATQPYWTHY (SEQ ID NO:83) QQNLAQSDQQFTATQPYWTHY (SEQ ID NO:84) QQNQAQSDQQYTATQPYWTHY (SEQ ID NO:85) LINEKGLHNAMCK (SEQ ID NO:86) TM3 Mutant: YTTAYYYTGYYGSTYYTTTTST (SEQ ID NO:87) DRYLAIVLAANSMNNRT (SEQ ID NO:88) TM4 mutant: VQHGTTTSQGTWAAATQVAAPQFMF (SEQ ID NO:89) VQHGVTTSQGTWAAATQTAAPQFMF (SEQ ID NO:90) VQHGTTTSQGVWAAATQTAAPQFMY (SEQ ID NO:91) VQHGTTTSQGTWAAAIQTAAPQFMY (SEQ ID NO:92) VQHGTTTSQGTWAAATQTAAPQFMF (SEQ ID NO:93) VQHGTTISQGTWAAATQTAAPQYMF (SEQ ID NO:94) VQHGTTTSQGTWAAATQTAAPQFMY (SEQ ID NO:95) TQHGTTTSQGTWAAATQTAAPQYMY (SEQ ID NO:96) TKQKENECLGDYPEVLQEIWPVLRNVET (SEQ ID NO:97) TM5 mutant: NFLGFQQPQQIMSYCYFRIT (SEQ ID NO:98) NFQGFLQPQQTMSYCYFRIT (SEQ ID NO:99) NFQGFLQPQQTMSYCYFRTT (SEQ ID NO: 100) NFQGFQQPQQTMSYCYYRIT (SEQ ID NO: 101) NFQGFLQPQQTMSYCYYRTT (SEQ ID NO:102) NFQGYLQPQQTMSYCYFRTT (SEQ ID NO:103) NYQGFQQPQQTMSYCYFRTT (SEQ ID NO:104) NYQGYQQPQQTMSYCYYRTT (SEQ ID NO: 105) QTLFSCKNHKKAKAIK (SEQ ID NO:106) TM6 mutant: LIQQTTTTFYQFWTPYNTMTFQETL (SEQ ID NO: 107) LIQQTTTTFYQYWTPYNVMTFQETQ (SEQ ID NO: 108) LIQQTTTTYYQFWTPYNTMTFQETQ (SEQ ID NO: 109) QIQQTTTTFYQYWTPYNTMTFQETQ (SEQ ID NO: 110) LTQQTTTTYYQFWTPYNTMTFQETQ (SEQ ID NO:111) QIQQTTTTFFQYWTPYNTMTYQETQ (SEQ ID NO:112) QIQQTTTTFYQYWTPYNTMTYQETQ (SEQ ID NO:113) QTQQTTTTYYQYWTPYNTMTYQETQ (SEQ ID NO:114) KLYDFFPSCDMRKDLRL (SEQ ID NO:115) TM7 mutant: ALSVTETVAFSHCCQNPQIYAFAG (SEQ ID NO:116) AQSVTETTAFSHCCQNPLIYAFAG (SEQ ID NO:117) ALSVTETVAFSHCCQNPQTYAYAG (SEQ ID NO:118) AQSVTETTAFSHCCQNPQIYAYAG (SEQ ID NO:119) ALSVTETTAFSHCCQNPQTYAYAG (SEQ ID NO:120) ALSTTETTAYSHCCQNPQIYAFAG (SEQ ID NO:121) ALSVTETTAYSHCCQNPQTYAYAG (SEQ ID NO:122) AQSTTETTAYSHCCQNPQTYAYAG (SEQ ID NO:123) EKFRRYLYHLYGKCLAVLCGRSVHVDFSSSESQRSRHGSVLSSNFTYHTSDGDALLLL (SEQ ID NO: 124)

[0174] As in Example 1 above, the sequences before, between and after each list of transmembrane domain mutants are the N', middle and C' intracellular or extracellular regions, respectively.

[0175] The sequences were then used to generate coding sequences suitable for expression in an expression system, in this case yeast, as known in the art, which were then recombinantly expressed to generate a library containing multiple proteins having SEQ ID NOs: 68, 77, 86, 88, 97, 106 and 115, each of which contains one transmembrane domain mutant in each mutant list between its respective intracellular and extracellular domains.

[0176] The library so generated was then assayed for binding to the CX3CR1 cognate ligand (CXCL1) in aqueous medium as described in Example 1. Ligand binding was detected and the samples were then sequenced. Seven mutants were sequenced. The results are shown in Figure 6.

[0177] Example 3: CCR3 Mutants The method of Example 1 was repeated for chemokine receptor type 3 isoform 3. [Table 1]

[0178] All or substantially all of the hydrophobic amino acids L, I, V and F in the transmembrane domain are replaced with Q, T and Y (respectively) to give the following sequence (lower line) aligned to the wild type (upper line): [ka]

[0179] Each predicted transmembrane region is underlined, illustrating a fully modified domain of the invention. Thus, for example, the invention includes a transmembrane domain comprising the underlined amino acids of SEQ ID NO: 126. Preferably, a protein comprising TM1 herein comprises one or more (e.g., all) of the extracellular and intracellular loop sequences (non-underlined sequences) of SEQ ID NO: 126. Additionally or alternatively, a protein comprising TM1 herein comprises one or more additional transmembrane regions (underlined sequences) within SEQ ID NO: 126 or a homologous sequence that retains one, two, three, or possibly four or more of the native V, L, I, and F amino acids set forth in SEQ ID NO: 125.

[0180] The native protein sequence of CCR3 was again subjected to the method (note the differences in the N-terminal sequence). The program output divided the native sequence into extracellular and intracellular regions and selected eight transmembrane domain mutants for each of the transmembrane domains. The results are shown in the table below. MTTSLDTVETFGTTSYYDDVGLLCEKADTRALMA (SEQ ID NO: 127) TM1 mutant: QFVPPQYSQTFTTGQQGNVTVTMTQIKY (SEQ ID NO:128) QFVPPQYSQTFTTGQQGNTTVTMTQIKY (SEQ ID NO:129) QFVPPQYSQTYTTGQQGNTTVTMTQIKY (SEQ ID NO: 130) QFTPPQYSQTYTTGQQGNVTTTMTQIKY (SEQ ID NO:131) QFTPPQYSQTYTTGQQGNTVTTMTQIKY (SEQ ID NO: 132) QFTPPQYSQTYTTGQQGNTTVTMTQIKY (SEQ ID NO: 133) QFTPPQYSQTYTTGQQGNTTTTMTQIKY (SEQ ID NO: 134) QYTPPQYSQTYTTGQQGNTTTTMTQTKY (SEQ ID NO:135) RRLRIMTNIY (SEQ ID NO:136) TM2 mutant: LLNQATSDQQFQVTQPFWIHY (SEQ ID NO:137) LQNQAISDQLFQTTQPFWTHY (SEQ ID NO:138) QQNLAISDQQFQTTQPFWTHY (SEQ ID NO:139) QLNQAISDQQFQTTQPYWTHY (SEQ ID NO:140) QQNLAISDQQYQVTQPYWTHY (SEQ ID NO:141) LQNQATSDQLFQTTQPYWTHY (SEQ ID NO:142) QQNQAISDQQYQVTQPYWTHY (SEQ ID NO:143) QQNQATSDQQYQTTQPYWTHY (SEQ ID NO:144) VRGHNWVFGHGMCK (SEQ ID NO:145) TM3 Mutant: LQSGFYHTGQYSETFFTTQQTT (SEQ ID NO:146) QLSGFYHTGQYSETFFTTQQTT (SEQ ID NO:147) QLSGFYHTGQYSETFYTTQQTT (SEQ ID NO:148) QLSGFYHTGQYSETYFTTQQTT (SEQ ID NO:149) QLSGYYHTGQYSETFFTTQQTT (SEQ ID NO:150) QQSGFYHTGQYSETFFTTQQTT (SEQ ID NO:151) QQSGFYHTGQYSETFYTTQQTT (SEQ ID NO:152) QQSGYYHTGQYSETYYTTQQTT (SEQ ID NO:153) DRYLAIVHAVFALRART (SEQ ID NO:154) TM4 mutant: TTFGTTTSTVTWGQAVQAAQPEFIF (SEQ ID NO:155) TTFGTTTSTTTWGQAVQAAQPEFIF (SEQ ID NO:156) TTYGTTTSTTTWGQAVQAAQPEFIF (SEQ ID NO:157) TTYGTTTSTTTWGQAVQAAQPEFTF (SEQ ID NO:158) TTYGTTTSTTTWGQATQAAQPEFIF (SEQ ID NO:159) TTFGTTTSTTTWGQATQAAQPEFIY (SEQ ID NO:160) TTYGTTTSTTTWGQATQAAQPEFIY (SEQ ID NO:161) TTYGTTTSTTTWGQATQAAQPEYTY (SEQ ID NO:162) YETEELFEETLCSALYPEDTVYSWRHFHTLRM (SEQ ID NO:163) TM5 mutant: TIFCQVQPQQTMATCYTGTT (SEQ ID NO:164) TIFCQTQPQQVMATCYTGTT (SEQ ID NO:165) TIFCQTQPQQTMATCYTGIT (SEQ ID NO:166) TIFCQTQPQQTMATCYTGTI (SEQ ID NO: 167) TTFCQVQPQQVMATCYTGTT (SEQ ID NO:168) TIYCQVQPQQVMATCYTGTT (SEQ ID NO:169) TIFCQTQPQQTMATCYTGTT (SEQ ID NO:170) TTYCQTQPQQTMATCYTGTT (SEQ ID NO:171) KTLLRCPSKKKYKAIR (SEQ ID NO:172) TM6 mutant: QTYTTMATYYTYWTPYNTATQQSSY (SEQ ID NO:173) QSILFGNDCERSKHLDL (SEQ ID NO:174) TM7 mutant: VMQVTEVTAYSHCCMNPVTYAFTG (SEQ ID NO:175) VMQVTEVTAYSHCCMNPTTYAYVG (SEQ ID NO:176) VMLTTEVTAYSHCCMNPTTYAFTG (SEQ ID NO:177) VMQVTETTAYSHCCMNPVTYAYTG (SEQ ID NO:178) TMQVTETIAYSHCCMNPTTYAFTG (SEQ ID NO:179) TMQVTETTAYSHCCMNPTTYAFVG (SEQ ID NO: 180) VMQTTETIAYSHCCMNPTTYAYTG (SEQ ID NO:181) TMQTTETTAYSHCCMNPTTYAYTG (SEQ ID NO:182) ERFRKYLRHFFHRHLLMHLGRYIPFLPSEKLERTSSVSPSTAEPELSIVF (SEQ ID NO: 183)

[0181] As in Example 1 above, the sequences before, between and after each list of transmembrane domain mutants are the N', middle and C' intracellular or extracellular regions, respectively.

[0182] The sequences were then used to generate coding sequences suitable for expression in an expression system, in this case yeast, as known in the art, which were then recombinantly expressed to generate a library containing multiple proteins having SEQ ID NOs: 127, 136, 145, 154, 163, 172, 174, and 183, each of which contains one transmembrane domain mutant in each mutant list between its respective intracellular and extracellular domains.

[0183] The library so generated was then assayed for binding to the CCR3 cognate ligand, i.e., CCL3, in aqueous media as described in Example 1. Ligand binding was detected and the samples were then sequenced. Eleven mutants were sequenced. The results are shown in FIG. 7.

[0184] Example 4: CCR5 Mutants The method of Example 1 was repeated for chemokine receptor type 5 isoform 3. [Table 2]

[0185] All or substantially all of the hydrophobic amino acids L, I, V and F in the transmembrane domain are replaced with Q, T and Y (respectively) to give the following sequence (lower line) aligned to the wild type (upper line): [ka]

[0186] Each predicted transmembrane region is underlined, illustrating a fully modified domain of the invention. Thus, for example, the invention includes a transmembrane domain comprising the underlined amino acids of SEQ ID NO: 185. Preferably, a protein comprising TM1 herein comprises one or more (e.g., all) of the extracellular and intracellular loop sequences (non-underlined sequences) of SEQ ID NO: 185. Additionally or alternatively, a protein comprising TM1 herein comprises one or more additional transmembrane regions (underlined sequences) within SEQ ID NO: 185 or a homologous sequence that retains one, two, three, or possibly four or more of the native V, L, I, and F amino acids set forth in SEQ ID NO: 184.

[0187] The native protein sequence of CCR5 is again subjected to the method (note the differences in the N-terminal sequence). The program output separates the native sequence into extracellular and intracellular regions and selects eight transmembrane domain mutants for each of the transmembrane domains. The results are shown in the table below. MDYQVSSPIYDINYYTSEPCQKINVKQIAA (SEQ ID NO:186) TM1 mutant: RLQPPQYSQTFTFGFTGNMQVTQTQINC (SEQ ID NO:187) RLQPPQYSQTFTFGYTGNMQVTQTQINC (SEQ ID NO:188) RQQPPQYSQTFTFGFTGNMQTTQTQINC (SEQ ID NO:189) RQQPPQYSQTFTYGFTGNMQTTQTQINC (SEQ ID NO: 190) RQQPPQYSQTYTFGFTGNMQTTQTQINC (SEQ ID NO:191) RQQPPQYSQTFTFGYTGNMQTTQTQINC (SEQ ID NO:192) RQQPPQYSQTYTFGYTGNMQTTQTQINC (SEQ ID NO:193) RQQPPQYSQTYTYGYTGNMQTTQTQTNC (SEQ ID NO:194) KRLKSMTDIY (SEQ ID NO:195) TM2 mutant: LQNQAISDQFFQQTVPFWAHY (SEQ ID NO:196) LQNQAISDQFFQQTTPFWAHY (SEQ ID NO:197) LQNQAISDQFFQQTTPYWAHY (SEQ ID NO: 198) LQNQAISDQFYQQTTPYWAHY (SEQ ID NO: 199) LQNQAISDQYFQQTTPYWAHY (sequence number 200) LQNQATSDQFFQQTTPYWAHY (Sequence number 201) LQNQAISDQYYQQTTPYWAHY (SEQ ID NO: 202) QQNQATSDQYYQQTTPYWAHY (SEQ ID NO: 203) AAAQWDFGNTMCQ (SEQ ID NO: 204) TM3 Mutant: QQTGQYFTGYYSGTYYTTQQTT (SEQ ID NO: 205) QQTGQYYTGYYSGTYYTTQQTT (SEQ ID NO: 206) DRYLAVVHAVFALKART (SEQ ID NO:207) TM4 mutant: TTYGTTTSTTTWTTATYASQPGTTY (sequence number 208) TRSQKEGLHYTCSSHFPYSQYQFWKNFQTLKI (SEQ ID NO: 209) TM5 mutant: VIQGQVQPQQVMVTCYSGIQ (SEQ ID NO: 210) VIQGQVQPQQVMTTCYSGIQ (SEQ ID NO:211) VIQGQVQPQQTMTTCYSGIQ (SEQ ID NO:212) VTQGQVQPQQTMVTCYSGTQ (SEQ ID NO:213) TIQGQVQPQQVMTTCYSGTQ (SEQ ID NO:214) TIQGQVQPQQTMVTCYSGTQ (SEQ ID NO:215) TTQGQVQPQQVMTTCYSGTQ (SEQ ID NO:216) TTQGQTQPQQTMTTCYSGTQ (SEQ ID NO:217) KTLLRCRNEKKRHRAVR (SEQ ID NO:218) TM6 mutant: QTFTTMTTYYQFWAPYNIVQQLNTF (SEQ ID NO: 219) QTFTTMTTYYQFWAPYNTVQQLNTF (SEQ ID NO: 220) QTFTTMTTYYQYWAPYNTVQQLNTF (SEQ ID NO:221) QTFTTMTTYYQYWAPYNTVQQQNTF (SEQ ID NO:222) QTYTTMTTYYQYWAPYNTVQQLNTF (SEQ ID NO:223) QTFTTMTTYYQYWAPYNTTQQLNTF (SEQ ID NO: 224) QTYTTMTTYYQYWAPYNTVQQQNTF (SEQ ID NO: 225) QTYTTMTTYYQYWAPYNTTQQQNTY (SEQ ID NO: 225) QEFFGLNNCSSSNRLDQ (SEQ ID NO:226) TM7 mutant: AMQVTETQGMTHCCINPIIYAFVG (SEQ ID NO:227) AMQVTETLGMTHCCTNPIIYAFTG (SEQ ID NO:228) AMQVTETQGMTHCCINPTIYAYVG (SEQ ID NO:229) AMQTTETQGMTHCCINPITYAFTG (SEQ ID NO:230) AMQTTETQGMTHCCINPTIYAFTG (SEQ ID NO:231) AMQVTETQGMTHCCTNPTIYAYVG (SEQ ID NO:232) AMQTTETQGMTHCCINPTTYAYVG (SEQ ID NO:233) AMQTTETQGMTHCCTNPTTYAYTG (SEQ ID NO:234) EKFRNYLLVFFQKHIAKRFCKCCSIFQQEAPERASSVYTRSTGEQEISVGL (SEQ ID NO: 235)

[0188] As in Example 1 above, the sequences before, between and after each list of transmembrane domain mutants are the N', middle and C' intracellular or extracellular regions, respectively.

[0189] The sequences were then used to generate coding sequences suitable for expression in an expression system, in this case yeast, as known in the art, which were then recombinantly expressed to generate a library containing multiple proteins having SEQ ID NOs: 186, 195, 204, 207, 209, 218, 226 and 235, each of which contains one transmembrane domain mutant in each mutant list between its respective intracellular and extracellular domains.

[0190] The library so generated was then assayed for binding to the CCR5 cognate ligand, CCL5, in aqueous media as described in Example 1. Ligand binding was detected and the samples were then sequenced. One mutant was sequenced. The results are shown in Figure 8.

[0191] Example 5: CXCR3 Mutants The method of Example 1 was repeated for CXC chemokine receptor type 3 isoform 2, with all or substantially all of the hydrophobic amino acids L, I, V and F in its transmembrane domain substituted with Q, T and Y (respectively) to give the following sequence (SEQ ID NO:325, lower line), aligned to the wild type (SEQ ID NO:324, upper line). [ka]

[0192] Each predicted transmembrane region is underlined, and a fully modified domain of the invention is illustrated. Preferably, the TM1-containing proteins herein include one or more (e.g., all) of the extracellular and intracellular loop sequences (sequences not underlined). Additionally or alternatively, the TM1-containing proteins herein include one or more additional transmembrane regions (sequences underlined) in SEQ ID NO:325 or a homologous sequence that retains one, two, three, or possibly four or more of the native V, L, I, and F amino acids set forth in SEQ ID NO:324.

[0193] As described above, the native protein sequence of CXCR3 was subjected to the method. The program output divided the native sequence into extracellular and intracellular regions and selected eight transmembrane domain mutants for each of the transmembrane domains. The results are shown in the table below. MVLEVSDHQVLNDAEVAALLENFSSSYDYGENESDSCCTSPPCPQDFSLNFDR (SEQ ID NO: 235) TM1 mutant: AFLPALYSQQFQQGQQGNGAVAATQLS (SEQ ID NO:236) AFQPALYSQQFQQGQQGNGAVAAVQQS (SEQ ID NO: 237) AFQPAQYSQQFLQGQQGNGAVAATQQS (SEQ ID NO:238) AYQPALYSLQYQQGQQGNGATAAVQQS (SEQ ID NO:239) AYQPALYSQLFQQGQQGNGATAATQQS (SEQ ID NO: 240) AFQPALYSLQYQQGQQGNGATAATQQS (SEQ ID NO:241) AYQPAQYSLQYQQGQQGNGATAAVQQS (SEQ ID NO:242) AYQPAQYSQQYQQGQQGNGATAATQQS (SEQ ID NO:243) RRTALSSTD (SEQ ID NO:244) TM2 mutant: TFLQHLAVADTQQVQTLPQWA (SEQ ID NO:245) TFLQHQAVADTQLVQTQPQWA (SEQ ID NO:246) TFQQHLAVADTQQVQTQPQWA (SEQ ID NO:247) TYLQHQAVADTQQVQTQPQWA (SEQ ID NO:248) TYQLHQAVADTQQVQTQPQWA (SEQ ID NO:249) TYQQHLAVADTQQVQTQPQWA (SEQ ID NO:250) TYQQHQAVADTQQVQTQPQWA (SEQ ID NO:251) TYQQHQATADTQQTQTQPQWA (SEQ ID NO:252) VDAAVQWVFGSGLCK (SEQ ID NO:253) TM3 Mutant: TAGAQYNTNFYAGAQQQACISF (SEQ ID NO:254) TAGAQYNTNFYAGAQLQACTSF (SEQ ID NO:255) TAGAQYNTNFYAGAQQLACTSF (SEQ ID NO:256) TAGAQFNTNYYAGAQQQACISF (SEQ ID NO:257) TAGAQYNTNYYAGAQQQACISF (SEQ ID NO:258) TAGAQYNTNYYAGAQLQACTSF (SEQ ID NO:259) TAGAQYNTNYYAGAQQLACTSF (SEQ ID NO:260) TAGAQYNTNYYAGAQQQACTSY (SEQ ID NO:261) DRYLNIVHATQLYRRGPPARVT (SEQ ID NO:262) TM4 mutant: LTCQAVWGQCQQFAQPDFIF (SEQ ID NO:263) QTCQAVWGQCQQFAQPDFIF (SEQ ID NO:264) QTCQATWGQCQQFAQPDFIF (SEQ ID NO:265) QTCQATWGQCQQYAQPDFIF (SEQ ID NO:266) QTCQATWGQCQQFAQPDFTF (SEQ ID NO:267) QTCQATWGQCQQFAQPDYIF (SEQ ID NO:268) QTCQATWGQCQQYAQPDYIF (SEQ ID NO:269) QTCQATWGQCQQYAQPDYTY (SEQ ID NO:270) LSAHHDERLNATHCQYNFPQVGR (SEQ ID NO:271) TM5 mutant: TAQRTQQQTAGYQQPQQTMAY (SEQ ID NO:272) CYAHILAVLLVSRGQRRLRAMR (SEQ ID NO:273) TM6 mutant: QVTTTTVAFAQCWTPYHQVVQV (SEQ ID NO:274) QVTTTTVAFAQCWTPYHQTVQV (SEQ ID NO:275) QVTTTTTAFAQCWTPYHQTVQV (SEQ ID NO:276) QVTTTTTAYAQCWTPYHQTVQV (SEQ ID NO:277) QVTTTTTAFAQCWTPYHQTTQV (SEQ ID NO:278) QTTTTTVAFAQCWTPYHQTTQV (SEQ ID NO:279) QVTTTTTAYAQCWTPYHQTTQV (SEQ ID NO:280) QTTTTTTAYAQCWTPYHQTTQT (SEQ ID NO:281) DILMDLGALARNCGRESRVDV (SEQ ID NO:282) TM7 mutant: AKSVTSGQGYMHCCLNPLQYAFV (SEQ ID NO:283) AKSVTSGQGYMHCCLNPQLYAFT (SEQ ID NO:284) AKSVTSGQGYMHCCLNPLQYAFT (SEQ ID NO:285) AKSTTSGQGYMHCCLNPQQYAFV (SEQ ID NO:286) AKSTTSGQGYMHCCQNPLQYAFV (SEQ ID NO:287) AKSTTSGQGYMHCCQNPQLYAFV (SEQ ID NO:288) AKSTTSGQGYMHCCQNPLQYAFT (SEQ ID NO:289) AKSTTSGQGYMHCCQNPQQYAYT (SEQ ID NO:290) GVKFRERMWMLLLRLGCPNQRGLQRQPSSSRRDSSWSETSEASYSGL (SEQ ID NO:291)

[0194] The above sequences can be used to generate coding sequences suitable for expression in an expression system, in this case yeast, as known in the art, which are then recombinantly expressed to generate a library containing a plurality of proteins with intracellular and extracellular loops, each of which contains one transmembrane domain mutant from each mutant list between its respective intracellular and extracellular domains.

[0195] The libraries so generated can then be assayed in aqueous medium for binding to their cognate ligands as described in Example 1.

[0196] Example 6: (CCR-1) CC chemokine receptor type 1 Example 1 was repeated for the title protein. [Table 3]

[0197] All or substantially all of the hydrophobic amino acids L, I, V and F in the transmembrane domain are replaced with Q, T and Y (respectively) to give the following sequence (lower line, SEQ ID NO:293) aligned to the wild type (upper line, SEQ ID NO:292). [ka]

[0198] Each predicted transmembrane region is underlined, and fully modified domains of the invention are illustrated. Thus, for example, the invention includes transmembrane domains that include each of the underlined domains. Preferably, proteins comprising TM1 herein include one or more (e.g., all) of the extracellular and intracellular loop sequences (sequences not underlined). Additionally or alternatively, proteins comprising TM1 herein include one or more additional transmembrane regions (sequences underlined) within the depicted protein or a homologous sequence that retains one, two, three, or possibly four or more of the native V, L, I, and F amino acids set forth in the wild-type sequence.

[0199] The wild-type sequence can be subjected to the methods described above to select for additional transmembrane domain mutants as described in Example 1. Coding sequences can be designed and recombinantly expressed proteins. The expressed proteins can be assayed for ligand binding as described herein.

[0200] Example 7: (CCR-2) CC chemokine receptor type 2 isoform A Example 1 was repeated for the title protein, substituting each of the hydrophobic amino acids L, I, V and F with Q, T and Y (respectively) in its transmembrane domain to give the following sequence (lower line, SEQ ID NO:295) aligned to the wild type (upper line, SEQ ID NO:294). [ka]

[0201] Each predicted transmembrane region is underlined, and fully modified domains of the invention are illustrated. Thus, for example, the invention includes transmembrane domains that include each of the underlined domains. Preferably, proteins comprising TM1 herein include one or more (e.g., all) of the extracellular and intracellular loop sequences (sequences not underlined). Additionally or alternatively, proteins comprising TM1 herein include one or more additional transmembrane regions (sequences underlined) within the depicted protein or a homologous sequence that retains one, two, three, or possibly four or more of the native V, L, I, and F amino acids set forth in the wild-type sequence.

[0202] The wild-type sequence can be subjected to the methods described above to select for additional transmembrane domain mutants as described in Example 1. Coding sequences can be designed and recombinantly expressed proteins. The expressed proteins can be assayed for ligand binding as described herein.

[0203] Example 8: (CCR-4) CC chemokine receptor type 4 Example 1 was repeated for the title protein, with substitution of all or substantially all of the hydrophobic amino acids L, I, V and F in its transmembrane domain with Q, T and Y (respectively) to give the following sequence (lower line, SEQ ID NO:297) aligned to the wild type (upper line, SEQ ID NO:296). [ka]

[0204] Each predicted transmembrane region is underlined, and fully modified domains of the invention are illustrated. Thus, for example, the invention includes transmembrane domains that include each of the underlined domains. Preferably, proteins comprising TM1 herein include one or more (e.g., all) of the extracellular and intracellular loop sequences (sequences not underlined). Additionally or alternatively, proteins comprising TM1 herein include one or more additional transmembrane regions (sequences underlined) within the depicted protein or a homologous sequence that retains one, two, three, or possibly four or more of the native V, L, I, and F amino acids set forth in the wild-type sequence.

[0205] The wild-type sequence can be subjected to the methods described above to select for additional transmembrane domain mutants as described in Example 1. Coding sequences can be designed and recombinantly expressed proteins. The expressed proteins can be assayed for ligand binding as described herein.

[0206] Example 9: (CCR-6) CC chemokine receptor type 6 Example 1 was repeated for the title protein, with substitution of all or substantially all of the hydrophobic amino acids L, I, V and F in its transmembrane domain with Q, T and Y (respectively) to give the following sequence (lower line, SEQ ID NO:299) aligned to the wild type (upper line, SEQ ID NO:298). [ka]

[0207] Each predicted transmembrane region is underlined, illustrating a fully modified domain of the invention. Thus, for example, the invention includes a transmembrane domain that includes each of the underlined domains. Preferably, the TM1-containing proteins herein include one or more (e.g., all) of the extracellular and intracellular loop sequences (non-underlined sequences). Additionally or alternatively, the TM1-containing proteins herein include one or more additional transmembrane regions (underlined sequences) within the depicted protein or a homologous sequence that retains one, two, three, or possibly four or more of the native L, I, V, and F amino acids set forth in the wild-type sequence.

[0208] The wild-type sequence can be subjected to the methods described above to select for additional transmembrane domain mutants as described in Example 1. Coding sequences can be designed and recombinantly expressed proteins. The expressed proteins can be assayed for ligand binding as described herein.

[0209] Example 10: (CCR-7) CC chemokine receptor type 7 precursor Example 1 was repeated for the title protein, with substitution of all or substantially all of the hydrophobic amino acids L, I, V and F in its transmembrane domain with Q, T and Y (respectively) to give the following sequence (lower line, SEQ ID NO:301) aligned to the wild type (upper line, SEQ ID NO:300). [ka]

[0210] Each predicted transmembrane region is underlined, illustrating a fully modified domain of the invention. Thus, for example, the invention includes a transmembrane domain that includes each of the underlined domains. Preferably, the TM1-containing proteins herein include one or more (e.g., all) of the extracellular and intracellular loop sequences (non-underlined sequences). Additionally or alternatively, the TM1-containing proteins herein include one or more additional transmembrane regions (underlined sequences) within the depicted protein or a homologous sequence that retains one, two, three, or possibly four or more of the native L, I, V, and F amino acids set forth in the wild-type sequence.

[0211] The wild-type sequence can be subjected to the methods described above to select for additional transmembrane domain mutants as described in Example 1. Coding sequences can be designed and recombinantly expressed proteins. The expressed proteins can be assayed for ligand binding as described herein.

[0212] Example 11: (CCR-8) CC chemokine receptor type 8 Example 1 was repeated for the title protein, with substitution of all or substantially all of the hydrophobic amino acids L, I, V and F in its transmembrane domain with Q, T and Y (respectively) to give the following sequence (lower line, SEQ ID NO:303) aligned to the wild type (upper line, SEQ ID NO:302). [ka]

[0213] Each predicted transmembrane region is underlined, illustrating a fully modified domain of the invention. Thus, for example, the invention includes a transmembrane domain that includes each of the underlined domains. Preferably, the TM1-containing proteins herein include one or more (e.g., all) of the extracellular and intracellular loop sequences (non-underlined sequences). Additionally or alternatively, the TM1-containing proteins herein include one or more additional transmembrane regions (underlined sequences) within the depicted protein or a homologous sequence that retains one, two, three, or possibly four or more of the native L, I, V, and F amino acids set forth in the wild-type sequence.

[0214] The wild-type sequence can be subjected to the methods described above to select for additional transmembrane domain mutants as described in Example 1. Coding sequences can be designed and recombinantly expressed proteins. The expressed proteins can be assayed for ligand binding as described herein.

[0215] Example 12: (CCR-9) CC Chemokine Receptor Type 9 Isoform B Example 1 was repeated for the title protein, with all or substantially all of the hydrophobic amino acids L, I, V and F in its transmembrane domain substituted with Q, T and Y (respectively) to give the following sequence (lower line, SEQ ID NO:305) aligned to the wild type (upper line, SEQ ID NO:304). [ka]

[0216] Each predicted transmembrane region is underlined, illustrating a fully modified domain of the invention. Thus, for example, the invention includes a transmembrane domain that includes each of the underlined domains. Preferably, the TM1-containing proteins herein include one or more (e.g., all) of the extracellular and intracellular loop sequences (non-underlined sequences). Additionally or alternatively, the TM1-containing proteins herein include one or more additional transmembrane regions (underlined sequences) within the depicted protein or a homologous sequence that retains one, two, three, or possibly four or more of the native L, I, V, and F amino acids set forth in the wild-type sequence.

[0217] The wild-type sequence can be subjected to the methods described above to select for additional transmembrane domain mutants as described in Example 1. Coding sequences can be designed and recombinantly expressed proteins. The expressed proteins can be assayed for ligand binding as described herein.

[0218] Example 13: (CCR-10) CC chemokine receptor type 10 Example 1 was repeated for the title protein, substituting each of the hydrophobic amino acids L, I, V and F with Q, T and Y (respectively) in its transmembrane domain to give the following sequence (lower line, SEQ ID NO:307) aligned to the wild type (upper line, SEQ ID NO:306). [ka]

[0219] Each predicted transmembrane region is underlined, illustrating a fully modified domain of the invention. Thus, for example, the invention includes a transmembrane domain that includes each of the underlined domains. Preferably, the TM1-containing proteins herein include one or more (e.g., all) of the extracellular and intracellular loop sequences (non-underlined sequences). Additionally or alternatively, the TM1-containing proteins herein include one or more additional transmembrane regions (underlined sequences) within the depicted protein or a homologous sequence that retains one, two, three, or possibly four or more of the native L, I, V, and F amino acids set forth in the wild-type sequence. The wild-type sequence can be subjected to the methods described above to select additional transmembrane domain mutants as described in Example 1. Coding sequences can be designed and recombinantly expressed proteins. The expressed proteins can be assayed for ligand binding as described herein.

[0220] Example 14: (CXCR1) Chemokine receptor type 1 Example 1 was repeated for the title protein, with substitution of all or substantially all of the hydrophobic amino acids L, I, V and F in its transmembrane domain with Q, T and Y (respectively) to give the following sequence (lower line, SEQ ID NO:309) aligned to the wild type (upper line, SEQ ID NO:308). [ka]

[0221] Each predicted transmembrane region is underlined, illustrating a fully modified domain of the invention. Thus, for example, the invention includes a transmembrane domain that includes each of the underlined domains. Preferably, the TM1-containing proteins herein include one or more (e.g., all) of the extracellular and intracellular loop sequences (non-underlined sequences). Additionally or alternatively, the TM1-containing proteins herein include one or more additional transmembrane regions (underlined sequences) within the depicted protein or a homologous sequence that retains one, two, three, or possibly four or more of the native L, I, V, and F amino acids set forth in the wild-type sequence.

[0222] The wild-type sequence can be subjected to the methods described above to select for additional transmembrane domain mutants as described in Example 1. Coding sequences can be designed and recombinantly expressed proteins. The expressed proteins can be assayed for ligand binding as described herein.

[0223] Example 15: (CXR1) CXR chemokine receptor type 1 Example 1 was repeated for the title protein, substituting each of the hydrophobic amino acids L, I, V and F with Q, T and Y (respectively) in its transmembrane domain to give the following sequence (lower line, SEQ ID NO:311) aligned to the wild type (upper line, SEQ ID NO:310). [ka]

[0224] Each predicted transmembrane region is underlined, and fully modified domains of the invention are illustrated. Thus, for example, the invention includes transmembrane domains that include each of the underlined domains. Preferably, proteins comprising TM1 herein include one or more (e.g., all) of the extracellular and intracellular loop sequences (sequences not underlined). Additionally or alternatively, proteins comprising TM1 herein include one or more additional transmembrane regions (sequences underlined) within the depicted protein or a homologous sequence that retains one, two, three, or possibly four or more of the native V, L, I, and F amino acids set forth in the wild-type sequence.

[0225] The wild-type sequence can be subjected to the methods described above to select for additional transmembrane domain mutants as described in Example 1. Coding sequences can be designed and recombinantly expressed proteins. The expressed proteins can be assayed for ligand binding as described herein.

[0226] Example 16: (CXCR2) Chemokine receptor type 2 Example 1 was repeated for the title protein, with all or substantially all of the hydrophobic amino acids L, I, V and F in its transmembrane domain substituted with Q, T and Y (respectively) to give the following sequence (lower line, SEQ ID NO:313) aligned to the wild type (upper line, SEQ ID NO:312). [ka]

[0227] Each predicted transmembrane region is underlined, illustrating a fully modified domain of the invention. Thus, for example, the invention includes a transmembrane domain that includes each of the underlined domains. Preferably, the TM1-containing proteins herein include one or more (e.g., all) of the extracellular and intracellular loop sequences (non-underlined sequences). Additionally or alternatively, the TM1-containing proteins herein include one or more additional transmembrane regions (underlined sequences) within the depicted protein or a homologous sequence that retains one, two, three, or possibly four or more of the native L, I, V, and F amino acids set forth in the wild-type sequence.

[0228] The wild-type sequence can be subjected to the methods described above to select for additional transmembrane domain mutants as described in Example 1. Coding sequences can be designed and recombinantly expressed proteins. The expressed proteins can be assayed for ligand binding as described herein.

[0229] Example 17: (CCR-10) CC chemokine receptor type 10 Example 1 was repeated for the title protein, substituting each of the hydrophobic amino acids L, I, V and F with Q, T and Y (respectively) in its transmembrane domain to give the following sequence (lower line, SEQ ID NO:315) aligned to the wild type (upper line, SEQ ID NO:314). [ka]

[0230] Each predicted transmembrane region is underlined, illustrating a fully modified domain of the invention. Thus, for example, the invention includes a transmembrane domain that includes each of the underlined domains. Preferably, the TM1-containing proteins herein include one or more (e.g., all) of the extracellular and intracellular loop sequences (non-underlined sequences). Additionally or alternatively, the TM1-containing proteins herein include one or more additional transmembrane regions (underlined sequences) within the depicted protein or a homologous sequence that retains one, two, three, or possibly four or more of the native L, I, V, and F amino acids set forth in the wild-type sequence.

[0231] The wild-type sequence can be subjected to the methods described above to select for additional transmembrane domain mutants as described in Example 1. Coding sequences can be designed and recombinantly expressed proteins. The expressed proteins can be assayed for ligand binding as described herein.

[0232] Example 18: (CXCR6) Chemokine receptor type 6 Example 1 was repeated for the title protein, substituting each of the hydrophobic amino acids L, I, V and F with Q, T and Y (respectively) in its transmembrane domain to give the following sequence (lower line, SEQ ID NO:317) aligned to the wild type (upper line, SEQ ID NO:316). [ka]

[0233] Each predicted transmembrane region is underlined, illustrating a fully modified domain of the invention. Thus, for example, the invention includes a transmembrane domain that includes each of the underlined domains. Preferably, the TM1-containing proteins herein include one or more (e.g., all) of the extracellular and intracellular loop sequences (non-underlined sequences). Additionally or alternatively, the TM1-containing proteins herein include one or more additional transmembrane regions (underlined sequences) within the depicted protein or a homologous sequence that retains one, two, three, or possibly four or more of the native L, I, V, and F amino acids set forth in the wild-type sequence.

[0234] The wild-type sequence can be subjected to the methods described above to select for additional transmembrane domain mutants as described in Example 1. Coding sequences can be designed and recombinantly expressed proteins. The expressed proteins can be assayed for ligand binding as described herein.

[0235] Example 19: (CXCR7) Chemokine receptor type 7 Example 1 was repeated for the title protein, with substitution of all or substantially all of the hydrophobic amino acids L, I, V and F in its transmembrane domain with Q, T and Y (respectively) to give the following sequence (lower line, SEQ ID NO:319) aligned to the wild type (upper line, SEQ ID NO:318). [ka]

[0236] Each predicted transmembrane region is underlined, illustrating a fully modified domain of the invention. Thus, for example, the invention includes a transmembrane domain that includes each of the underlined domains. Preferably, the TM1-containing proteins herein include one or more (e.g., all) of the extracellular and intracellular loop sequences (non-underlined sequences). Additionally or alternatively, the TM1-containing proteins herein include one or more additional transmembrane regions (underlined sequences) within the depicted protein or a homologous sequence that retains one, two, three, or possibly four or more of the native L, I, V, and F amino acids set forth in the wild-type sequence.

[0237] The wild-type sequence can be subjected to the methods described above to select for additional transmembrane domain mutants as described in Example 1. Coding sequences can be designed and recombinantly expressed proteins. The expressed proteins can be assayed for ligand binding as described herein.

[0238] Example 20: (CLR-1a) Chemokine-like receptor 1 isotype a Example 1 was repeated for the title protein, with substitution of all or substantially all of the hydrophobic amino acids L, I, V and F in its transmembrane domain with Q, T and Y (respectively) to give the following sequence (lower line, SEQ ID NO:321) aligned to the wild type (upper line, SEQ ID NO:320). [ka]

[0239] Each predicted transmembrane region is underlined, illustrating a fully modified domain of the invention. Thus, for example, the invention includes a transmembrane domain that includes each of the underlined domains. Preferably, the TM1-containing proteins herein include one or more (e.g., all) of the extracellular and intracellular loop sequences (non-underlined sequences). Additionally or alternatively, the TM1-containing proteins herein include one or more additional transmembrane regions (underlined sequences) within the depicted protein or a homologous sequence that retains one, two, three, or possibly four or more of the native L, I, V, and F amino acids set forth in the wild-type sequence.

[0240] The wild-type sequence can be subjected to the methods described above to select for additional transmembrane domain mutants as described in Example 1. Coding sequences can be designed and recombinantly expressed proteins. The expressed proteins can be assayed for ligand binding as described herein.

[0241] Example 21: DARIA Duffy antigen / chemokine receptor isotype a Example 1 was repeated for the title protein, substituting each of the hydrophobic amino acids L, I, V and F with Q, T and Y (respectively) in its transmembrane domain to give the following sequence (lower line, SEQ ID NO:323) aligned to the wild type (upper line, SEQ ID NO:322). [ka]

[0242] Each predicted transmembrane region is underlined, illustrating a fully modified domain of the invention. Thus, for example, the invention includes a transmembrane domain that includes each of the underlined domains. Preferably, the TM1-containing proteins herein include one or more (e.g., all) of the extracellular and intracellular loop sequences (non-underlined sequences). Additionally or alternatively, the TM1-containing proteins herein include one or more additional transmembrane regions (underlined sequences) within the depicted protein or a homologous sequence that retains one, two, three, or possibly four or more of the native L, I, V, and F amino acids set forth in the wild-type sequence. The wild-type sequence can be subjected to the methods described above to select additional transmembrane domain mutants as described in Example 1. The coding sequence can be designed and recombinantly expressed to produce a protein. The expressed protein can be assayed for ligand binding as described herein.

[0243] Example 22: CD81 antigen CD81 may play an important role in regulating lymphoma cell proliferation and interacts with the 16 kDa Leu-13 protein to form a complex that may be involved in signal transduction. CD81 may function as a viral receptor for HCV.

[0244] Example 1 was repeated for the title protein, substituting each of the hydrophobic amino acids L, I, V and F with Q, T and Y (respectively) in its transmembrane domain to give the following sequence (lower line, SEQ ID NO:325) aligned to the wild type (upper line, SEQ ID NO:324). [ka]

[0245] The predicted transmembrane regions are exemplary of modified domains of the present invention and include the following (SEQ ID NOs: 326, 327, 328, 329, 330, 331, 332, 333, respectively). [ka]

[0246] Thus, for example, the invention includes transmembrane domains, each of which comprises a modified domain or "mt" domain. Preferably, the TM1-containing proteins herein include one or more (e.g., all) of the extracellular and intracellular loop sequences (sequences not underlined). Additionally or alternatively, the TM1-containing proteins herein include one or more additional transmembrane regions (sequences underlined) within the depicted protein or a homologous sequence that retains one, two, three, or possibly four or more of the native V, L, I, and F amino acids set forth in the wild-type sequence.

[0247] The wild-type sequence can be subjected to the methods described above to select for additional transmembrane domain mutants as described in Example 1. Coding sequences can be designed and recombinantly expressed proteins. The expressed proteins can be assayed for ligand binding as described herein.

[0248] Example 23: E. coli expression of QTY mutants and CXCR4-QTY mutants

[0249] 1. Large-scale production of CXCR4-QTY in E. coli BL21(DE3) The water-soluble GPCR CXCR4 was produced in E. coli with an estimated yield of approximately 20 mg of purified protein per liter of routinely used LB medium. The estimated production cost is approximately $0.25 per milligram. Advantageously, gram quantities of water-soluble GPCRs can be readily obtained using this technique, which can then facilitate their structural determination.

[0250] 2. Determining the location where water-soluble CXCR4-QTY is produced in E. coli cells Water-soluble CXCR4-QTY was cloned into pET vector. We first performed a small-scale E. coli culture study to evaluate the location of the produced CXCR4-QTY protein (150 ml culture). After culturing the cells induced with IPTG at 24°C for 4 hours, we harvested and sonicated the cells, and separated them into two fractions by centrifugation at 14,637×g (12,000 rpm). We then detected the location of CXCR4-QTY protein using Western blot analysis of specific anti-rho tag monoclonal antibody. We observed that CXCR4-QTY protein was in the supernatant fraction, and no protein was in the pellet fraction, thus suggesting that the protein is completely water-soluble.

[0251] 3. Estimated yield of CXCR4-QTY produced in the soluble fraction of E. coli cells We then performed another 150 ml culture and obtained about 6 mg of 1D4 monoclonal antibody purified CXCR4-QTY. Because we underestimated the yield (we did not expect such a surprisingly high yield), we did not use enough affinity rho-1D4 tag monoclonal antibody beads to capture the produced CXCR4-QTY. Thus, no significant amount of CXCR4-QTY protein bound to the beads because not enough beads were added during purification and the protein was in the flow-through lane and was washed away. Despite the significant loss, we can still obtain about 6 mg for 150 ml culture, as can be seen from lanes 8-10 (elution fraction).

[0252] 4. Measurement of Thermal Stability of Purified Water-soluble CXCR4-QTY Protein In most cases, the structure determines the function of a protein. Therefore, it is important to know whether the purified CXCR4-QTY protein produced in E. coli is still correctly folded into a typical α-helical structure with about 50% α-helix. We performed secondary structure measurement using circular dichroism (CD). We observed the CD spectrum of the purified CXCR4-QTY protein at various temperatures. We measured the thermal stability of the purified CXCR4-QTY protein. We observed that the purified CXCR4-QTY protein was relatively stable up to 55°C, and the protein was only partially denatured gradually, with a CD signal reduction of about 15%. At 55°C to 65°C, the denaturation increased toward 65°C, and a denaturation transition occurred between 65°C and 75°C, at which the protein was almost completely denatured.

[0253] We obtained the melting temperature (Tm) of the purified water-soluble CXCR4-QTY protein by plotting temperature against ellipticity at 222 nm. From this plot, we estimated the Tm of the purified CXCR4-QTY protein to be about 67° C. This Tm suggests that the purified water-soluble CXCR4-QTY protein is very stable compared to many other soluble proteins. This thermal stability property makes it easier to obtain diffracting crystals, since it is known that the better the thermal stability, the better the crystal lattice packing, and therefore the more chances of obtaining a structure.

[0254] 5. Additional G Protein-Coupled Receptors The inventors selected ten G protein-coupled receptors (GPCRs) to design water soluble forms thereof using the QTY method described in U.S. Patent Publication No. 2012 / 0252719A, entitled "Water Soluble Membrane Proteins and Methods for the Preparation and Use Thereof" to Zhang et al. ("Zhang"). Alternatively, proteins described herein can be selected.

[0255] 6. Molecular cloning of genes We successfully confirmed the native and QTY genes of GPCRs in the cell-free protein expression plasmid vector pIVex2.3d and E. coli pET28a and pET-duet-1 plasmid vectors.

[0256] 7. Production of Soluble GPCRs We produced several native and QTY proteins. When we produced native GPCR in a cell-free system, the detergent Brij35 was required, and without detergent, the protein precipitated as soon as it was produced. On the other hand, we tested QTY mutants in the presence and absence of detergent. Without detergent, the cell-free system produced soluble protein.

[0257] We cloned the QTY mutants into E. coli in vivo expression system pET28a and pET-duet-1 plasmid vectors for E. coli cell protein production in E. coli BL21(DE3) strain. We purified several soluble GPCR proteins, including CXCR4 and CCR5, which we used for secondary structure analysis. We performed ligand binding studies for CXCR4 with its natural ligand CCL12 (SDF1a). We performed E. coli production and purification of the soluble GPCR CCR5e mutant. The CCR5e mutant had 58 amino acid changes (about 18% change). The soluble GPCR CCR5e mutant was purified to homogeneity using a specific monoclonal antibody rhodopsin tag. The blue strain showed a single band on SDS gel indicating its purity. It appears to be a pure homodimer (the native membrane-bound CXCR4 crystal structure was a dimer) as estimated from the size marker of the protein. Western blot confirmed the existence of monomers and homodimers of common CCR5e variants in GPCRs.

[0258] 8. Secondary structure study of QTY CCR5e The present inventors obtained a water-soluble QTY mutant of GPCR CCR5e. Then, the present inventors carried out secondary structure analysis using Aviv model 410 circular dichroism instrument, and confirmed that the GPCR QTY CCR5-e mutant has a typical α-helical structure. The present inventors also carried out experiments at various temperatures to determine the Tm of the CCR5e mutant, i.e., the thermal stability of the water-soluble CCR5e mutant. From these experiments, the present inventors determined that the Tm of the CCR5e mutant is about 46°C. This Tm is good for crystal screening experiments.

[0259] 9. Ligand binding study of CXCR4 using CCL12 (SDF1a) To ensure that the designed water-soluble QTY GPCRs still maintain their biological functions, i.e., recognize and bind their natural ligands, we first studied water-soluble CXCR4 with its natural ligand CCL12 (also called SDF1a) using ELISA measurements. Assay concentrations range from 50 nM to 10 μM. The measured Kd is about 80 nM. The Kd of native membrane-bound CXCR4 with SDF1a is about 100 nM. Thus, the Kd of water-soluble CXCR4 is within an acceptable range. Further experiments using more sensitive SPR or other measurements may be performed to generate a more accurate Kd.

[0260] While the present invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and detail can be made therein without departing from the scope of the invention as encompassed by the appended claims.

[0261] This application may include the following inventions. (1) 1. A computer-implemented method for performing a procedure for selecting water-soluble variants of a G protein-coupled receptor (GPCR), comprising: inputting the sequence of said GPCR for analysis; Obtaining a mutant of said GPCR in which multiple hydrophobic amino acid substitutions are made within the transmembrane (TM) domain alpha helix segment ("TM region") of said GPCR, (a) the hydrophobic amino acid is selected from the group consisting of leucine (L), isoleucine (I), valine (V) and phenylalanine (F); (b) each leucine (L) is independently replaced with glutamine (Q), asparagine (N), or serine (S); (c) the isoleucine (I) and the valine (V) are each independently replaced with threonine (T), asparagine (N), or serine (S); and (d) each phenylalanine is replaced with a tyrosine (Y) and then obtaining alpha helix secondary structure results for said variants and confirming maintenance of alpha helix secondary structure in said variants; obtaining a transmembrane domain result for said variant to confirm the water solubility of said variant; thereby selecting a water-soluble mutant of the GPCR. (2) The method according to (1), wherein step (3) is carried out before, simultaneously with, or after step (4). (3) The method of claim 1 or 2, wherein in step (2), one subset of the plurality of hydrophobic amino acids in the same TM region of one of the GPCRs is substituted to generate one member of a mutant candidate library, and one or more different subsets of the plurality of hydrophobic amino acids are substituted to generate further members of the library. (4) The method of claim 3, further comprising a step of ranking all members of the library based on a combined score, the combined score being a weighted combination of the alpha helix secondary structure prediction result and the membrane spanning region prediction result. (5) The method of (1), further comprising the step of ranking the variants using a ranking function. (6) The method according to (1), further comprising the step of carrying out the method using a data processor. (7) 9. The method of claim 6, further comprising a memory coupled to the data processor. (8) The method according to (5), wherein the ranking function includes a secondary structure component and a water solubility component. (9) The method according to (8), wherein the ranking function comprises weighting values ​​of the secondary structure components and / or the water-soluble components. (10) The method of (4), further comprising the step of selecting the N members having the highest combination scores to form a first library of mutant candidates for the TM region, where N is a predetermined integer (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more). (11) The method of (10), further comprising the step of generating a library of mutant candidates for one, two, three, four, five or all six other TM regions of the GPCR. (12) The method according to (11), further comprising the step of replacing two or more TM regions of the GPCR with corresponding TM regions in the mutant candidate library to generate a combinatorial mutant library. (13) The method according to any one of (1) to (12), wherein substantially all (eg, all) of the leucines are substituted with glutamine. (14) The method according to any one of (1) to (13), wherein substantially all (eg, all) of the isoleucines are substituted with threonines. (15) The method according to any one of (1) to (14), wherein substantially all (eg, all) of the valines are substituted with threonines. (16) The method according to any one of (1) to (15), wherein substantially all (eg, all) of the phenylalanines are substituted with tyrosine. (17) The method according to any one of (1) to (16), wherein one or more (e.g., one, two or three) of the leucines are unsubstituted. (18) The method according to any one of (1) to (17), wherein one or more (e.g., one, two or three) of the isoleucines are unsubstituted. (19) The method according to any one of (1) to (18), wherein one or more (e.g., one, two or three) of the valines are unsubstituted. (20) The method according to any one of (1) to (19), wherein one or more (e.g., one, two, or three) of the phenylalanines are unsubstituted. (twenty one) The method according to any one of (1) to (20), further comprising a step of producing / expressing the combination mutant. (twenty two) The method according to any one of (1) to (21), further comprising a step of testing the combination mutants for ligand binding (e.g., by yeast two-hybrid assay), in which those having substantially the same ligand binding as compared to the GPCR are selected. (twenty three) The method according to any one of (1) to (22), further comprising a step of testing the combination mutants for the biological function of the GPCR, in which one having substantially the same biological function as the GPCR is selected. (twenty four) The method of any one of (1) to (23), wherein the combinatorial mutant library comprises less than about 2 million members. (twenty five) The method according to any one of (1) to (24), wherein the sequence of the GPCR comprises information regarding the TM region of the GPCR. (26) The method according to any one of (1) to (25), wherein the sequence of the GPCR is obtained from a protein structure database (e.g., PDB, UniProt). (27) The method according to any one of (1) to (26), wherein the TM region of the GPCR is predicted based on the sequence of the GPCR. (28) The method according to (27), wherein the TM region of the GPCR is predicted using the TMHMM2.0 (Transmembrane Prediction Using Hidden Markov Models) software module. (29) The method according to (28), wherein the TMHMM2.0 software module utilizes a dynamic baseline for peak searching. (30) The method according to any one of (1) to (29), further comprising the step of providing a polynucleotide sequence for each of the mutants of the GPCR. (31) The method of (30), wherein the polynucleotide sequence is codon optimized for expression in a host (e.g., a bacterium such as E. coli, a yeast such as Saccharomyces cerevisiae or Schizosaccharomyces pombe, an insect cell such as an Sf9 cell, a non-human mammalian cell, or a human cell). (32) 32. The method according to any one of claims 1 to 31, wherein the scripting step includes a VBA script. (33) The method according to any one of (1) to (32), wherein the scripting procedure is operable by a Linux (registered trademark) system (e.g., Ubuntu 12.04 LTS), a Unix (registered trademark) system, a Microsoft Windows operating system, an Android operating system, or an Apple iOS operating system. (34) 1. A water-soluble mutant of a G protein-coupled receptor (GPCR) comprising multiple hydrophobic amino acid substitutions within a transmembrane (TM) domain alpha helical segment ("TM region") of the GPCR, (a) the hydrophobic amino acid is selected from the group consisting of leucine (L), isoleucine (I), valine (V) and phenylalanine (F); (b) each said leucine (L) is independently replaced with glutamine (Q), asparagine (N), or serine (S); (c) said isoleucine (I) and said valine (V) are each independently replaced with threonine (T), asparagine (N) or serine (S); and (d) each phenylalanine is replaced with a tyrosine (Y), followed by all seven TM regions of the mutant maintain an α-helical secondary structure; and No predicted transmembrane domain A water-soluble mutant characterized in that (35) The water-soluble mutant according to (34), comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 4 to 11, 13 to 20, 22 to 29, 31 to 38, 40 to 47, 49 to 56, and 58 to 64. (36) The water-soluble variant according to (35), further comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 3, 12, 21, 30, 39, 48 and 57. (37) A water-soluble mutant according to (35) or (36), which binds to a CXCR4 ligand. (38) The water-soluble mutant according to (34), comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 69 to 76, 78 to 85, 87, 89 to 96, 98 to 105, 107 to 114, and 116 to 123. (39) The water-soluble variant according to (38), further comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 68, 77, 86, 88, 97, 106, 115 and 124. (40) A water-soluble mutant according to (38) or (40), which binds to a CX3CR1 ligand. (41) The water-soluble mutant according to (34), comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 128 to 135, 137 to 144, 146 to 153, 155 to 162, 164 to 171, 173, and 175 to 182. (42) The water-soluble variant according to (41), further comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 127, 136, 145, 154, 163, 172, 174 and 183. (43) A water-soluble mutant according to (41) or (42), which binds to a CCR3 ligand. (44) The water-soluble mutant according to (34), comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 187 to 194, 196 to 203, 205 to 206, 208, 210 to 217, 219 to 225, and 227 to 234. (45) The water-soluble variant according to (44), further comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 186, 195, 204, 207, 209, 218, 226 and 235. (46) A water-soluble mutant according to (44) or (45), which binds to a CCR5 ligand. (47) The water-soluble mutant according to (34), comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 236 to 243, 245 to 252, 254 to 261, 263 to 270, 272, 274 to 281, and 283 to 290. (48) The water-soluble variant according to (47), further comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 235, 244, 253, 262, 271, 273, 282 and 291. (49) A water-soluble mutant according to (47) or (48), which binds to a CXCR3 ligand. (50) 34. A water-soluble variant according to claim 34, comprising one or more transmembrane domains as set forth in any one of SEQ ID NOs: 2, 67, 126, 185, 327, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, or 325. (51) The water-soluble mutant according to (50), which is water-soluble and binds to a ligand of a homologous native transmembrane protein. (52) (a) culturing the bacteria in a growth medium under conditions suitable for protein production; (b) fractionating the bacterial lysate to produce a soluble fraction and an insoluble pellet fraction; (c) isolating the protein from the soluble fraction. Including, (1) The protein is a mutant of a G protein-coupled receptor (GPCR) according to any one of (29) to (46), (2) the protein yield is at least 20 mg / L (e.g., 30 mg / L, 40 mg / L, 50 mg / L or more) of growth medium; A method for producing a protein in a bacterium (e.g., Escherichia coli), comprising: (53) The method according to (47), wherein the bacterium is Escherichia coli BL21 and the growth medium is LB medium. (54) The method according to (47) or (48), wherein the protein is encoded by a plasmid in the bacterium. (55) The method according to any one of (47) to (49), wherein expression of the protein is under the control of an inducible promoter. (56) The method according to (50), wherein the inducible promoter is inducible by IPTG. (57) The method according to any one of (47) to (51), wherein the lysate is produced by ultrasonic treatment. (58) The method according to any one of (47) to (52), wherein the lysate is centrifuged at 14,500 × g or more to produce the soluble fraction. (59) A non-transitory computer-readable medium having stored thereon a series of instructions for performing a method according to any one of (1) to (33). (60) A data processing system operative to select water-soluble variants of a membrane protein, the system comprising a data processor operative to perform amino acid substitutions and to rank the protein variants according to a ranking function including secondary structure components and water-solubility components. (61) The system of (60), further comprising a library of membrane proteins for processing by the system. (62) 6. The system of claim 60, further comprising a memory coupled to the data processor storing coded instructions for executing a permutation processor. (63) The system described in (60) , which operates in accordance with steps (a), (b), (c) and (d) of the method described in (1). (64) The system of claim 60, further comprising a ranking function that is a weighted combination based on the secondary structure components. (65) The system according to (60), which communicates with an external program via a network. (66) The system according to (60), further comprising a database for storing water-soluble variants. (67) 61. The system of claim 60, further comprising instructions for performing dynamic baseline processing. (68) The system of claim 60, further comprising an interface for selecting parameters of the method. (69) The system according to (60), further comprising the step of inputting the sequence according to (35) to (50). (70) 1. A computer-implemented method for carrying out a procedure for selecting water-soluble mutants, comprising: processing the data to identify membrane protein sequences for analysis; obtaining a mutant of said membrane protein, wherein a plurality of hydrophobic amino acids in the transmembrane (TM) domain α-helical segment ("TM region") of said membrane protein are substituted; Including, The data processor: determining the secondary structure of the α-helix of the mutant to confirm maintenance of the secondary structure of the α-helix in the mutant; determining the transmembrane domain identity of the variant to confirm the water solubility of the variant; and Selecting a water-soluble mutant of the membrane protein A method comprising: (71) The substitution is (a) selecting the hydrophobic amino acid from the group consisting of leucine (L), isoleucine (I), valine (V) and phenylalanine (F); (b) independently replacing leucine (L) with glutamine (Q), asparagine (N), or serine (S); (c) independently replacing each of the isoleucine (I) and valine (V) with threonine (T), asparagine (N) or serine (S); and (d) replacing each said phenylalanine with a tyrosine (Y). The method according to (70), comprising: (72) The method of claim 71, wherein a subset of said plurality of hydrophobic amino acids of the same TM region of a GPCR is substituted to generate one member of a mutant candidate library, and one or more different subsets of said plurality of hydrophobic amino acids are substituted to generate additional members of said library. (73) The method of claim 70, further comprising a step of ranking all members of the library based on a combined score, the combined score being a weighted combination of the alpha helix secondary structure prediction results and the transmembrane domain prediction results. (74) 7. The method of claim 70, further comprising the step of ranking the variants using a ranking function. (75) 7. The method of claim 70, further comprising a memory coupled to the data processor. (76) The method according to (74), wherein the ranking function comprises a secondary structure component and a water solubility component. (77) The method according to (76), wherein the ranking function comprises weighting values ​​of the secondary structure components and / or the water-soluble components. (78) The method of (73), further comprising the step of selecting the N members having the highest combination scores to form a first library of mutant candidates for the TM region, where N is a predetermined integer (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more). (79) The method of (78), further comprising generating one library of mutant candidates for one, two, three, four, five, or all six other TM regions of the GPCR. (80) The method of (79), further comprising the step of replacing two or more TM regions of the GPCR with corresponding TM regions in the mutant candidate library to generate a combinatorial mutant library.

Claims

1. 1. A computer-implemented method for performing a procedure for selecting water-soluble mutants of a G protein-coupled receptor (GPCR), comprising: inputting the sequence of said GPCR for analysis; Obtaining a mutant of said Fc receptor having a substitution of multiple hydrophobic amino acids within a transmembrane (TM) domain alpha helix segment ("TM region") of said Fc receptor, (a) the hydrophobic amino acid is selected from the group consisting of leucine (L), isoleucine (I), valine (V) and phenylalanine (F); (b) each leucine (L) is independently replaced with glutamine (Q), asparagine (N), or serine (S); (c) the isoleucine (I) and the valine (V) are each independently replaced with threonine (T), asparagine (N), or serine (S); and (d) each phenylalanine is replaced with a tyrosine (Y); and then obtaining alpha helix secondary structure results for said variants and confirming maintenance of alpha helix secondary structure in said variants; obtaining a transmembrane domain result for said variant to confirm the water solubility of said variant; thereby selecting a water-soluble mutant of the GPCR.

2. 2. The method of claim 1, wherein step (3) is carried out before, simultaneously with, or after step (4).

3. 3. The method of claim 1 or 2, wherein in step (2), one subset of the plurality of hydrophobic amino acids in the same TM region of one of the GPCRs is substituted to generate one member of a mutant candidate library, and one or more different subsets of the plurality of hydrophobic amino acids are substituted to generate further members of the library.

4. The method of claim 3, further comprising the step of ranking all members of the library based on a combined score, the combined score being a weighted combination of the alpha helix secondary structure prediction results and the transmembrane domain prediction results.

5. The method of claim 1 , further comprising the step of ranking the variants using a ranking function.

6. The method of claim 1 further comprising the step of performing the method using a data processor.

7. The method of claim 6 further comprising a memory coupled to the data processor.

8. The method of claim 5 , wherein the ranking function comprises a secondary structure component and a water solubility component.

9. The method of claim 8 , wherein the ranking function comprises weighting values ​​of the secondary structure components and / or the water-soluble components.

10. 5. The method of claim 4, further comprising the step of selecting the N members having the highest combination scores to form a first library of mutant candidates for the TM region, where N is a predetermined integer (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more).

11. 11. The method of claim 10, further comprising generating one library of mutant candidates for one, two, three, four, five or all six other TM regions of the GPCR.

12. 12. The method of claim 11, further comprising replacing two or more TM regions of the GPCR with corresponding TM regions in the mutant candidate library to generate a combinatorial mutant library.

13. The method of any one of claims 1 to 12, wherein substantially all (eg all) of the leucines are substituted with glutamine.

14. 14. The method of any one of claims 1 to 13, wherein substantially all (e.g. all) of the isoleucines are replaced with threonines.

15. 15. The method of any one of claims 1 to 14, wherein substantially all (e.g. all) of the valines are replaced with threonines.

16. 16. The method of any one of claims 1 to 15, wherein substantially all (e.g. all) of the phenylalanines are substituted with tyrosine.

17. 17. The method of any one of claims 1 to 16, wherein one or more (e.g. 1, 2 or 3) of the leucines are unsubstituted.

18. 18. The method of any one of claims 1 to 17, wherein one or more (e.g. one, two or three) of the isoleucines are unsubstituted.

19. 19. The method of any one of claims 1 to 18, wherein one or more (e.g., one, two or three) of the valines are unsubstituted.

20. 20. The method of any one of claims 1 to 19, wherein one or more (e.g. 1, 2 or 3) of the phenylalanines are unsubstituted.

21. The method of any one of claims 1 to 20, further comprising the step of producing / expressing said combination mutant.

22. 22. The method of any one of claims 1 to 21, further comprising testing the combination mutants for ligand binding (e.g. in a yeast two-hybrid assay), wherein those having substantially the same ligand binding compared to the GPCR are selected.

23. The method of any one of claims 1 to 22, further comprising a step of testing the combination mutants for the biological function of the GPCR, wherein those having substantially the same biological function compared to the GPCR are selected.

24. 24. The method of any one of claims 1 to 23, wherein the combinatorial mutant library comprises less than about 2 million members.

25. A method according to any one of claims 1 to 24, wherein the sequence of the GPCR comprises information about the TM region of the GPCR.

26. A method according to any one of claims 1 to 25, wherein the sequence of the GPCR is obtained from a protein structure database (e.g. PDB, UniProt).

27. The method according to any one of claims 1 to 26, wherein the TM region of the GPCR is predicted based on the sequence of the GPCR.

28. 28. The method of claim 27, wherein the TM region of the GPCR is predicted using the TMHMM2.0 (Transmembrane Prediction using Hidden Markov Models) software module.

29. 30. The method of claim 28, wherein the TMHMM2.0 software module utilizes a dynamic baseline for peak searching.

30. A method according to any one of claims 1 to 29, further comprising the step of providing a polynucleotide sequence for each mutant of said GPCR.

31. 31. The method of claim 30, wherein the polynucleotide sequence is codon optimized for expression in a host (e.g., a bacterium such as E. coli, a yeast such as Saccharomyces cerevisiae or Schizosaccharomyces pombe, an insect cell such as an Sf9 cell, a non-human mammalian cell, or a human cell).

32. The method of any one of claims 1 to 31, wherein the scripted procedure comprises a VBA script.

33. The method of any one of claims 1 to 32, wherein the scripting procedure is operable with a Linux system (e.g. Ubuntu 12.04 LTS), a Unix system, a Microsoft Windows operating system, an Android operating system or an Apple iOS operating system.

34. 1. A water-soluble mutant of a G protein-coupled receptor (GPCR) comprising multiple hydrophobic amino acid substitutions within a transmembrane (TM) domain α-helical segment ("TM region") of the GPCR, (a) the hydrophobic amino acid is selected from the group consisting of leucine (L), isoleucine (I), valine (V) and phenylalanine (F); (b) each leucine (L) is independently replaced with glutamine (Q), asparagine (N), or serine (S); (c) said isoleucine (I) and said valine (V) are each independently replaced with threonine (T), asparagine (N) or serine (S); and (d) each phenylalanine is replaced with a tyrosine (Y), followed by all seven TM regions of the mutant maintain an α-helical secondary structure; and No predicted transmembrane domain A water-soluble mutant characterized in that

35. 35. The water-soluble variant of claim 34, comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 4-11, 13-20, 22-29, 31-38, 40-47, 49-56 and 58-64.

36. 36. The water-soluble variant of claim 35, further comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 3, 12, 21, 30, 39, 48 and 57.

37. 37. A water-soluble variant of claim 35 or 36, which binds to a CXCR4 ligand.

38. 35. The water-soluble variant of claim 34, comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 69-76, 78-85, 87, 89-96, 98-105, 107-114 and 116-123.

39. 39. The water-soluble variant of claim 38, further comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 68, 77, 86, 88, 97, 106, 115 and 124.

40. 41. A water-soluble variant of claim 38 or 40 which binds to a CX3CR1 ligand.

41. 35. The water-soluble variant of claim 34, comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 128-135, 137-144, 146-153, 155-162, 164-171, 173 and 175-182.

42. 42. The water-soluble variant of claim 41, further comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 127, 136, 145, 154, 163, 172, 174 and 183.

43. 43. A water-soluble variant of claim 41 or 42 which binds to a CCR3 ligand.

44. The water-soluble variant of claim 34, comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 187-194, 196-203, 205-206, 208, 210-217, 219-225, and 227-234.

45. 45. The water-soluble variant of claim 44, further comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 186, 195, 204, 207, 209, 218, 226 and 235.

46. 46. ​​A water-soluble variant of claim 44 or 45, which binds to a CCR5 ligand.

47. 35. The water-soluble variant of claim 34, comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 236-243, 245-252, 254-261, 263-270, 272, 274-281 and 283-290.

48. 48. The water-soluble variant of claim 47, further comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 235, 244, 253, 262, 271, 273, 282 and 291.

49. 49. A water-soluble variant of claim 47 or 48, which binds to a CXCR3 ligand.

50. 35. The water-soluble variant of claim 34, comprising one or more transmembrane domains set forth in any one of SEQ ID NOs: 2, 67, 126, 185, 327, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323 or 325.

51. 51. The water-soluble variant of claim 50, wherein the water-soluble variant is water-soluble and binds to a ligand of a homologous native transmembrane protein.

52. (a) culturing bacteria in a growth medium under conditions suitable for protein production; (b) fractionating the bacterial lysate to produce a soluble fraction and an insoluble pellet fraction; (c) isolating the protein from the soluble fraction; Including, (1) The protein is a mutant of a G protein-coupled receptor (GPCR) according to any one of claims 29 to 46, (2) the protein yield is at least 20 mg / L (e.g., 30 mg / L, 40 mg / L, 50 mg / L or more) of growth medium; 1. A method for producing a protein in a bacterium (e.g., Escherichia coli), comprising:

53. 48. The method of claim 47, wherein the bacterium is E. coli BL21 and the growth medium is LB medium.

54. 49. The method of claim 47 or 48, wherein the protein is encoded by a plasmid within the bacterium.

55. The method of any one of claims 47 to 49, wherein expression of the protein is under the control of an inducible promoter.

56. 51. The method of claim 50, wherein the inducible promoter is inducible by IPTG.

57. 52. The method of any one of claims 47 to 51, wherein the lysate is produced by sonication.

58. 53. The method of any one of claims 47-52, wherein the lysate is centrifuged at 14,500 x g or more to generate the soluble fraction.

59. A non-transitory computer readable medium having stored thereon a set of instructions for performing the method according to any one of claims 1 to 33.

60. A data processing system operative to select water-soluble variants of a membrane protein, the system comprising a data processor operative to perform amino acid substitutions and to rank the protein variants according to a ranking function including secondary structure components and water-solubility components.

61. 61. The system of claim 60, further comprising a library of membrane proteins for processing by the system.

62. 61. The system of claim 60, further comprising a memory coupled to the data processor storing coded instructions for executing a permutation processor.

63. 61. The system of claim 60, operative for steps (a), (b), (c) and (d) of the method of claim 1.

64. 61. The system of claim 60, further comprising a ranking function that is a weighted combination based on the secondary structure components.

65. 61. The system of claim 60, which communicates with an external program over a network.

66. 61. The system of claim 60, further comprising a database for storing water-soluble variants.

67. 61. The system of claim 60, further comprising instructions for performing dynamic baseline processing.

68. 61. The system of claim 60, further comprising an interface for selecting parameters of the method.

69. The system according to claim 60, further comprising the step of inputting a sequence according to any one of claims 35 to 50.

70. 1. A computer-implemented method for carrying out a procedure for selecting water-soluble mutants, comprising: processing the data to identify membrane protein sequences for analysis; obtaining a mutant of said membrane protein, in which a plurality of hydrophobic amino acids in the transmembrane (TM) domain α-helical segment ("TM region") of said membrane protein have been substituted; Including, The data processor: determining the secondary structure of the α-helix of the mutant to confirm maintenance of the secondary structure of the α-helix in the mutant; determining the transmembrane domain identity of the variant to confirm the water solubility of the variant; and Selecting a water-soluble mutant of the membrane protein A method comprising:

71. The substitution is (a) selecting the hydrophobic amino acid from the group consisting of leucine (L), isoleucine (I), valine (V) and phenylalanine (F); (b) independently replacing leucine (L) with glutamine (Q), asparagine (N), or serine (S); (c) independently replacing each of isoleucine (I) and valine (V) with threonine (T), asparagine (N) or serine (S); and (d) replacing each said phenylalanine with a tyrosine (Y).

71. The method of claim 70, comprising:

72. 72. The method of claim 71 , wherein one subset of said plurality of hydrophobic amino acids of the same TM region of a GPCR is substituted to generate one member of a mutant candidate library, and one or more different subsets of said plurality of hydrophobic amino acids are substituted to generate further members of said library.

73. 71. The method of claim 70, further comprising the step of ranking all members of the library based on a combined score, the combined score being a weighted combination of the alpha helix secondary structure prediction results and the transmembrane domain prediction results.

74. 71. The method of claim 70, further comprising ranking the variants using a ranking function.

75. 71. The method of claim 70, further comprising a memory coupled to the data processor.

76. 75. The method of claim 74, wherein the ranking function comprises a secondary structure component and a water solubility component.

77. 77. The method of claim 76, wherein the ranking function comprises weighting values ​​of the secondary structure components and / or the water-soluble components.

78. 74. The method of claim 73, further comprising selecting the N members having the highest combination scores to form a first library of mutant candidates for the TM region, where N is a predetermined integer (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more).

79. 79. The method of claim 78, further comprising generating one library of mutant candidates for one, two, three, four, five or all six other TM regions of the GPCR.

80. 80. The method of claim 79, further comprising replacing two or more TM regions of said GPCR with corresponding TM regions in said mutant candidate library to generate a combinatorial mutant library.

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