WATER-SOLUBLE TRANS-MEMBRANE PROTEINS AND METHODS FOR THE PREPARATION AND USE THEREOF

1 . A computer implemented method for executing a procedure to select a water-soluble variant of a G Protein-Coupled Receptor (GPCR), the method comprising:

(1) entering a sequence of the GPCR for analysis;

(2) obtaining a variant of the GPCR, wherein a plurality of hydrophobic amino acids in the transmembrane (TM) domain alpha-helical segments (“TM regions”) of the GPCR are substituted, wherein:

(a) said hydrophobic amino acids are selected from the group consisting of Leucine (L), Isoleucine (I), Valine (V), and Phenylalanine (F);

(b) each said Leucine (L) is independently substituted by Glutamine (Q), Asparagine (N), or Serine (S);

(c) each said Isoleucine (I) and said Valine (V) are independently substituted by Threonine (T), Asparagine (N), or Serine (S); and,

(d) each said Phenylalanine is substituted by Tyrosine (Y); and, subsequently,

(3) obtaining an α-helical secondary structure result for the variant to verify maintenance of α-helical secondary structures in the variant; and

(4) obtaining a trans-membrane region result for the variant to verify water solubility of the variant,

thereby selecting the water-soluble variant of the GPCR.

2 . The method of claim 1 , wherein step (3) is performed prior to, concurrently with, or after step (4).

3 . The method of claim 1 , wherein in step (2), one subset of said plurality of hydrophobic amino acids in one and the same TM region of the GPCR are substituted to generate one member of a library of potential variants, and one or more different subsets of said plurality of hydrophobic amino acids are substituted to generate additional members of the library.

4 . The method of claim 3 , further comprising ranking all members of said library based on a combined score, wherein the combined score is a weighed combination of the α-helical secondary structure prediction result and the trans-membrane region prediction result.

5 . The method of claim 1 further comprising ranking the variant using a ranking function.

6 - 7 . (canceled)

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

9 . The method of claim 8 wherein the ranking function includes a weighting value for the secondary structure component and/or the water solubility component.

10 . The method of claim 4 , further comprising: selecting N members with the highest combined scores to form a first library of potential variants for said TM region, wherein N is a pre-determined 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 claim 10 , further comprising generating one library of potential variants for 1, 2, 3, 4, 5, or all 6 other TM regions of the GPCR.

12 . The method of claim 11 further comprising: replacing two or more TM regions of the GPCR with corresponding TM regions from the libraries of potential variants, to create a library of combinatory variants.

13 . The method of claim 1 , wherein substantially all said leucines are substituted by glutamines, substantially all said isoleucines are substituted by threonines, substantially all said valines are substituted by threonines, or substantially all said phenylalanines are substituted by tyrosines.

14 - 16 . (canceled)

17 . The method of claim 1 , wherein one or more said leucines are not substituted, one or more said isoleucines are not substituted, one or more said valines are not substituted, or one or more said phenylalanines are not substituted.

18 - 20 . (canceled)

21 . The method of claim 1 , further comprising producing/expressing said combinatory variants.

22 . The method of claim 1 , further comprising testing said combinatory variants for ligand binding (e.g., in yeast two-hybrid system), wherein those having substantially the same ligand binding compared to that of the GPCR are selected.

23 . The method of claim 1 , further comprising testing said combinatory variants for a biological function of the GPCR, wherein those having substantially the same biological function compared to that of the GPCR are selected.

24 - 26 . (canceled)

27 . The method of claim 1 , wherein the TM regions of the GPCR are predicted based on the sequence of the GPCR.

28 . The method of claim 27 , wherein the TM regions of the GPCR are predicted using TMHMM 2.0 (TransMembrane prediction using Hidden Markov Models) software module.

29 - 33 . (canceled)

34 . A water-soluble variant of a G Protein-Coupled Receptor (GPCR), wherein:

a plurality of hydrophobic amino acids in the transmembrane (TM) domain alpha-helical segments (“TM regions”) of the GPCR are substituted, wherein:

(a) said hydrophobic amino acids are selected from the group consisting of Leucine (L), Isoleucine (I), Valine (V), and Phenylalanine (F);

(b) each said Leucine (L) is independently substituted by Glutamine (Q), Asparagine (N), or Serine (S);

(c) each said Isoleucine (I) and said Valine (V) are independently substituted by Threonine (T), Asparagine (N), or Serine (S); and,

(d) each said Phenylalanine is substituted by Tyrosine (Y); and, subsequently, all seven TM regions of the variant maintains α-helical secondary structures; and, there is no predicted trans-membrane region.

35 - 58 . (canceled)

59 . A non-transitory computer readable medium having stored thereon a sequence of instructions to perform the method of any of claims claim 1 .

60 . A data processing system operative to select a water soluble variant of a G protein coupled receptor comprising:

a data processor operative to perform substitution of amino acids as claimed in claim 1 wherein the processor ranks a protein variant with a ranking function; and

a memory connected to the data processor that stores a substituted amino acid structure and a rank.