Claims:1. A method for identifying functionally interacting amino acid residues in a protein of interest, said method comprising:
a. obtaining a first library of single mutation variants of said protein of interest, wherein said each variant contains an amino acid substitution at a single residue position, and at least 70% of each possible amino acid substitution at each residue position of said protein of interest is represented in said first library;
b. identifying a first subpopulation of variants, wherein said first subpopulation comprises a population enriched in variants, each of which decreases the activity of said protein of interest compared to wild-type levels;
c. introducing at least five mutations at different residue positions from said first subpopulation singly in to each variant of the first library to obtain a second library of variants having two or one amino acid substitution in said protein of interest;
d. identifying a second subpopulation from said second library, comprising variants, each of which has two amino acid substitutions;
e. identifying a third subpopulation, which is a subset of the said second subpopulation of said second library, wherein in each variant of said third subpopulation, the mutation as identified from each variant of said first library suppresses the altered activity of the variant of the first subpopulation,
wherein the two substituted amino acids in each variant of the said third subpopulation are indicative that the two amino acids at corresponding positions in wild-type said protein of interest are functionally interacting.

2. A method for identifying suppressors of single amino acid mutants in a protein of interest, said method comprising:
a. obtaining a first library of single mutation variants of said protein of interest, wherein said each variant contains an amino acid substitution at a single residue position, and at least 70% of each possible amino acid substitution at each residue position of said protein of interest is represented in said first library;
b. identifying a first subpopulation of variants, wherein said first subpopulation comprises a population enriched in variants, each of which alters the activity of said protein of interest compared to wild-type levels;
c. introducing at least five mutations at different residue positions from said first subpopulation singly in to each variant of the first library to obtain a second library of variants having two or one amino acid substitution in said protein of interest;
d. identifying a second subpopulation from said second library, comprising variants, each of which has two amino acid substitutions;
e. identifying a third subpopulation, which is a subset of the said second subpopulation of said second library, wherein in each variant of said third subpopulation, the mutation as identified from each variant of said first library suppresses the altered activity of the variant of the first subpopulation,
wherein the two substituted amino acids in each variant of said third subpopulation are indicative that the substituted amino acid of variant member of first library is a suppressor of the substituted amino acid of first subpopulation.

3. The method as claimed in claim 2, wherein in each variant of the third subpopulation, substituted amino acid of variant member of first library is a distal suppressor, said distal suppressor is able to suppress the altered activity of more than one variant comprising a single substituted amino acid as identified in the said first subpopulation, wherein the said variants of the first subpopulation have amino acid substitutions in different residue positions, and said amino acid substitutions in different residues do not suppress each other.

4. The method as claimed in claim 2, wherein in each variant of the third subpopulation the substituted amino acid of variant member of first library is a proximal suppressor, wherein said proximal suppressor is able to suppress the altered activity of at least one variant comprising a single substituted amino acid as identified in the said first subpopulation.

5. A method for predicting the structure of a protein of interest, said method comprising:
a. obtaining a first library of single mutation variants of said protein of interest, wherein said each variant contains an amino acid substitution at a single residue position, and at least 70% of each possible amino acid substitution at each residue position of said protein of interest is represented in said first library;
b. identifying a first subpopulation of variants, wherein said first subpopulation comprises a population enriched in variants, each of which alters the activity of said protein of interest compared to wild-type levels;
c. introducing at least five mutations at different residue positions from said first subpopulation singly in to each variant of the first library to obtain a second library of variants having two or one amino acid substitution in said protein of interest;
d. identifying a second subpopulation from said second library, comprising variants, each of which has two amino acid substitutions;
e. identifying a third subpopulation, which is a subset of the said second subpopulation of said second library, wherein in each variant of said third subpopulation, the mutation as identified from each variant of said first library suppresses the altered activity of the variant of the first subpopulation; and
f. generating a predictive structure of said protein of interest based on identification of pairs of interacting amino acid residues in each variant of the third subpopulation of claim 4.

6. A method for identification of amino acid residues and residue positions which modulate thermal stability of a protein of interest, said method comprising:
a. obtaining a first library of single mutation variants of said protein of interest, wherein said each variant contains an amino acid substitution at a single residue position, and at least 70% of each possible amino acid substitution at each residue position of said protein of interest is represented in said first library;
b. identifying a first subpopulation of variants, wherein said first subpopulation comprises a population enriched in variants, each of which alters the activity of said protein of interest compared to wild-type levels;
c. introducing at least five mutations at different residue positions from said first subpopulation singly in to each variant of the first library to obtain a second library of variants having two or one amino acid substitution in said protein of interest;
d. identifying a second subpopulation from said second library, comprising variants, each of which has two amino acid substitutions;
e. identifying a third subpopulation, which is a subset of the said second subpopulation of said second library, wherein in each variant of said third subpopulation, the mutation as identified from each variant of said first library suppresses the altered activity of the variant of the first subpopulation, and in each variant of the third subpopulation, the substituted amino acid of variant member of first library is a distal suppressor, said distal suppressor is able to suppress the altered activity of more than one variant comprising a single substituted amino acid as identified in the said first subpopulation, wherein the said variants of the first subpopulation have amino acid substitutions in different residue positions, and said amino acid substitutions in different residues do not suppress each other,
wherein said mutation as identified from each variant of said first library is an amino acid which at the particular residue position modulates thermal stability of said protein of interest.

7. The method as claimed in any of the claims 1-6, wherein said protein of interest can be a single protein, or a multi-protein complex.

8. The method as claimed in any of the claims 1-6, wherein said amino acid substitution is a naturally occurring amino acid, or a synthetic amino acid.

9. The method as claimed in any of the claims 1-8, wherein said protein of interest has amino acid sequence selected from the group consisting of SEQ ID NO: 1, and SEQ ID NO: 2.

10. The method as claimed in claim 1, wherein said first library of single mutation variants is as described in the instant specification
, Description:As Attached