Method of residue proximity information and protein model discrimination using saturation-suppressor mutagenesis

A method for identifying functionally interacting amino acid residues in a protein of interest, wherein the method identifies suppressors of single amino acid mutants in the 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 from the first library, wherein said first subpopulation comprises a population enriched in single amino acid substitution 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;
• (d) identifying a second subpopulation from said second library, comprising variants, each of which has two amino acid substitutions in said protein of interest;
• (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 a variant member of first library is a suppressor of the substituted amino acid of first subpopulation.