Phi Value Analysis

Phi value analysis is an experimental protein engineering method used to study the structure of the folding transition state in small protein domains that fold in a two-state manner. Since the folding transition state is by definition a transient and partially unstructured state, its structure is difficult to determine by traditional methods such as protein NMR or X-ray crystallography. In phi-value analysis, the folding kinetics and conformational folding stability of the wild-type protein are compared with those of one or more point mutants. This comparison yields a phi value (defined below) that seeks to measure the mutated residue's energetic contribution to the folding transition state (and thus the degree of native structure around the mutated residue in the transition state) from the relative free energies of the unfolded state, the folded state and the transition state for the wild-type and mutant proteins.

Typically, a high fraction of the protein's residues are mutated one by one to identify clusters of residues that are well-ordered in the folded transition state. The interactions of these residues can be validated using double-mutant-cycle phi analysis, in which the effects of the single mutants are compared with those of the double mutant. In general, the mutations are conservative and replace the original residue with a smaller one (cavity-creating mutations), most commonly alanine; however, others such as tyrosine-to-phenylalanine, isoleucine-to-valine and threonine-to-serine mutations are also used. Examples of proteins that have been studied by phi value analysis include chymotrypsin inhibitor, SH3 domains, individual domains of proteins L and G, ubiquitin, and barnase.