Phi value analysis, analysis, or -value analysis is an experimental protein engineering technique for studying the structure of the folding transition state of small protein domains that fold in a two-state manner. The structure of the folding transition state is hard to find using methods such as protein NMR or X-ray crystallography because folding transitions states are mobile and partly unstructured by definition. In -value analysis, the folding kinetics and conformational folding stability of the wild-type protein are compared with those of point mutants to find phi values. These measure the mutant residue's energetic contribution to the folding transition state, which reveals the degree of native structure around the mutated residue in the transition state, by accounting for the relative free energies of the unfolded state, the folded state, and the transition state for the wild-type and mutant proteins.
The protein's residues are mutated one by one to identify residue clusters that are well-ordered in the folded transition state. These residues' interactions can be checked by double-mutant-cycle analysis, in which the single-site mutants' effects are compared to the double mutants'. Most mutations are conservative and replace the original residue with a smaller one (cavity-creating mutations) like alanine, though tyrosine-to-phenylalanine, isoleucine-to-valine and threonine-to-serine mutants can be used too. Chymotrypsin inhibitor, SH3 domains, WW domain, individual domains of proteins L and G, ubiquitin, and barnase have all been studied by analysis.