Phi value analysis

Phi value analysis, ϕ {displaystyle phi } analysis, or ϕ {displaystyle phi } -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 like protein NMR or X-ray crystallography because folding transitions states are mobile and partly unstructured by definition. In ϕ {displaystyle phi } -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.Phi is defined thus:Alan Fersht pioneered phi value analysis in his study of the small bacterial protein barnase. Using molecular dynamics simulations, he found that the transition state between folding and unfolding looks like the native state and is the same no matter the reaction direction. Phi varied with the mutation location as some regions gave values near zero and others near one. The distribution of ϕ {displaystyle phi }   values throughout the protein's sequence agreed with all of the simulated transition state but one helix which folded semi-independently and made native-like contacts with the rest of the protein only once the transition state had formed fully. Such variation in the folding rate in one protein makes it hard to interpret ϕ {displaystyle phi }   values as the transition state structure must otherwise be compared to folding-unfolding simulations which are computationally expensive.Other 'kinetic perturbation' techniques for studying the folding transition state have appeared recently. Best known is the psi ( ψ {displaystyle psi }  ) value which is found by engineering two metal-binding amino acid residues like histidine into a protein and then recording the folding kinetics as a function of metal ion concentration, though Fersht thought this approach difficult. A 'cross-linking' variant of the ϕ {displaystyle phi }  -value was used to study segment association in a folding transition state as covalent crosslinks like disulfide bonds were introduced.The error in equilibrium stability and aqueous (un)folding rate measurements may be large when values of ϕ {displaystyle phi }   for solutions with denaturants must be extrapolated to aqueous solutions that are nearly pure or the stability difference between the native and mutant protein is 'low', or less than 7 kJ/mol. This may cause ϕ {displaystyle phi }   to fall beyond the zero-one range. Calculated values ϕ {displaystyle phi }   depend strongly on how many data point are available and lab conditions.