Phi value analysis
-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.
-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.
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.
is the difference in energy between the wild-type protein's transition and denatured state,
bits are the differences in energy between the native and denatured state.
Conservative substitutions on the protein's surface often give phi values near one.
is well between zero and one, it is less informative as it doesn't tell us which is the case: Alan Fersht pioneered phi value analysis in his study of the small bacterial protein barnase.
[4][5] 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.
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
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.
) value[6][7] 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,[8] though Fersht thought this approach difficult.
-value was used to study segment association in a folding transition state as covalent crosslinks like disulfide bonds were introduced.
-value analysis to measure the response of mutants as a function of temperature to separate enthalpic and entropic contributions to the transition state free energy.
[11] The error in equilibrium stability and aqueous (un)folding rate measurements may be large when values of
[13] A study of 78 mutants of WW domain with up to four mutations per residue has quantified what types of mutations avoid interference from native state flexibility, solvation, and other effects, and statistical analysis shows that reliable information about transition state perturbation can be obtained from large mutant screens.
