|
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. ==Mathematical definition== The phi value is defined as:〔Daggett V, Fersht AR. (2000). Transition states in protein folding. In ''Mechanisms of Protein Folding'' 2nd ed, editor RH Pain. Oxford University Press.〕 where represents the energy difference between the transition state and the denatured state for the wild-type protein, represents this energy difference for the mutant protein, and the terms represent the energy difference between the native state and the denatured state. Thus, the phi value represents the ratio of the energetic destabilization introduced by the mutation to the transition state versus that introduced to the native folded state. The phi value should range from 0 to 1, but also negative phi values can be observed.〔http://www.ncbi.nlm.nih.gov/pubmed/16142914〕 A phi value of 0 implies that the mutation has no effect on the structure of the rate-limiting transition state on the folding pathway, while a phi value of 1 indicates that the degree to which the transition state is destabilized by the mutation is exactly equal to the degree to which the folded state is destabilized. A phi value near 0 suggests that the region surrounding the mutation is relatively unfolded or unstructured in the transition state, while a value near 1 implies that the local structure around the mutation site in the transition state closely resembles the structure in the native state. It is generally the case that conservative substitutions on the surface of a protein yield phi values near 1. When the phi value is intermediate between 0 and 1, the method cannot directly distinguish between the alternative hypotheses that the transition state is partially structured, or that there are two populations of mostly-unfolded and mostly-folded states. 抄文引用元・出典: フリー百科事典『 ウィキペディア（Wikipedia）』 ■ウィキペディアで「Phi value analysis」の詳細全文を読む スポンサード リンク
|