Identifying critical residues in protein folding: Insights from phi-value and Pfold analysis

Abstract

We apply a simulational proxy of the phi-value analysis and perform extensive mutagenesis experiments to identify the nucleating residues in the folding reactions of two small lattice Go polymers with different native geometries. These results are compared with those obtained from an accurate analysis based on the reaction coordinate folding probability Pfold, and on structural clustering methods. For both protein models, the transition state ensemble is rather heterogeneous and splits-up into structurally different populations. For the more complex geometry the identified subpopulations are actually structurally disjoint. For the less complex native geometry we found a broad transition state with microscopic heterogeneity. For both geometries, the identification of the folding nucleus via the Pfold analysis agrees with the identification of the folding nucleus carried out with the phi-value analysis. For the most complex geometry, however, the apllied methodologies give more consistent results than for the more local geometry. The study of the transition state' structure reveals that the nucleus residues are not necessarily fully native in the transition state. Indeed, it is only for the more complex geometry that two of the five critical residues show a considerably high probability of having all its native bonds formed in the transition state. Therefore, one concludes that in general the phi-value correlates with the acceleration/deceleration of folding induced by mutation, rather than with the degree of nativeness of the transition state, and that the traditional interpretation of phi-values may provide a more realistic picture of the structure of the transition state only for more complex native geometries.