title: Insights into protein folding mechanisms from large scale analysis of mutational effects.
abstract: Protein folding mechanisms are probed experimentally using single-point mutant perturbations. The relative effects on the folding (phi-values) and unfolding (1 - phi) rates are used to infer the detailed structure of the transition-state ensemble (TSE). Here we analyze kinetic data on > 800 mutations carried out for 24 proteins with simple kinetic behavior. We find two surprising results: (i) all mutant effects are described by the equation: DeltaDeltaG(double dagger)(unfold)=0.76DeltaDeltaG(eq) +/- 1.8kJ/mol. Therefore all data are consistent with a single phi-value (0.24) with accuracy comparable to experimental precision, suggesting that the structural information in conventional phi-values is low. (ii) phi-values change with stability, increasing in mean value and spread from native to unfolding conditions, and thus cannot be interpreted without proper normalization. We eliminate stability effects calculating the phi-values at the mutant denaturation midpoints; i.e., conditions of zero stability (phi(0)). We then show that the intrinsic variability is phi(0) = 0.36 +/- 0.11, being somewhat larger for beta-sheet-rich proteins than for alpha-helical proteins. Importantly, we discover that phi(0)-values are proportional to how many of the residues surrounding the mutated site are local in sequence. High phi(0)-values correspond to protein surface sites, which have few nonlocal neighbors, whereas core residues with many tertiary interactions produce the lowest phi(0)-values. These results suggest a general mechanism in which the TSE at zero stability is a broad conformational ensemble stabilized by local interactions and without specific tertiary interactions, reconciling phi-values with many other empirical observations.
authors.affiliation: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, Madrid 28040, Spain.
authors.forename: Athi N
authors.initials: AN
authors.lastname: Naganathan
authors.forename: Victor
authors.initials: V
authors.lastname: Muñoz
mesh_terms: D030562:Databases, Protein; D009154:Mutation / Q000235:genetics*; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D011506:Proteins