期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2010
卷号：107
期号：19
页码：8611-8616
DOI：10.1073/pnas.1000988107
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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: [IMG]/medium/pnas.1000988107eq1.gif" ALT="Formula ">. 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.
