Résumé
Here we introduce an approach to mapping the folding transition state ensemble of proteins based on the pressure dependence of protein stability. Previously, we have shown that the activation volume for folding of wild type (WT) SNase is large and positive, and hence that the rate-limiting step in folding involves significant dehydration. In contrast, variants bearing buried ionizable residues at position 66 were shown recently to fold through a highly hydrated transition state ensemble (TSE). We present the effects on the pressure-jump folding kinetics of Lys substitutions in different internal positions throughout the structure. We calculate the Vi value of the variants as the activation volume for folding relative to that of the wild type. We find that the structure of the SNase WT includes part of the beta-barrel and part of the first alpha-helix. The unique advantage of Vi-value analysis is that it conveys direct information about the state of hydration of the TSE, which has been recognized as a key factor in the protein folding transition.