V_i -Value Analysis:  A Pressure-Based Method for Mapping the Folding Transition State Ensemble of Proteins

Abstract

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 V i 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 β-barrel and part of the first α-helix. The unique advantage of V i -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.

Simulated pressure denaturation thermodynamics of ubiquitin

“…Our results also indicate that the volume difference is dominated by changes in the polar and positively charged residues. These results are consistent with some of the findings of Mitra et al that, in general, at ambient temperatures a negative sign comes about by a positive contribution from nonpolar groups that is outweighed by a negative contribution from polar groups as well as the hydration of cavities (not explicitly investigated here, but accounted for) [55]. However, the use of a fully extended denatured state clouds this comparison.…”

Remodeling of the Folding Free Energy Landscape of Staphylococcal Nuclease by Cavity-Creating Mutations

“…We have shown that the conformations at the folding barrier for WT and Δ+PHS resemble the main intermediate, with a collapsed and solvent excluded central core and a disrupted C-terminal helix. 7 This TSE must also be destabilized in I92A, and thus I92A likely folds through multiple alternate pathways involving the intermediates detected here.…”

Desolvation Barrier Effects Are a Likely Contributor to the Remarkable Diversity in the Folding Rates of Small Proteins

“…84 Recent explicit-water simulations of two-helix systems 78 has revealed an intimate relationship between the enthalpic contribution to the overall folding barrier and the activation volume of folding transition state determined from pressure-based experimental methods. 85 The The schematic molecular drawings illustrate the distances between the methane molecules (full circles) at the cm, db, and ssm positions vis-à-vis the size of a water molecule (dashed circles). For the example in this figure, the r cm distance, the db height ɛ db , and the ssm depth ɛ ssm in U(r) (continuous curve) are shown with values equal to those in the methane-methane PMF from atomic simulation (dashed curve).…”

“…Thus, formation of the helix dimer entails surmounting an "activation volume" (peak of volume increase as the two helices approach each other from large separation) of ≈ 55 mL/mol and ≈ 150 mL/mol, respectively, for a pair of 20-residue polyalanine and polyleucine helices (Fig. 3 85), suggesting that the extent of dehydration at the folding rate-limiting step of this protein may be similar to that typified by the dimerization of two rigid 20-residue polyalanine helices. This comparison between activation volume data from pressure experiments and from explicit-water simulation of many-body hydrophobic interactions provided further support to the hypothesis that the ratelimiting step of folding for some proteins likely involves large-scale, near-simultaneous hydrophobic burial.…”