Hydration of the Folding Transition State Ensemble of a Protein†

Ludovic Brun, Daniel G. Isom, Priya Velu, Bertrand García-Moreno, Catherine Ann Royer
2006 Biochemistry  
A complete description of the mechanisms of protein folding requires knowledge of the structural and physical character of the folding transition state ensembles (TSE). A key question remains, concerning the role of hydration of the hydrophobic core in determining folding mechanisms. To address this we probed the state of hydration of the TSE of staphylococcal nuclease (SNase) by examining the fluorescence-detected pressure-jump relaxation behavior of six SNase variants in which a residue in
more » ... hydrophobic core, Val-66, was replaced with polar or ionizable residues (Lys, Arg, His, Asp, Glu, Asn). Owing to a large positive activation volume for folding, the major effect of pressure on the wild type protein is to decrease the folding rate. By the time wild type SNase reaches the folding transition state, most water has already been expelled from its hydrophobic core. In contrast, the major effect of pressure on the variant proteins is an increase of the unfolding rate due to a large negative activation volume for unfolding. This results from a significant increase in the hydration of the TSE when an internal ionizable group is present. These data confirm that the role of water in the folding reaction can differ from protein to protein, and that even a single substitution in a critical position can modulate significantly the properties of the TSE. The exponential nature of protein folding kinetics suggests the existence of rate-limiting energy barriers between the folded and the unfolded states. The lack of detailed understanding of this barrier and of the attendant rate-limiting ensemble of states still limits our ability to describe mechanisms of folding in detail. Although use of ϕ-value analysis (1-4) to describe the character of the folding transition state ensembles (TSE) remains controversial (5;6), a self-consistent view is emerging (7-12). Considerable differences in the TSE of different proteins have been observed (13;14), but in general, native-like structures are thought to dominate the TSE, possibly even with many side chains already fully dehydrated but with few of them satisfying their native contacts (5;6;15). Computational analyses of ϕ values have influenced significantly the prevailing views on the structural character of the TSE (16-28), which is thought to be determined primarily by the topology of the protein, not by detailed energetic factors (17;24;29;30). Some computational studies suggest that TSEs consist mostly of expanded and distorted native
doi:10.1021/bi052638z pmid:16533028 pmcid:PMC4442614 fatcat:bjet2yeeznc4vh2e4ypvllku64