A complete description of the mechanisms of protein folding requires knowledge of the structural and physical character of the folding transition state ensembles (TSEs). A key question concerning the role of hydration of the hydrophobic core in determining folding mechanisms remains. 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 the hydrophobic core, Val-66, was replaced with polar or ionizable residues (Lys, Arg, His, Asp, Glu, and Asn). Because of 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 in the unfolding rate due to a large negative activation volume for unfolding. This results from a significant increase in the level of 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.
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