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Abstract

The cold-shock protein CspB folds rapidly in a N reversible arrow U two-state reaction via a transition state that is about 90% native in its interactions with denaturants and water. This suggested that the energy barrier to unfolding is overcome by processes occurring in the protein itself, rather than in the solvent. Nevertheless, CspB unfolding depends on the solvent viscosity. We determined the activation volumes of unfolding and refolding by pressure-jump and high-pressure stopped-flow techniques in the presence of various denaturants. The results obtained by these methods agree well. The activation volume of unfolding is positive (DeltaV(NU)double dagger = 16(+/-4) ml/mol) and virtually independent of the nature and the concentration of the denaturant. We suggest that in the transition state the protein is expanded and water molecules start to invade the hydrophobic core. They have, however, not yet established favorable interactions to compensate for the loss of intra-protein interactions. The activation volume of refolding is positive as well (DeltaV(UN)double dagger = 53(+/- 6) ml/mol) and, above 3 M urea, independent of the concentration of the denaturant. At low concentrations of urea or guanidinium thiocyanate, DeltaV(UN)double dagger decreases significantly, suggesting that compact unfolded forms become populated under these conditions. (C) 2002 Elsevier Science Ltd. All rights reserve