Denaturant-induced movement of the transition state of protein folding revealed by high-pressure stopped-flow measurements

摘要

The small all-p protein tendamistat folds and unfolds with two-state kinetics. We determined the volume changes associated with the folding process by performing kinetic and equilibrium measurements at variable pressure between 0.1 and 100 MPa (1 to 1,000 bar). GdmCl-induced equilibrium unfolding transitions reveal that the volume of the native state is increased by 41 .4 ± 2.0 cm~3/mol relative to the unfolded state. This value is virtually independent of denatur- ant concentration. The use of a high-pressure stopped-flow instru- ment enabled us to measure the activation volumes for the refolding and unfolding reaction over a broad range of GdmCl concentrations. The volume of the transition state is 60% native-like in the absence of denaturant, indicating partial solvent accessibility of the core residues. The volume of the transition state increases linearly with denaturant concentration and exceeds the volume of the native state above 6 M GdmCl. This result argues for a largely desolvated transition state with packing defi- ciencies at high denaturant concentrations and shows that the struc- ture of the transition state depends strongly on the experimental conditions.