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

The small all-beta protein tendamistat folds and unfolds with two-state kin etics. We determined the volume changes associated with the folding process by performing kinetic and equilibrium measurements at variable pressure be tween 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 denaturant concentration, The use of a high-pressure stoppe d-flow instrument enabled us to measure the activation volumes for the refo lding (Delta V(f)(0)double dagger) and unfolding reaction (Delta V(u)(0)dou ble dagger) over a broad range of GdmCl concentrations. The volume of the t ransition state is 60% native-like (Delta V(f)(0)double dagger = 25.0 +/- 1 .2 cm(3)/mol) in the absence of denaturant, indicating partial solvent acce ssibility of the core residues, The volume of the transition state increase s linearly with denaturant concentration and exceeds the volume of the nati ve state above 6 M GdmCl. This result argues for a largely desolvated trans ition state with packing deficiencies at high denaturant concentrations and shows that the structure of the transition state depends strongly on the e xperimental conditions.