Folding of a pressure-denatured model protein.

The noncovalent complex formed by the association of two fragments of chymotrypsin inhibitor-2 is reversibly denatured by pressure in the absence of chemical denaturants. On pressure release, the complex returned to its original conformation through a biphasic reaction, with first-order rate constants of 0.012 and 0.002 s-1, respectively. The slowest phase arises from an interconversion of the pressure-denatured state, as revealed by double pressure-jump experiments. Below 5 microM, the process was concentration dependent with a second-order rate constant of 1,700 s-1 M-1. Fragment association at atmospheric pressure showed a similar break in the order of the reaction above 5 microM, but both first- and second-order folding/association rates are 2.5 times faster than those for the refolding of the pressure-denatured state. Although the folding rates of the intact protein and the association of the fragments displayed nonlinear Eyring behavior for the temperature dependence, refolding of the pressure-denatured complex showed a linear response. The negligible heat capacity of activation reflects a balance of minimal change in the burial of residues from the pressure-denatured state to the transition state. If we add the higher energy barrier in the refolding of the pressure-denatured state, the rate differences must lie in the structure of this state, which has to undergo a structural rearrangement. This clearly differs from the conformational flexibility of the isolated fragments or the largely unfolded denatured state of the intact protein in acid and provides insight into denatured states of proteins under folding conditions.