Folding of a pressure-denatured model protein

The noncovalent complex formed by the association of two fragments of chymo trypsin inhibitor-2 is reversibly denatured by pressure in the absence of c hemical denaturants, On pressure release, the complex returned to its origi nal conformation through a biphasic reaction, with first-order rate constan ts 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 pre ssure-jump experiments. Below 5 mu M, the process was concentration depende nt with a second-order rate constant of 1,700 s(-1) M-1. Fragment associati on at atmospheric pressure showed a similar break in the order of the react ion above 5 mu M, but both first- and second-order folding/association rate s are 2.5 times faster than those for the refolding of the pressure-denatur ed state. Although the folding rates of the intact protein and the associat ion of the fragments displayed nonlinear Eyring behavior for the temperatur e dependence, refolding of the pressure-denatured complex showed a linear r esponse. The negligible heat capacity of activation reflects a balance of m inimal change in the burial of residues from the pressure-denatured state t o the transition state. If we add the higher energy barrier in the refoldin g of the pressure-denatured state, the rate differences must lie in the str ucture of this state, which has to undergo a structural rearrangement. This clearly differs from the conformational flexibility of the isolated fragme nts or the largely unfolded denatured state of the intact protein in acid a nd provides insight into denatured states of proteins under folding conditi ons.