Distinct unfolding and refolding pathways of ribonuclease a revealed by heating and cooling temperature jumps.

Heating and cooling temperature jumps (T-jumps) were performed using a newly developed technique to trigger unfolding and refolding of wild-type ribonuclease A and a tryptophan-containing variant (Y115W). From the linear Arrhenius plots of the microscopic folding and unfolding rate constants, activation enthalpy (DeltaH(#)), and activation entropy (DeltaS(#)) were determined to characterize the kinetic transition states (TS) for the unfolding and refolding reactions. The single TS of the wild-type protein was split into three for the Y115W variant. Two of these transition states, TS1 and TS2, characterize a slow kinetic phase, and one, TS3, a fast phase. Heating T-jumps induced protein unfolding via TS2 and TS3; cooling T-jumps induced refolding via TS1 and TS3. The observed speed of the fast phase increased at lower temperature, due to a strongly negative DeltaH(#) of the folding-rate constant. The results are consistent with a path-dependent protein folding/unfolding mechanism. TS1 and TS2 are likely to reflect X-Pro(114) isomerization in the folded and unfolded protein, respectively, and TS3 the local conformational change of the beta-hairpin comprising Trp(115). A very fast protein folding/unfolding phase appears to precede both processes. The path dependence of the observed kinetics is suggestive of a rugged energy protein folding funnel.