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ABSTRACT
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 (ΔH^sup #^), and activation entropy (ΔS^sup #^) 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 ΔH^sup #^ 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^sup 114^ isomerization in the folded and unfolded protein, respectively, and TS3 the local conformational change of the β-hairpin comprising Trp^sup 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.
INTRODUCTION
How do proteins morph into their folded, active forms? By which mechanisms do they unfold? Despite decades of computational and experimental research efforts, these processes remain elusive. To study folding and unfolding kinetics, proteins are usually forced into a nonequilibrium state by a rapid change of pressure, temperature, or concentration of chemical denaturant. The analysis of the relaxation kinetics toward the new equilibrium position can then inform about the reaction mechanism and the transition state (TS1) ensemble.
Most often, these processes are triggered by rapid flow techniques, involving turbulent mixing with high concentrations of a chemical denaturant, such as urea or guanidine hydrochloride. Such studies are generally confined to timescales of 1 ms or longer. An alternative technique is the laserinduced temperature jump (T-jump) (1-9). Although the T-jump is used much less often, it provides two main advantages: 1), it does not require the introduction of extraneous reagent into the sample; and 2), it permits the observation of conformational changes on the nanosecond timescale. However, these advantages are hampered...