Kinetic analysis of the reversible folding reactions of small proteins: Application to the folding of lysozyme and cytochrome c

Abstract

AbstractA general kinetic analysis for unimolecular three‐species models is presented. Criteria are established for each model in order to allow selection among alternative models on the basis of their amplitude behavior. These criteria are applied to the previously reported kinetic data for the folding transitions of lysozyme and cytochrome c in guanidine hydrochloride. The degree of success in applying these criteria is found to depend on the extent to which the apparent rate constants, λ1 and λ2, approach each other in certain conditions. Ikai and Tanford have presented a similar kinetic analysis, although these authors considered only the case where no intermediate kinetic species was populated initially. For one protein system, namely, ribonuclease A (RNase A), at least one stable intermediate exists after unfolding outside of the equilibrium transition zone. The criteria developed by Ikai and Tanford cannot be applied to RNase A kinetics and, therefore, a more general analysis is presented. Previously published studies of the reversible folding kinetics of lysozyme and cytochrome c are reexamined using the current amplitude analysis, and it is found that both of these folding transitions can be described by the three‐species model, used previously to describe the folding kinetics of RNase A (U1 and U2 are two forms of unfolded enzyme and N is the native species). These results suggest that this three‐species model may provide a fairly general description of the reversible folding kinetics of small proteins. As an outgrowth of the current analysis, several suggestions for experimental design in studying protein folding will be presented and discussed.