A sequential model of nucleation-dependent protein folding: Kinetic studies of ribonuclease A

A simple sequential model of nucleation-dependent protein folding is presented here, together with some kinetic studies of the reversible thermal unfolding of ribonuclease A. The relevant properties of the simple sequential model are as follows. (1) Nucleation limits the rate of in vitro refolding; (2) there is a definite sequence of steps in the normal pathway of unfolding and refolding; (3) unfolding and refolding are co-operative reactions: the co-operativity depends on the magnitude of the equilibrium constant for nucleation and on the number of steps in folding; (4) by several criteria, unfolding by the simple sequential model approximates a two-state unfolding even when there are sizeable concentrations of stable, partly folded intermediates; and (5) a critical prediction of the simple sequential model (see Appendix) is the approximate division of the kinetics of unfolding into two phases: a rapid transient phase, followed by a slow steady-state unfolding with a single exponential time course. If unfolding is strongly co-operative, the transient phase is predicted to have a small amplitude and to be faster by several orders of magnitude than the steady-state unfolding. The kinetic studies have measured the slow unfolding and refolding reactions of ribonuclease A (-SS- bridges intact) by the change in absorbance at 287 nm, which reflects the exposure to solvent of buried tyrosine groups. The slow reaction follows a single exponential time course at neutral pH, although equilibrium studies by others show significant deviations from two-state behavior in this pH range. This observation can be explained by the model, and provides the chief reason for presenting it. At pH 1.3 some complexity can be detected in the slow unfolding reaction within the first one-third of the thermal transition zone (in agreement with the 1963 results of Scott & Scheraga), although approximate two-state kinetic behavior is found by us in the upper two-thirds of the thermal transition zone. These results are not explained by the simple sequential model, and indicate that more complex models will have to be considered. Equilibrium studies by others have shown a close correspondence to a two-state reaction throughout the thermal transition zone in the pH range 1 to 2.