Multiple pathways for regenerating ribonuclease A.

Abstract

This paper is concerned with the pathways for the regeneration of RNase A from the reduced protein by a mixture of GSSG and GSH. Experimental work on the regeneration has led to the identification of several different pathways, depending on the concentrations of GSH and GSSG, and an energetic analysis has provided information about the stabilities of the various intermediates. The equilibrium and kinetic data for the regeneration process have led to two models of protein-folding pathways. The intermediates in the regeneration process were trapped without chemical modification, and were fractionated on a carboxymethyl-cellulose column. The regeneration pathway(s) could be represented in terms of two simple reactions (Eqs. (1) and (2)). The system rapidly reaches a pre-equilibrium among the intermediates prior to the rate-limiting steps, and the concentrations of the intermediates (and hence the equilibrium constants among them) were determined. The regeneration process was also re-started from several of the isolated intermediates, and led to the predicted distribution of intermediates in the pre-equilibrium. Kinetic data, obtained by following the time dependence of the regain of enzymatic activity, together with the distributions of the intermediates at pre-equilibrium, led to the identification of the rate limiting steps, which differed according to the concentrations of GSH and GSSG. The relative apparent standard state conformational chemical potentials of the intermediates were estimated by using data for the apparent equilibrium constants (among the species in pre-equilibrium) and for the redox potentials of cysteine/cystine and GSH/GSSG. The two models deduced from the equilibrium and kinetic data are designated as growth-type and rearrangement-type models. In the growth-type model, nucleation of the native-like structure occurs in the folding process, in the rate-limiting step(s), and subsequent folding around the nucleation sites proceeds smoothly to form the native disulfide bonds and conformation. In the rearrangement-type model, proper nucleation does not occur in the folding process; instead, non-native interactions play a significant role in the folding pathways and lead to metastable intermediate species. Such non-native interactions must be disrupted or rearranged to nucleate the native interactions (in the rate limiting step(s)) for the protein to fold. Other protein foldings, reported in the literature, can be shown to conform to this model.