Coupling of conformational folding and disulfide-bond reactions in oxidative folding of proteins

The oxidative folding of proteins consists of conformational folding and di sulfide-bond reactions. These two processes are coupled significantly in fo lding-coupled regeneration steps, in which a single chemical reaction (the "forward" reaction) converts a conformationally unstable precursor species into a conformationally stable, disulfide-protected successor species. Two limiting-case mechanisms for folding-coupled regeneration steps are describ ed. In the folded-precursor mechanism, the precursor species is preferentia lly folded at the moment of the forward reaction. The (transient) native st ructure increases the effective concentrations of the reactive thiol and di sulfide groups, thus favoring the forward reaction. By contrast, in the qua si-stochastic mechanism, the forward reaction occurs quasi-stochastically i n an unfolded precursor; i.e., reactive groups encounter each other with a probability determined primarily by loop entropy, albeit modified by confor mational biases in the unfolded state. The resulting successor species is i nitially unfolded, and its folding competes with backward chemical reaction s to the unfolded precursors. The folded-precursor and quasi-stochastic mec hanisms may be distinguished experimentally by the dependence of their kine tics on factors affecting the rates of thiol-disulfide exchange and conform ational (un)folding. Experimental data and structural and biochemical argum ents suggest that the quasi-stochastic mechanism is more plausible than the folded-precursor mechanism for most proteins.