Secondary structure in ribonuclease. II. Relations between folding kinetics and secondary structure elements

The kinetics of regain of 2′-CMP binding are monitored during renaturation of RNAase S. Experiments were performed by mixing equimolar amounts of S-peptide with S-protein. The S-protein fragment was incubated initially (i.e. before mixing with S-peptide) at pH 6.2 or 1.7 and various guanidine hydrochloride (GuHCl) concentrations. Three well-resolved phases are observed. The fastest phase is second-order. The reciprocal half-time increases linearly with fragment concentration and is independent of initial conditions for the S-protein fragment. An apparent on rate of k_on = 2 × 10⁵m^?1s^?1 is measured in 0.5 m-GuHCl (pH 6.2) and 20 ° C. Identical association kinetics are observed by changes in tyrosine absorbance. The fraction of native RNAase S formed in this second-order reaction strictly equals the fraction of S-protein molecules with intact β-sheet in initial conditions. The relation holds for different pH values, GuHCl concentrations and temperatures. The fraction of apparent helical content of S-protein in initial conditions may also vary but this is not reflected by the association reaction. We interpret this to mean that the β-sheet but not the α-helices must be preformed in initial conditions in order to generate the high-affinity peptide binding site of S-protein. Furthermore, it is concluded that the S-protein moiety β-sheet forms or unfolds in a single one-step reaction. 2′-CMP binding reports, additionally, two slower phases of renaturation. These are produced by S-protein molecules that have their β-sheet unfolded in initial conditions. It is observed that a unique dependence of these two folding rates exists for RNAase A, RNAase S and S-protein as function of t_m, the temperature of half-completion of thermal denaturation as measured by unfolding of the β-sheet in the respective compound in final conditions. The t_m value varies with changing pH, with GuHCl concentration and (for RNAase S) with changing fragment concentration. The findings are interpreted to argue in favor of a sequential mechanism of folding, where the stability of a structural precursor determines the rate of folding.