Kinetic mechanism of a partial folding reaction. 1. Properties of the reaction and effects of denaturants

The bimolecular association rate constant (k(on)) and dissociation rate constant (k(off)) of the complex between fluorescein-labeled S-peptide analogues and folded S-protein are reported, This is the first kinetic study of a protein folding reaction in which most of the starting material is already folded and only a small part (one additional helix) becomes ordered; it provides a folding landscape with a small conformational entropy barrier, and one in which kinetic traps are unlikely. Refolding and unfolding are measured under identical strongly native conditions, and, the reaction is found to be two-state at low reactant concentrations. The dissociation constant (K-d) Of the complex and the properties of the transition state may be calculated from the rate constants without extrapolation. The folded complex is formed fast (k(on) = 1.8 x 10(7) M-1 s(-1)) and is very stable (K-d = 6 pM) at 10 degrees C, 10 mM MOPS, pH 6.7. Charge interactions stabilize the complex by 1.4 kcal mol(-1). The charge effect enters in the refolding reaction: increasing the salt concentration reduces k(on) dramatically and has little effect on k(off). Urea and GdmCl destabilize the complex by decreasing k(on) and increasing k(off). The slopes (nr-values) of plots of In K-d VS [cosolvent] are 0.75 +/- 0.04 and 2.8 +/- 0.3 kcal mol(-1) M-1 for urea and GdmCl, respectively. The ratio m(on)/(m(on) + m(off)) is 0.54 +/- 0.04 for urea and 0.57 +/- 0.1 for GdmCl, where m(on) is the In-value for k(on) and m(off) is the m-value for k(off), indicating that more than half of the sites for interaction with either cosolvent are buried in the ensemble of structures present at the transition state.