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 rat e constant (k(off)) of the complex between fluorescein-labeled S-pepti de analogues and folded S-protein are reported, This is the first kine tic 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 confor mational 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 th e properties of the transition state may be calculated from the rate c onstants without extrapolation. The folded complex is formed fast (k(o n) = 1.8 x 10(7) M-1 s(-1)) and is very stable (K-d = 6 pM) at 10 degr ees C, 10 mM MOPS, pH 6.7. Charge interactions stabilize the complex b y 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 de creasing 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) i s the In-value for k(on) and m(off) is the m-value for k(off), indicat ing that more than half of the sites for interaction with either cosol vent are buried in the ensemble of structures present at the transitio n state.