Kinetic mechanism of a partial folding reaction. 2. Nature of the transition state

The effects of mutations, temperature, and solvent viscosity on the bimolecular association rate constant (kon) and dissociation rate constant (koff) of the complex (RNaseS*) formed by S-peptide analogues and folded S-protein are reported. An important advantage of this system is that both kon and koff may be measured under identical strongly native conditions, and Kd for the complex may be calculated from the ratio koff/kon (preceding article). The side chains of S-peptide residues His-12 and Met-13 contribute a large fraction of the total interface with S-protein. Changing these residues, either singly or in a double mutant, destabilizes RNaseS* by up to 6 orders of magnitude, but causes no more than a 3-fold decrease in kon. Therefore, nativelike side-chain interactions between these residues and S-protein are not present in the transition state for folding. The absence of side-chain interactions in the transition state is surprising, since it has buried 55% of the total surface area that is buried upon forming RNaseS*, as estimated from the denaturant dependences of kon and koff (preceding article). The temperature dependence of the refolding rate suggests that the transition state for complex formation is stabilized by hydrophobic interactions: 66% of the change in heat capacity on forming RNaseS* occurs in the association reaction, consistent with the estimate of surface area burial from the denaturant studies. The solvent viscosity is varied to determine if the folding reaction is diffusion limited. Because kon, koff, and Kd all can be measured under the same native conditions, the viscosity effect on reaction rates can be separated from the effect of sucrose on the stability of RNaseS*. Both kon and koff are found to be inversely proportional to the solvent viscosity, indicating that the association and dissociation kinetics are diffusion controlled. The stabilizing effect of sucrose on RNaseS* appears as a reduction in koff.