KINETIC MECHANISM OF A PARTIAL FOLDING REACTION - 2 - NATURE OF THE TRANSITION-STATE

The effects of mutations, temperature, and solvent viscosity on the bi molecular association rate constant (k(on)) and dissociation rate cons tant (k(off)) of the complex (RNaseS) formed by S-peptide analogues a nd folded S-protein are reported. An important advantage of this syste m is that both k(on) and k(off) may be measured under identical strong ly native conditions, and K-d for the complex may be calculated from t he ratio k(off)/k(on) (preceding article), The side chains of S-peptid e 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 k(on). Therefore, native like side-chain interactions between these residues and S-protein are not present in the transition state for folding, The absence of side-c hain interactions in the transition state is surprising, since it has buried 55% of the total surface area that is buried upon forming RNase S. as estimated from the denaturant dependences of k(on) and k(off) ( preceding article), The temperature dependence of the refolding rate s uggests that the transition state for complex formation is stabilized by hydrophobic interactions: 66% of the change in heat capacity on for ming RNaseS occurs in the association reaction, consistent with the e stimate of surface area burial from the denaturant studies. The solven t viscosity is varied to determine if the folding reaction is diffusio n limited, Because k(on), k(off), and K-d all can be measured under th e same native conditions, the viscosity effect on reaction rates can b e separated from the effect of sucrose on the stability of RNaseS. Bo th K-on and k(off) are found to be inversely proportional to the solve nt viscosity, indicating that the association and dissociation kinetic s are diffusion controlled. The stabilizing effect of sucrose on RNase S appears as a reduction in k(off).