Mapping the transition state of the WW domain beta-sheet

The folding kinetics of a three-stranded antiparallel P-sheet (MnN domain) have been measured by temperature jump relaxation. Folding and activation f ree energies were determined as a function of temperature for both the wild -type and the mutant domain, W39F, which modifies the beta(2)-beta(3) hydro phobic interface. The folding rate decreases at higher temperatures as a re sult of the increase in the activation free energy for folding. Phi-Values were obtained for thermal perturbations allowing the primary features of th e folding free energy surface to be determined. The results of this analysi s indicate a significant shift from an "early" (Phi(T) = 0.4) to a "late" ( Phi(T) = 0.8) transition state with increasing temperature. The temperature -dependent Phi-value analysis of the wild-type WW domain and of its more st able W39F hydrophobic cluster mutant reveals little partici pation of resid ue 39 in the transition state at lower temperature. As the temperature is r aised, hydrophobic interactions at the beta(2)-beta(3) interface gain impor tance in the transition state and the barrier height of the wild-type, whic h contains the larger tryptophan residue, increases more slowly than the ba rrier height of the mutant. (C) 2000 Academic Press.