The transition state transit time of WW domain folding is controlled by energy landscape roughness.

Protein folding barriers can be so low that a substantial protein population diffusing in the transition state region can be detected. The very fast kinetic phase contributed by transition state transit is the molecular phase. We detect the molecular phase of the beta-sheet protein FiP35 from 60 to 83 degrees C by T-jump relaxation experiments. The molecular phase actually slows down slightly with increasing temperature. Thus the friction that controls the prefactor in Kramers' transition state model does not scale with solvent viscosity. Instead, we postulate that an increase in the energy landscape roughness as the hydrophobic effect strengthens with increasing temperature explains the slowing of the molecular phase. We measured that the duration tau(m) of the molecular phase depends slightly on the size of the T-jump, in agreement with this explanation. The tau(m) measured here provides the best current estimate for the transit time from folded to unfolded state of a single protein molecule. We confirm this by directly comparing relaxation and single molecule signals computed by using Langevin trajectory models on a realistic FiP35 free energy surface.