Probing the bottom of a folding funnel using conformationally gated electron transfer reactions

The effect of global stability on the kinetics of interconversion between the native (N) and a compact, partially unfolded form (I) of iso-1-cytochrome c stabilized by His73-heme ligation is investigated using a novel conformationally gated ET method. For the K73H variant and the 2-fold less stable AcH73 variant, the N and I conformers are of nearly equal stability at pH 7.5. The pH jump kinetic data yield k_obs = k_NI + k_IN of 35-40 s^-1 at final pH values from 6 to 8 for the AcH73 variant, about 3-fold faster than for the more stable K73H variant. Gated ET measurements give k_NI = 28 s^-1 and k_IN = 13 s^-1 for the AcH73 variant, 10- and 2-fold greater than that for the more stable K73H variant. Thus, funneled landscapes have evolved such that loss of global stability lowers barriers at the bottom of a folding funnel, still allowing for efficient folding.