Structural origin of slow diffusion in protein folding

Interactions that slow protein folding As proteins fold, they diffuse over an energy barrier that separates unfolded and folded states. The transition path defines how a single protein crosses the barrier and so contains key information on the mechanism of folding. Transition paths have not yet been experimentally observed, but Chung et al. have discovered which structural features of the protein affect the duration of the transition. As the protein folds, non-native salt bridges form and break, slowing diffusion along the transition path. Science, this issue p. 1504 Single-molecule experiments and simulations show how molecular interactions can direct protein folding by slowing diffusion. Experimental, theoretical, and computational studies of small proteins suggest that interresidue contacts not present in the folded structure play little or no role in the self-assembly mechanism. Non-native contacts can, however, influence folding kinetics by introducing additional local minima that slow diffusion over the global free-energy barrier between folded and unfolded states. Here, we combine single-molecule fluorescence with all-atom molecular dynamics simulations to discover the structural origin for the slow diffusion that markedly decreases the folding rate for a designed α-helical protein. Our experimental determination of transition path times and our analysis of the simulations point to non-native salt bridges between helices as the source, which provides a quantitative glimpse of how specific intramolecular interactions influence protein folding rates by altering dynamics and not activation free energies.