Role of unfolded state heterogeneity and en‐route ruggedness in protein folding kinetics

In order to improve our understanding of the physical bases of protein folding, there is a compelling need for better connections between experimental and computational approaches. This work addresses the role of unfolded state conformational heterogeneity and en‐route intermediates, as an aid for planning and interpreting protein folding experiments. The expected kinetics were modeled for different types of energy landscapes, including multiple parallel folding routes, preferential paths dominated by one primary folding route, and distributed paths with a wide spectrum of microscopic folding rate constants. In the presence of one or more preferential routes, conformational exchange among unfolded state populations slows down the observed rates for native protein formation. We find this to be a general phenomenon, taking place even when unfolded conformations interconvert much faster than the “escape” rate constants to folding. Dramatic kinetic deceleration is expected in the presence of an increasing number of folding‐incompetent unfolded conformations. This argues for the existence of parallel folding paths involving several folding‐competent unfolded conformations, during the early stages of protein folding. Deviations from single‐exponential behavior are observed for unfolded conformations exchanging at comparable rates or more slowly than folding events. Analysis of the effect of en‐route (on‐path) intermediate formation and landscape ruggedness on folding kinetics leads to the following unexpected conclusions: (1) intermediates, which often retard native state formation, may in some cases accelerate folding, and (2) rugged landscapes, usually associated with stretched exponentials, display single‐exponential behavior in the presence of late high‐friction paths.