Cooperative Native Contact Formation Facilitates Free Energy Barrier Crossing in Protein Folding

Understanding the folding mechanisms of biomolecules, such as proteins and nucleic acids, requires probing time-dependent changes of conformations, including secondary and tertiary structure formation. All these mechanism-relevant structural transitions, typically seen as coarse-grained descriptions in molecular dynamics simulations, occur in the tiny fraction of a molecular trajectory called the transition path (TP). However, TPs have been inaccessible for most proteins due to the limited time resolution of single-molecule techniques. Here, we measure the TP times of eight two-state single-domain proteins in aqueous solution using nanophotonics-enhanced single-molecule fluorescence spectroscopy. We found that the TP times are extremely short, ranging from 0.7 to 4  μs, and insensitive to most protein properties. Surprisingly, however, the extracted diffusion coefficient at the free energy barrier on the number of native contacts coordinate increases with protein length. This facilitation of diffusion in larger proteins is explained by increased cooperative native contact formation during folding, which reduces the roughness of the energy landscape. Our results reveal that evolutionary optimization of the energy landscape is much more efficient for protein folding than for other biomolecular processes.