Probing microscopic conformational dynamics in folding reactions by measuring transition paths

Transition paths comprise those parts of a folding trajectory where the molecule passes through the high-energy transition states separating folded and unfolded conformations. The transition states determine the folding kinetics and mechanism but are difficult to observe because of their brief duration. Single-molecule experiments have in recent years begun to characterize transition paths in folding reactions, allowing the microscopic conformational dynamics that occur as a molecule traverses the energy barriers to be probed directly. Here we review single-molecule fluorescence and force spectroscopy measurements of transition-path properties, including the time taken to traverse the paths, the local velocity along them, the path shapes, and the variability within these measurements reflecting differences between individual barrier crossings. We discuss how these measurements have been related to theories of folding as diffusion over an energy landscape to deduce properties such as the diffusion coefficient, and how they are being combined with simulations to obtain enhanced atomistic understanding of folding. The richly detailed information available from transition path measurements holds great promise for improved understanding of microscopic mechanisms in folding.