LAUSR

The formation of complex structures in biomolecules typically involves thermally-activated crossing of an energy barrier. The unstable transition states in the barrier region dominate the folding dynamics and are thus of critical importance for understanding folding mechanisms. Because of their brevity it has not heretofore been possible to observe them directly, hence their properties could only be deduced indirectly. Recent work has begun to probe the properties of the transition paths—the paths taken through the barrier region—such as the average transition-path time. Using optical tweezers to measure the folding of single molecules held under tension by monitoring changes in their end-to-end extension, we previously measured the time required for individual transition-path crossings directly. Here we investigate for the first time the dynamics within the transition states. From folding trajectories of DNA hairpins, we measured the variations in the velocity as the molecule moved through the barrier region between folded and unfolded states, as well as the locations and durations of pauses within the barrier. The velocity distribution proved directly that folding is a diffusive phenomenon, agreeing well with simple one-dimensional theories. We also observed brief but ubiquitous pauses in the motion, reflecting microwells throughout the barrier region that allowed transient high-energy transition states to be observed directly. Remarkably, the transition-state dynamics agreed quantitatively with the predictions of a microscopic theory of folding, allowing the diffusion coefficient governing the timescale to be calculated directly from the pausing statistics. The result matched the value found from macroscopic kinetics, showing that the folding dynamics can be described quantitatively in a consistent way across all observable timescales.