Measurement of Average Transition-Path Time for Protein Folding in Single Molecule FRET Experiments

Abstract

The transition-path is the tiny fraction of an equilibrium molecular trajectory when a transition occurs between two states, and appears as an instantaneous jump in the measured signal in single molecule force or fluorescence experiments. Transition-paths are readily observed in atomistic molecular dynamics simulations for systems with fast kinetics, but have never been observed experimentally for any system in the condensed phase. The importance of the transition-path in protein folding is that it contains all the mechanistic information on how a protein folds and unfolds and is predicted from both theory and simulations to be heterogeneous. As a first step toward observing transition-paths in protein folding, we previously estimated an upper bound of ∼200 microseconds for the transition-path time of protein G using single molecule FRET spectroscopy, 10,000 times shorter than the average unfolded-state waiting-time of ∼2 seconds (Chung et al., PNAS 2009). The biggest obstacle to resolving a transition-path is to detect a sufficient number of photons during a single transition-path. To overcome this problem, we employed a fully-automated data acquisition system to collect a very large number of photon trajectories at high illumination intensities, and carried out a collective photon-by-photon analysis of the transitions between the folded and unfolded states using a maximum likelihood method (Chung et al., JPC A 2011). We determined a transition-path time of ∼2 microseconds for a WW domain that folds in ∼100 microseconds and an upper bound of ∼15 microseconds for protein GB1 that folds in ∼2 seconds. The transition-path times for the two proteins differ by less than 10-fold while the folding rates differ by a factor of 20,000. This result shows that a slow-folding protein can fold almost as fast as a fast-folding protein when folding actually occurs!