Transition paths are a uniquely single molecule property not yet observed for any molecular process in solution. The duration of transition paths is the tiny fraction of the time in an equilibrium single molecule trajectory when the process actually happens. We determined an upper bound for the transition path time for protein folding from photon-by-photon trajectories. FRET trajectories were measured on single molecules of the dye-labeled, 56-residue two-state protein GB1, immobilized on a glass surface via a biotin-streptavidin-biotin linkage. Characterization of individual emitted photons by their wavelength, polarization, and absolute and relative time of arrival following picosecond excitation allowed the determination of distributions of FRET efficiencies, donor and acceptor lifetimes, steady state polarizations, and waiting times in the folded and unfolded states. Acquisition of single molecule spectra enabled a clear distinction between jumps in the FRET efficiency due to folding or unfolding transitions of the polypeptide and those correspond-ing to a previously unknown photophysical change of the com-monly-used donor dye, Alexa 488. Comparison with the results for freely diffusing molecules showed that immobilization has no detectable effect on the structure or dynamics of the unfolded protein and only a small effect on the folding/unfolding kinetics. Analysis of the photon-by-photon trajectories yields a transition path time less than 200 microsec, more than 10,000 times shorter than the mean waiting time in the unfolded state (the inverse of the folding rate coefficient). A. Szabos theory for diffusive transition paths shows that the upper bound for the transition path time is consistent with previous estimates of the Kramers pre-exponential factor for the rate coefficient. The theory also predicts that for smooth free energy barriers the transition path time is remarkably insensitive to the folding rate, with only a 2-fold difference for rate coefficients that differ by 100,000-fold.
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