Measuring ultrafast protein folding rates from photon-by-photon analysis of single molecule fluorescence trajectories.
Folding and unfolding rates for the ultrafast folding villin subdomain were determined from a photon-by-photon analysis of fluorescence trajectories in single molecule FRET experiments. One of the obstacles to measuring fast kinetics in single molecule fluorescence experiments is blinking of the fluorophores on a timescale that is not well separated from the process of interest. By incorporating acceptor blinking into a two-state kinetics model, we show that it is possible to extract accurate rate coefficients on the microsecond time scale for folding and unfolding using the maximum likelihood method of I.V. Gopich and A. Szabo. This method yields the most likely parameters of a given model that can reproduce the observed photon trajectories. The extracted parameters agree with both the decay rate of the donor-acceptor cross correlation function and the results of ensemble equilibrium and kinetic experiments using nanosecond laser temperature jump.
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892-0520, USA.
Hoi Sung
HS
Chung
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892-0520, USA.
Troy
T
Cellmer
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892-0520, USA.
John M
JM
Louis
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892-0520, USA.
William A
WA
Eaton