Analysis of Fluorescence Lifetime and Energy Transfer Efficiency in Single-Molecule Photon Trajectories of Fast-Folding Proteins

In single-molecule Forster resonance energy transfer (FRET) spectroscopy, the dynamics of molecular processes are usually determined by analyzing the fluorescence intensity of donor and acceptor dyes. Since FRET efficiency is related to fluorescence lifetimes, additional information can be extracted by analyzing fluorescence intensity and lifetime together. For fast processes where individual states are not well separated in a trajectory, it is not easy to obtain the lifetime information. Here, we present analysis methods to utilize fluorescence lifetime information from single-molecule FRET experiments, and apply these methods to three fast-folding, two-state proteins. By constructing 2D FRET efficiency-lifetime histograms, the correlation can be visualized between the FRET efficiency and fluorescence lifetimes in the presence of the submicrosecond to millisecond dynamics. We extend the previously developed method for analyzing delay times of donor photons to include acceptor delay times. To determine the kinetics and lifetime parameters accurately, we used a maximum likelihood method. We found that acceptor blinking can lead to inaccurate parameters in the donor delay time analysis. This problem can be solved by incorporating acceptor blinking into a model. While the analysis of acceptor delay times is not affected by acceptor blinking, it is more sensitive to the shape of the delay time distribution resulting from a broad conformational distribution in the unfolded state.