Three-Color Single-Molecule FRET and Fluorescence Lifetime Analysis of Fast Protein Folding

Abstract

We describe the theory, experiment, and analysis of three-color Förster resonance energy transfer spectroscopy for probing conformational dynamics of a fast-folding protein, α3D. To site-specifically label fluorophores, a cysteine residue and an unnatural amino acid were labeled first, and then we appended a cysteine residue to the C-terminus of the protein by the sortase-mediated ligation for the third dye. All three FRET efficiencies were determined using alternating excitation of the donor and acceptor 1. Due to fast folding on a millisecond time scale, we used a maximum likelihood method that analyzes photon trajectories without binning the data. The extracted kinetic parameters agree well with the previously measured parameters with two-color FRET, suggesting the third fluorophore does not affect the folding dynamics of the protein. The FRET efficiencies for all three dye pairs were calculated after various corrections. They were compared with the FRET efficiencies obtained from the two-color segments collected in the same experiment. The FRET efficiencies of the folded state agree with those from the two-color segments, whereas the unfolded state FRET efficiencies are different. This happens because fluctuations of all three inter-dye distances contribute to the three-color FRET efficiency. This difference can be accounted for by using the Gaussian chain model with the parameters obtained from the two-color segments. Therefore, three-color FRET provides additional information on the flexibility of molecules that cannot be obtained from a combination of two-color FRET experiments. Using the delay times of photons from the laser pulse, fluorescence lifetimes were determined using the maximum likelihood analysis. The correlation between FRET efficiencies and lifetimes of the donor, acceptor 1, and acceptor 2 was visualized in two-dimensional FRET efficiency-lifetime histograms, which can be used to demonstrate the presence of conformational dynamics in a protein.