Measurement of Single Molecule Folding/unfolding Trajectories

Abstract

We have measured folding/unfolding trajectories of single protein G (B1 domain) molecules, a simple two-state folder, by simultaneously measuring the fluorescence intensity, lifetime, and spectrum at various concentrations of denaturant. Protein molecules were labeled by a fluorescence resonance energy transfer (FRET) pair, Alexa Fluor 488 and Alexa Fluor 594 and were immobilized on a glass surface coated with polyethyleneglycol via streptavidin-biotin linkage. The vast majority of molecules (∼ 85%) exhibits simple two-state trajectories, with either high or low values of the FRET efficiency, corresponding to the folded and unfolded states, respectively, with unresolvable jumps between them. About 10% of the trajectories show transitions in the unfolded state that can be attributed to a ∼20 nm spectral shift of the donor, as revealed by measurements of their emission spectra. The mean FRET efficiency of immobilized molecules matches the value measured in free diffusion experiments. There is a distribution of these values beyond the width expected from shot noise, which can, however, be quantitatively accounted for by the distribution of acceptor lifetimes. In spite of these complications from photophysics, rate coefficients obtained from the exponential distribution of residence times in either the folded or unfolded state yield relaxation times that agree within a factor of 2 with those measured on the dye-labeled protein by stopped flow kinetics. In addition, no correlation is observed between the donor and acceptor intensity in the unfolded state from microseconds to seconds suggesting that structural averaging between unfolded conformations occurs on the nanosecond timescale, as expected from previous measurements by B. Schuler and coworkers (PNAS:104,2655,2007). All these results indicate that we have successfully immobilized the protein without significantly altering its structure, kinetics, or dynamics, and represent a major step forward toward the goal of “watching” individual molecules fold.