Dynamics and folding of single two-stranded coiled-coil peptides studied by fluorescent energy transfer confocal microscopy

We report single-molecule measurements on the folding and unfolding conform ational equilibrium distributions and dynamics of a disulfide crosslinked v ersion of the two-stranded coiled coil from GCN4. The peptide has a fluores cent donor and acceptor at the N termini of its two chains and a Cys disulf ide near its C terminus. Thus, folding brings the two N termini of the two chains close together, resulting in an enhancement of fluorescent resonant energy transfer. End-to-end distance distributions have thus been character ized under conditions where the peptide is nearly fully folded (0 M urea), unfolded (7.4 M urea), and in dynamic exchange between folded and unfolded states (3.0 M urea). The distributions have been compared for the peptide f reely diffusing in solution and deposited onto aminopropyl silanized glass. As the urea concentration is increased, the mean end-to-end distance shift s to longer distances both in free solution and on the modified surface. Th e widths of these distributions indicate that the molecules are undergoing millisecond conformational fluctuations. Under all three conditions, these fluctuations gave nonexponential correlations on 1- to 100-ms time scale. A component of the correlation decay that was sensitive to the concentration of urea corresponded to that measured by bulk relaxation kinetics. The tra jectories provided effective intramolecular diffusion coefficients as a fun ction of the end-to-end distances for the folded and unfolded states. Singl e-molecule folding studies provide information concerning the distributions of conformational states in the folded, unfolded, and dynamically intercon verting states.