Folding dynamics of single GCN-4 peptides by fluorescence resonant energy transfer confocal microscopy

We have prepared a bichromophoric crosslinked variant of GCN4-P1 for single-molecule fluorescence energy transfer experiments (GCN4-Pf). The folding and unfolding fluctuations of single GCN4-Pf molecules are measured in a two-channel confocal microscope with which donor and acceptor fluorescence trajectories are measured simultaneously. The energy transfer efficiency is thereby determined and its probability distributions as a function of added denaturant [urea] are calculated. The distributions indicate that single molecule GCN4-Pf is in dynamic folding equilibrium with the position of the equilibrium being altered by the concentration of urea.