Fig. 1
From: Transition path times of coupled folding and binding reveal the formation of an encounter complex
Observing coupled folding and binding by single-molecule FRET. a ACTR labeled with Cy3B as the donor fluorophore (green) is immobilized on a polyethyleneglycol (PEG)-coated quartz cover slide via a biotin−avidin−biotin linkage and excited by a laser beam. NCBD free in solution is labeled with CF660R as the acceptor fluorophore (red). Cartoon based on PDB entries 1KBH15 and 2KKJ17. b Schematic free-energy landscape of the reaction with a molecular trajectory of a binding transition. The molecules are in the unbound or bound states most of the time and only rarely cross the energy barrier. A binding transition path extends from the time when x0 is crossed until x1 is reached without returning to x0. c Examples of measured fluorescence time traces of binding events (ionic strength 108 mM, viscosity 1.28 cP), represented as binned (top) and single-photon data (bottom). The molecules start in the unbound state, indicated by high donor intensity (green) and low acceptor intensity (red). When NCBD binds to ACTR, the FRET efficiency increases in a rapid jump, corresponding to the transition path, with a concomitant increase in acceptor emission and a decrease in donor emission. In the single-photon representation of the transition-path region, donor photons are shown as green lines and acceptor photons as red lines. Segments of the trajectories identified as populating the transition path by the Viterbi algorithm are indicated by the gray shading in the single-photon time traces
