Fig. 3.

Representative fluorescence and photon trajectories, and FRET efficiency histograms of WW domain and protein GB1. (A and B) For the fluorescence trajectories - donor (green) and acceptor (red) - photons were collected in 50 μs bins for the WW domain and in 100 μs bins for protein GB1. Measurements were made at 293 K at high illumination intensity in 3 M GdmCl/50% glycerol for the WW domain (A) (20 kW/cm², ~ 650 photons/ms) and in 4 M urea for protein GB1 (B) (10 kW/cm², ~ 350 photons/ms). Strings of arrival times of donor and acceptor photons (photon trajectories) in the transition region (the 80 μs yellow shaded regions) are displayed below the binned fluorescence trajectories. Dashed vertical lines in the photon trajectories indicate the most probable transition interval found by the Viterbi algorithm (11). The absolute times refer to the start of data collection, which began at ~ 100 ms before the laser was turned on. (C and D) FRET efficiency histograms. The mean FRET efficiencies for the WW domain were calculated for each of 50 μs bins for the trajectories with the mean photon count rate > 400 ms⁻¹ (C) and for folded and unfolded segments of protein GB1 containing ~ 2500 photons (D).