Figure 5.

FRET efficiency histogram of immobilized proteins. (A) The distribution was obtained from the FRET efficiencies of the initial segments of the trajectories. The ranges of the states are E < 0.1 (green, donor only), 0.1 ≤ E < 0.3 (light green, donor only with Alexa 488^R), 0.3 ≤ E < 0.625 (yellow, unfolded), 0.625 ≤ E < 0.825 (light red, unfolded with Alexa 488^R), and E ≥ 0.825 (red, folded). These ranges are based on the results in panel B for the streptavidin-biotin immobilization, where the separate states from Alexa 488^R are clearly resolved. The equilibrium constant was calculated from the ratio of the fractions belonging to the folded and unfolded states (K = f_F/f_U). (Red and green dashed lines) Distributions due solely to shot noise for >1000 photons for unfolded and folded peaks, respectively. (Orange vertical lines) Mean FRET efficiencies of the unfolded state in free diffusion experiment in Fig. 2. (B) The result for streptavidin-biotin immobilization is shown for comparison (from (19)).