Extended Data Figure 3.
FRET efficiency histograms of α3D in 2.25 M GdmCl solution at different temperatures. The FRET efficiency histograms were constructed from 1 ms bins in the trajectories with the mean photon count rate > 40 ms-1. Wide and narrow bars are the experimental histograms and the histograms constructed from recolored photon trajectories using the parameters obtained from the maximum likelihood method with the two-state model (Extended Data Table 1), respectively. The agreement between the two histograms validates the description of α3D as a two-state folder8. The similar ratio of the integral of the folded (high FRET) and the unfolded (low FRET) distributions indicates that the equilibrium constant is unchanged over the temperature range of the measurement, as shown more precisely in the maximum likelihood analysis. At high temperature and low pH, where the 11 glutamates and 1 aspartate are protonated, more than two states are observed36,37.