Fig. 3
Multiparameter Fluorescence Detection of FRET and energy landscape of the PDZ1-2 tandem variants. Each panel plots two FRET indicators for each single-molecule event. The donor over acceptor fluorescence ratio (FD/FA) on the y-axis and the average fluorescence lifetime (〈τD(A)〉f) on the x-axis. Shown are the 2D histograms (orange contours) for (a) Q107C/H182C-DA, (b) D91C/M159C-DA, and (c) S142C/K202C-DA. The 1D frequency histograms are shown along the axes for each FRET indicator. The static FRET lines (Supplementary Eq. 10, Supplementary Table 3) are shown in red. The dynamic FRET lines (Supplementary Eq. 11, Supplementary Table 4) are shown in green and connect the states identified with eTCSPC. The mean interdye distances for each of the eTCSPC states was converted to FD/FA and shown as lines colored purple, blue, and cyan in decreasing order of population occupancy. d–f Histograms of the potential mean force (PMF) calculated from DMD simulations for the variants (d) Q107C/H182C-DA, (e) D91C/M159C-DA, and (f) S142C/K202C-DA. The remaining histograms are shown in Supplementary Fig. 6. Histograms show the interdomain distance between the center of mass of the two PDZ domains plotted against the interdye distance distribution, which was calculated as p(RDA)PMF = exp(−EPMF/kBT) where EPMF is the integrated free energy over interdomain distance as the function of the interdye distance, RDA; and kB is Boltzmann constant. Contours are scaled in kcal/mole. Lines represent the mean interdye distances for each of the states identified by eTCSPC and are colored purple, blue, and cyan in decreasing order of population occupancy. Interdomain distances for previously reported models of the PDZ tandem are marked by symbols and denoted beneath the panels. Two basins are clearly identified, with residual states that are heterogeneously distributed with respect to the interdomain distance
