Fig. 4

Single-molecule FRET dynamics. a Sequential frames (30 ms per frame) of part of a single NTS1 dimer trajectory observed by single-molecule FRET. Donor (green) and acceptor (red) channels together with their overlay (registered to nearest pixel) show temporal fluctuation in FRET in the dimer species (note, relative contrast is set to illustrate both channels in overlay). Scale bar: 5 μm. See also Supplementary Movie 5. b Representative NTS1 dimer fluorescence traces (green: donor, red: acceptor, see also Supplementary Fig. 9) illustrate temporal fluctuation within a single dimer trajectory; donor and acceptor fluorescence intensities are anti-correlated. c FRET efficiency histogram from 40 dimer trajectories shows a broad distribution of FRET efficiencies consistent with high and lower acceptor fluorescence intensity populations, suggesting multiple dimerisation interfaces. d Fluorescence intensity of the acceptor in a dimer species is shown for a single dimer trajectory (see also Supplementary Fig. 8). A bimodal acceptor fluorescence intensity distribution, showing high- and low-FRET states, was measured for NTS1 dimers in single-molecule experiments (Fig. 2b and Supplementary Fig. 8). Rather than observing dimer trajectories with acceptor fluorescence intensities at either low or high levels, the acceptor fluorescence intensity showed changes within trajectories, suggesting that the bimodal acceptor fluorescence intensity distribution (Fig. 2b) is a result of dynamic interconversion between different dimerisation interfaces. Interconversion between high- and low-FRET states was analysed. A conservative threshold was defined (166.5 counts—grey dashed line in d, see also Supplementary Fig. 8), above which intensities have <1% probability of belonging to the low-FRET state. e Lifetime measurements above and below this threshold show dimers exhibiting higher propensity for greater dwell-times in low-FRET (top panel) than in high-FRET (bottom panel) configurations, in keeping with the relative mixing proportions of the two component intensity distributions and FRET efficiency distribution. The lifetime of the low-FRET state is dependent upon it being an initial, mid-, or final transitional state, which could be suggestive of two or more low-FRET states exhibiting differing stability, with the capability to interconvert via a high-FRET state