Fig. 1. Effect of l-Phe on LeuT_WT dynamics.

a Intracellular-labeled LeuT_WT-7C/86C was imaged with single-molecule fluorescence resonance energy transfer (smFRET) in the presence of 5 mM Na⁺ and in the absence of substrates (top), in the presence of 1 μM l-Phe (middle) or in the presence of 300 μM l-Phe (bottom). Shown are representative smFRET traces (blue) with state assignment traces (red). b Transition density contour plots showing the mean FRET value in the dwell just prior to (bottom axis) and just after (left axis) each transition between distinct FRET states (high-FRET state with a FRET efficiency of ~0.8, intermediate-FRET state with a FRET efficiency of ~0.6 and low-FRET state with a FRET efficiency of ~0.4), summed into a histogram. The average transition rate between states is given at the bottom left of each panel. Transitions per second rate scale is shown on top. c Ensemble-averaged occupancy in the low- (blue circles), intermediate- (green triangles) and high-FRET states (red squares) were fitted to dose-response functions with the Hill coefficients fixed at 1.0 and EC₅₀ of 5.1 μM (lines). d Rate constants for each transition type from experiments conducted in the presence of the indicated concentration of l-Phe (bottom axis), relative to the absence of substrate. Shaded area indicates regime in which time averaging obscures transitions, resulting in erroneous rate estimates. Source data are provided as a Source Data file.