Extended Data Fig. 1: Salt and pH dependence of LIV-BP^WT affinity for leucine and single-molecule calibration curve of LIV-BP^SS.

a, Bulk acceptor fluorescence of LIV-BP^WT measured in the presence of varying leucine concentrations and buffer conditions, demonstrating a lack of dependence on salt and pH conditions. b, Surface-immobilized LIV-BP^SS incubated in the presence of varying concentrations of leucine. For each concentration, FRET efficiencies were summed across time to generate a histogram (symbols) and fit to Gaussian functions (lines) to obtain estimates of the mean FRET value. Data were collected at least three times with similar results. c, Mean FRET values for leucine (black circles), obtained as shown in a, fit with equation (1) in Supplementary Methods (black line) to obtain the K_d. Analogous data with mean FRET values are shown for each concentration for isoleucine (red), valine (blue) and leucine (black) were fit with equation (1) to determine the K_d. The grey data points and line (LeuL) indicate LIV-BP^SS encapsulated within the liposome with leucine allowed to equilibrate at each concentration for at least 10 min into the liposome lumen. This indicates that the binding activity of LIV-BP is unaffected by encapsulation within the liposome lumen. Mean ± s.e.m. (n = 3 separate days with 2 protein preparations).