Figure 5.

GFP-TS assay and comparing the lipid stabilization of bacterial versus eukaryotic transporters. (a) Box-and-whisker plots show the distribution of thermostabilities for eukaryotic transporter (red bars) and bacterial transporters (black bars) before and after purification as assessed by the GFP-TS assay; the median is shown as a line in the box, while bottom and top boundaries represent the lower and upper quartile, respectively. Whiskers indicate the minimum and maximum apparent T_m. (b) Schematic representation of the GFP-TS assay for monitoring ligand interactions, including lipids. (c) The GFP-TS melting curves for the bacterial monosaccharide transporter XylE (left) and rat GLUT5 (right) in crude detergent solubilized membranes (black circles) and as a purified fusion (cyan squares). Error bars show the range of two technical replicates, and the values reported for the apparent T_m are the mean ± SEM of the fit. (d) Supernatant fluorescence of detergent purified rat GLUT5-GFP before heating at apparent T_m +5 °C (nonfilled bars) and that remaining after heating and centrifugation (black bars) in the presence of listed lipids solubilized in the same detergent or detergent only (control); the asterisk indicates the most stabilizing lipid (bars show the range of two technical replicates). (e) GFP-TS melting curves for purified rat GLUT5 in the absence (black) and presence of brain lipids (cyan); apparent T_m were calculated as described in (b), and the values reported are the mean ± SEM of the fit. Reproduced with permission from ref (119). Copyright 2018 Nature.