Extended Data Figure 5: d-fructose binding monitored by tryptophan fluorescence quenching.

a, Cartoon representation of the outward-facing rGLUT5 structure, as viewed from the plane of the membrane with the colouring as shown in Fig. 1a. Atoms in all tryptophan residues are shown as spheres and tryptophan W419, whose fluorescence is quenched by substrate, is labelled. b, Emission fluorescence spectra for purified deglycosylated rGLUT5 wild-type-like mutant N50Y (referred to as WT), shown in the range of 320–360 nm with an excitation wavelength of 295 nm after the addition of 40 mM d-fructose (top), and 40 mM l-fructose (bottom). Emission fluorescence spectra for purified wild-type protein that had been previously incubated with the inhibitor HgCl₂ is also shown for d-fructose (middle). c, Tryptophan fluorescence quenching (excitation 295 nm; emission 338 nm) after incubation of purified rGLUT5 N50Y with either 40 mM d-fructose (filled bar) or l-fructose, d-glucose, d-mannose, d-xylose or d-galactose as labelled (open bars). Tryptophan fluorescence quenching for purified wild-type protein that had been previously incubated with the inhibitor HgCl₂ is also shown for d-fructose (open bar). d, As in c, but for rGLUT5 with a single tryptophan residue (W419), which contains the following mutations: N50Y, W70F, W191F, W239F, W265F, W275F, W338F and W370F. No tryptophan quenching was observed for d-fructose (5 mM HgCl₂), l-fructose, d-glucose or d-galactose. In all experiments errors bars indicate s.e.m.; n = 3.