Structural Studies of the Melibiose Permease ofEscherichia coliby Fluorescence Resonance Energy Transfer
Journal of Biological Chemistry
In the accompanying paper, we demonstrated the presence of a fluorescence resonance energy transfer (FRET) between the tryptophans of the melibiose permease (MelB) of Escherichia coli and a fluorescent sugar, 2-(N-5-dimethylaminonaphthalene-1-sulfonyl)aminoethyl-1-thio-␤-D-galactopyranoside (Dns 2 -S-Gal) bound at the sugar-binding site (Maehrel, C., Cordat, E., Mus-Veteau, I., and Leblanc, G. (1998) J. Biol. Chem. 273, 33192-33197). To identify the tryptophans that transfer their energy to the
... fluorescent sugar, we analyzed the FRET properties of MelB mutants carrying the replacement of each of the eight MelB tryptophans by a phenylalanine. The data indicate that Trp 64 , localized in loop 2-3 from the N-terminal domain, and Trp 299 , localized in helix IX in the C-terminal domain, are responsible for up to 80% of the FRET signal. Moreover, by assuming that only Trp 299 transfers energy to Dns 2 -S-Gal in mutant W64F, whereas only Trp 64 transfers energy to Dns 2 -S-Gal in mutant W299F, we calculated that Trp 299 and Trp 64 are about 14 and 20 Å away from the probe, respectively. In addition, we observed that mutating Trp 342 , localized in helix X of the C-terminal domain, produces a significant increase of the polarity of the fluorescent sugar environment, suggesting its proximity to the sugar-binding site. Taken together, these data provide additional support for the suggestion that (i) the sugarbinding site is localized in the C-terminal part of the transporter, probably close to membrane segments IX and X, and (ii) the N-terminal domain, and particularly cytoplasmic loop 2-3, is also close to the sugar-binding site.