Structural studies of the melibiose permease of Escherichia coli by fluorescence resonance energy transfer -: II.: Identification of the tryptophan residues acting as energy donors

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-beta-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 Angstrom 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 sugar-binding 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.