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 r esonance energy transfer (FRET) between the tryptophans of the melibiose pe rmease (MelB) of Escherichia coli and a fluorescent sugar, 2'-(N-5-dimethyl aminonaphthalene-1-sulfonyl)-aminoethyl-1-thio-beta-D-galactopyranoside (Dn s(2)-S-Gal) bound at the sugar-binding site (Maehrel, C., Cordat, E,, Mus-V eteau, I., and Leblanc, G. (1998) J. Biol. Chem. 273, 33192-33197). To iden tify the tryptophans that transfer their energy to the fluorescent sugar, w e analyzed the FRET properties of MelB mutants carrying the replacement of each of the eight MelB tryptophans by a phenylalanine. The data indicate th at 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 energ y 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. Tak en together, these data provide additional support for the suggestion that (i) the sugar-binding site is localized in the C-terminal part of the trans porter, probably close to membrane segments IX and X, and (ii) the N-termin al domain, and particularly cytoplasmic loop 2-3, is also close to the suga r-binding site.