title: Structural studies of the melibiose permease of Escherichia coli by fluorescence resonance energy transfer. II. Identification of the tryptophan residues acting as energy donors.
abstract: 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 (Dns2-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 Trp64, localized in loop 2-3 from the N-terminal domain, and Trp299, localized in helix IX in the C-terminal domain, are responsible for up to 80% of the FRET signal. Moreover, by assuming that only Trp299 transfers energy to Dns2-S-Gal in mutant W64F, whereas only Trp64 transfers energy to Dns2-S-Gal in mutant W299F, we calculated that Trp299 and Trp64 are about 14 and 20 A away from the probe, respectively. In addition, we observed that mutating Trp342, 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.
authors.affiliation: Laboratoire J. Maetz, Département de Biologie Cellulaire et Moléculaire du Commissariat à l'Energie Atomique and CNRS-ERS 1253, 06238 Villefranche sur Mer cedex, France.
authors.forename: E
authors.initials: E
authors.lastname: Cordat
authors.forename: I
authors.initials: I
authors.lastname: Mus-Veteau
authors.forename: G
authors.initials: G
authors.lastname: Leblanc
mesh_terms: D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology*; D026901:Membrane Transport Proteins / Q000737:chemistry*; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D027981:Symporters; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D026901:Membrane Transport Proteins; D027981:Symporters; D014364:Tryptophan; C021548:melibiose permease