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 (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.
Cordat
E
E
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.
Mus-Veteau
I
I
Leblanc
G
G
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*
D026901:Membrane Transport Proteins; D027981:Symporters; D014364:Tryptophan; C021548:melibiose permease