Structure of the cytoplasmic loop between putative helices II and III of the mannitol permease of Escherichia coli: a tryptophan and 5-fluorotryptophan spectroscopy study.
In this work, four single tryptophan (Trp) mutants of the dimeric mannitol transporter of Escherichia coli, EII(mtl), are characterized using Trp and 5-fluoroTrp (5-FTrp) fluorescence spectroscopy. The four positions, 97, 114, 126, and 133, are located in a region shown by recent studies to be involved in the mannitol translocation process. To spectroscopically distinguish between the Trp positions in each subunit of dimeric EII(mtl), 5-FTrp was biosynthetically incorporated because of its much simpler photophysics compared to those of Trp. The steady-state and time-resolved fluorescence methodologies used point out that all four positions are in structured environments, both in the absence and in the presence of a saturating concentration of mannitol. The fluorescence decay of all 5-FTrp-containing mutants was highly homogeneous, suggesting similar microenvironments for both probes per dimer. However, Stern-Volmer quenching experiments using potassium iodide indicate different solvent accessibilities for the two probes at positions 97 and 133. A 5 A two-dimensional (2D) projection map of the membrane-embedded IIC(mtl) dimer showing 2-fold symmetry is available. The results of this work are in better agreement with a 7 A projection map from a single 2D crystal on which no symmetry was imposed.
Vos
Erwin P P
EP
Department of Biophysical Chemistry, Groningen Biomolecular Science and Biotechnology Institute, The Netherlands.
Bokhove
Marcel
M
Hesp
Ben H
BH
Broos
Jaap
J
D000595:Amino Acid Sequence; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008353:Mannitol / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism
D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; C026927:5-fluorotryptophan; D008353:Mannitol; D014364:Tryptophan; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli