Abstract：
: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.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Vos EP,Bokhove M,Hesp BH,Broos Jdoi
10.1021/bi8020668subject
Has Abstractpub_date
2009-06-16 00:00:00pages
5284-90issue
23eissn
0006-2960issn
1520-4995journal_volume
48pub_type
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