title: Dynamics of the L-fucose/H+ symporter revealed by fluorescence spectroscopy.
abstract: FucP of Escherichia coli catalyzes L-fucose/H(+) symport, and a crystal structure in an outward-facing conformation has been reported. However, nothing is known about FucP conformational dynamics. Here, we show that addition of L-fucose to purified FucP in detergent induces ∼20% quenching of Trp fluorescence in a concentration-dependent manner without a shift in λ(max). Quenching is essentially abolished when both Trp38 and Trp278, which are positioned on opposing faces of the outward-facing cavity walls, are replaced with Tyr or Phe, and reduced quenching is observed when either Trp is mutated. Therefore, both Trp residues are involved in the phenomenon. Furthermore, replacement of either Trp38 or Trp278, predominantly Trp38, causes decreased quenching, decreased apparent affinity for L-fucose, and significant inhibition of active L-fucose transport, indicating that the two residues are likely involved directly in sugar binding. It is proposed that sugar binding induces a conformational change in which the outward-facing cavity in FucP closes, thereby bringing Trp38 and Trp278 into close proximity around the bound sugar to form an "occluded" intermediate. The location of these two Trp residues provides a unique method for analyzing structural dynamics in FucP.
authors.affiliation: Department of Physiology, University of California, Los Angeles, CA 90095, USA.
authors.forename: Junichi
authors.initials: J
authors.lastname: Sugihara
authors.forename: Linfeng
authors.initials: L
authors.lastname: Sun
authors.forename: Nieng
authors.initials: N
authors.lastname: Yan
authors.forename: H Ronald
authors.initials: HR
authors.lastname: Kaback
mesh_terms: D018360:Crystallography, X-Ray; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D005643:Fucose / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence / Q000379:methods*; D027981:Symporters / Q000235:genetics* / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; C088688:FucP protein, E coli; D027981:Symporters; D005643:Fucose