Dynamics of the L-fucose/H+ symporter revealed by fluorescence spectroscopy.

TitleDynamics of the L-fucose/H+ symporter revealed by fluorescence spectroscopy.
Publication TypeJournal Article
Year of Publication2012
AuthorsSugihara, J, Sun, L, Yan, N, H Kaback, R
JournalProc Natl Acad Sci U S A
Volume109
Issue37
Pagination14847-51
Date Published2012 Sep 11
ISSN1091-6490
KeywordsCrystallography, X-Ray, Escherichia coli, Escherichia coli Proteins, Fucose, Models, Molecular, Mutagenesis, Site-Directed, Plasmids, Protein Conformation, Spectrometry, Fluorescence, Symporters
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.

DOI10.1073/pnas.1213445109
Alternate JournalProc. Natl. Acad. Sci. U.S.A.
PubMed ID22930818
PubMed Central IDPMC3443143
Grant ListR56 DK051131 / DK / NIDDK NIH HHS / United States
DK069463 / DK / NIDDK NIH HHS / United States
DK51131 / DK / NIDDK NIH HHS / United States
R01 DK051131 / DK / NIDDK NIH HHS / United States
GM074929 / GM / NIGMS NIH HHS / United States
U54 GM074929 / GM / NIGMS NIH HHS / United States
R56 DK069463 / DK / NIDDK NIH HHS / United States
R01 DK069463 / DK / NIDDK NIH HHS / United States