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

The transmembrane protein FucP of Escherichia coli is a symporter that catalyzes the cotransport of L-fucose and H+ across cytoplasmic membrane, and a crystal structure in an outward-facing conformation has been reported. However, nothing is known about FucP conformational dynamics. Here, it is shown that addition of L-fucose to purified FucP in detergent induces ~20% quenching of tryptophan (Trp) fluorescence in a concentration-dependent manner without a shift in λmax. Quenching is essentially abolished when both Trp38 and Trp278, which are positioned on opposing faces of the outward-facing cavity walls, are replaced with tyrosine (Tyr) or phenylalanine (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 unique location of these two Trp residues provides a novel method for analyzing structural dynamics in FucP