A variety of integral membrane proteins have evolved to transport biologically important molecules across phospholipid membranes, and a majority of membrane proteins encoded in the human genome is transporters. The major facilitator superfamily (MFS) is arguably the largest group of secondary active transporters present in the kingdoms of life, yet only three X-ray crystal structures have been obtained for human members of the superfamily. Thus, many inferred structures of eukaryotic transporters have been derived from homology modeling using prokaryotic transporters with known structures, and studies on prokaryotic transporters have become exceedingly important. Furthermore, current structural information is insufficient to achieve a comprehensive understanding of the dynamics of these proteins, and application of biochemical and biophysical methods is vital for understanding of their mechanisms of action.

This thesis focuses on investigating dynamics of various MFS transporters, mainly L-fucose/H+ and sucrose/H+ symporters of Escherichia coli (FucP and CscB, respectively). FucP is a MFS transporter that catalyzes cotransport of L- fucose and H+ across the cytoplasmic membrane. Although its crystal structure in an outward (periplasmic)-open conformation has been reported, nothing is known about its conformational dynamics. In addition, CscB, another MFS member, catalyzes sucrose/H+ symport across the cytoplasmic membrane. The structural organization of key residues involved in sugar binding in CscB appears to be similar to that of the lactose permease of E. coli (LacY), arguably the best studied MFS member, which is highly specific for galactopyranosides. However, the determinants for sugar specificity in CscB, as well as structural dynamics, are unclear.

Conformational changes induced by sugar binding in FucP and CscB are analyzed by monitoring the fluorescence of Trp residues located on the walls of hydrophilic cavities in both symporters. The unique location of Trp residues provides a novel method for studying structural dynamics of membrane proteins purified in detergent. Also, the dynamics of FucP embedded in the native bacterial membrane is analyzed by examining site-directed alkylation of Cys- replacement mutants with the fluorescent alkylating agent, tetramethylrhodamine-5-maleimide. This method reveals sugar-induced changes in reactivity/accessibility of Cys replacements at given positions.

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.