UCLA

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.