OxlT, an oxalate/formate transporter from the bacterium Oxalobacter formigenes is a member of the widely distributed class of Major Facilitator Superfamily (MFS) of transport proteins that play an important role in regulation of oxalate in the human intestinal tract. This thesis describes studies of the functional mechanism, oligomeric state, folding, and stability of OxlT as an example of this important class of MFS transporters. Using triple-detector size exclusion chromatography with static light scattering (SEC-LS), we showed that OxlT is a monomer in solution when solubilized by a variety of detergents. OxlT also failed to crosslink through disulfides or through chemical crosslinkers both in solution and in the membrane. Finally, functional reconstitution of OxlT in proteoliposomes at low protein:lipid ratios confirmed that the protein retained the functional monomeric state in the membrane as well.
The folding and stability of membrane proteins is currently only poorly understood. We have employed several different spectroscopic techniques for understanding the folding and unfolding pathways of OxlT. Changes in intrinsic tryptophan fluorescence of OxlT showed a biphasic change upon addition of the denaturant SDS, indicative of the presence of a partially folded intermediate. We also developed protocols to label OxlT with Cy3-MTSEA and Cy5-MTSEA dyes that have provided significant enhancement of specificity of labeling compared to routinely used maleimide based dyes. Based on measurements of Fluorescence Resonance Energy Transfer (FRET), doubly labeled cysteine mutants of OxlT (F18C-A342C and G196C-A342C) also showed a biphasic decrease in energy transfer in the presence of increasing concentrations of SDS. Our experiments also suggested that OxlT unfolding is at least partially reversible. Initial single molecule FRET measurements of OxlT under conditions of free diffusion are also indicative of the presence of a folding intermediate. We have also documented FRET measurements from single OxlT molecules trapped inside a nanochannel (600 nm x 400 nm x 15000 nm). We show that this novel approach of trapping a molecule is suitable for studying important phenomena like membrane protein unfolding at the single molecule level without the need for surface tethering. Our approaches and observations with OxlT can be extended to other interesting transporters.
|Advisor:||Dumont, Mark E.|
|Commitee:||Foster, Thomas H., Gunter, Thomas E., Novotny, Lukas, Waugh, Richard E.|
|School:||University of Rochester|
|Department:||School of Medicine and Dentistry|
|School Location:||United States -- New York|
|Source:||DAI-B 74/07(E), Dissertation Abstracts International|
|Keywords:||Fluorescent labeling, Fret, Membrane protein folding, Membrane transport, Oxalate transporters, OxlT, Smfret|
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