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It transports newly translated proteins into the inner membrane, or across it and into the periplasm and outer membrane of gram-negative bacteria. Within this pathway, SecA is an essential ATPase necessary for protein secretion through the membrane-bound SecYEG channel protein. In the cytoplasm, SecA is largely dimeric, although it is in equilibrium with a small population of the monomer form. This equilibrium is influenced by translocation ligands, anionic phospholipids, temperature, and SecA and salt concentrations. The bacterial membrane is composed of 20-25% anionic phospholipids, which have been previously shown to monomerize SecA. As such, there is a significant gap in the present literature regarding the complex oligomeric and structural behavior of SecA in the membrane. To fill this gap, we investigated the oligomeric state of the SecA dimer using Förster Resonance Energy Transfer (FRET), using established mutants and FRET pairs. Our research found that membrane-bound SecA has a 12% dimeric component under near-physiological conditions. We also estimate the KD of membrane-bound SecA to be roughly 1000-fold tighter in solution at low salt than in the membrane. Finally, we provide evidence for a mechanism whereby SecA inserts into the membrane as a monomer, and then dimerizes once it is in the membrane. Our results clarify the oligomeric status of membrane-bound SecA, and we consider models where SecA monomer-dimer transitions within the membrane play roles in facilitating the protein translocation process.</p>"},{"label":"Digital Identifier","value":"m2022-n887"},{"label":"Thesis Advisor(s)","value":["Oliver, Donald B.","Mukerji, Ishita"]},{"label":"Digital Object Identifier (DOI)","value":"https://doi.org/10.14418/wes01.2.365"},{"label":"Discipline","value":["Molecular Biology and Biochemistry"]},{"label":"Date","value":"January 20, 2022"},{"label":"EDTF Date","value":"D:00 M:00 Y:2022"},{"label":"Extent","value":"125 pages : illustrations, charts ; 29 cm"},{"label":"File Format","value":["application/pdf"]},{"label":"Genre","value":["masters theses"]},{"label":"Digital Origin","value":["born digital"]},{"label":"Type","value":["Text"]}],"description":"Investigation of the oligomeric state of membrane-bound SecA using Förster resonance energy 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