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MdfA as a Model for Structural and Functional Characterization of Putative Multidrug Transporters



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Numerous members of the Major Facilitator Superfamily of membrane transporters are involved in multidrug resistance (MDR) of gram-positive bacteria, such as Staphylococcus aureus (S. aureus). Genome data indicate that MDR transporters (MDT) constitute up to 1.0% of total cell proteins, but the majority remain uncharacterized: substrates, transport rates, and structures remain unknown (Forrest et al., 2011; Tsai and Ziegler, 2010; Ward et al., 2001). The main goal of this project was to develop a technique for substrate profile analysis and transport kinetics measurement of putative MDT from S. aureus, and other bacteria, using a well-characterized transporter, the multidrug facilitator A (MdfA), from Escherichia coli (E. coli), as a model protein. To provide structural basis for drug transport and binding studies, we also conducted preliminary MdfA crystallization trials. MdfA was expressed and purified using a procedure previously established in our lab (O'Grady, 2010). MdfA couples substrate transport across the cell membrane to the counterflow of protons. To test substrate transport by MdfA, we developed an assay based on monitoring proton transport in membrane vesicles using 9-amino-6-chloro-2-methoxy acridine (ACMA), a pH-sensitive fluorophore. We confirmed MdfA activity in membrane vesicles by this assay. In proteoliposomes containing co-reconstituted MdfA and FoF1 ATPase, addition of ATP generates a transmembrane pH gradient, which can be used as the driving force for MdfA-mediated substrate transport. Substrate binding to MdfA was investigated by nuclear magnetic resonance (NMR) experiments with 13C-labeled chloramphenicol. We observed specific interactions between chloramphenicol, a known MdfA substrate, and MdfA with an estimated dissociation constant (Kd) on the order of 10 μM. Detergents that have been previously successfully used for structural studies of membrane proteins by NMR and X-ray crystallography were tested for structural studies of MdfA. We determined that MdfA is active; however, further work is required to optimize the substrate profile assay, confirm substrate-binding data, and obtain crystals suitable for structural studies.



Multidrug Transporters, MdfA, Antibiotic Resistance



Master of Science (M.Sc.)






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