title: E. coli multidrug transporter MdfA is a monomer.
abstract: MdfA is a 410-residue-long secondary multidrug transporter from E. coli. Cells expressing MdfA from a multicopy plasmid exhibit resistance against a diverse group of toxic compounds, including neutral and cationic ones, because of active multidrug export. As a prerequisite for high-resolution structural studies and a better understanding of the mechanism of substrate recognition and translocation by MdfA, we investigated its biochemical properties and overall structural characteristics. To this end, we purified the beta-dodecyl maltopyranoside (DDM)-solubilized protein using a 6-His tag and metal affinity chromatography, and size exclusion chromatography (SE-HPLC). Purified MdfA was analyzed for its DDM and phospholipid (PL) content, and tetraphenylphosphonium (TPP+)-binding activity. The results are consistent with MdfA being an active monomer in DDM solution. Furthermore, an investigation of two-dimensional crystals by electron crystallography and 3D reconstruction lent support to the notion that MdfA may also be monomeric in reconstituted proteoliposomes.
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: Nadejda
authors.initials: N
authors.lastname: Sigal
authors.forename: Oded
authors.initials: O
authors.lastname: Lewinson
authors.forename: Sharon G
authors.initials: SG
authors.lastname: Wolf
authors.forename: Eitan
authors.initials: E
authors.lastname: Bibi
mesh_terms: D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003460:Crystallization; D003902:Detergents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D026901:Membrane Transport Proteins / Q000737:chemistry*; D008854:Microscopy, Electron
chemical_list: D003902:Detergents; D029968:Escherichia coli Proteins; C118438:Mdfa protein, E coli; D026901:Membrane Transport Proteins