E. coli Multidrug Transporter MdfA Is a Monomer

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 struct...

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Bibliographic Details
Published in:Biochemistry (Easton) 2007-05, Vol.46 (17), p.5200-5208
Main Authors: Sigal, Nadejda, Lewinson, Oded, Wolf, Sharon G, Bibi, Eitan
Format: Article
Language:English
Subjects:
Chromatography, Affinity
Chromatography, Gel
Chromatography, High Pressure Liquid
Crystallization
Detergents - chemistry
Electrophoresis, Polyacrylamide Gel
Escherichia coli
Escherichia coli - chemistry
Escherichia coli Proteins - chemistry
Membrane Transport Proteins - chemistry
Microscopy, Electron
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Summary: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 β-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.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi602405w