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 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.
Additional Information
© 2007 American Chemical Society. Received 21 November 2006. Published online 4 April 2007. Published in print 1 May 2007. This work was supported by a grant from the Israel Cancer Research Foundation (ICRF) and the Israel Science Foundation (ISF). We thank Werner Kuhlbrandt and Thomas Walz for their help and advice in the initial stages of the 2D crystallization project, Daniel Tal for his help during the SE-HPLC studies, and Lubov Ginzburg for help in the phospholipid assays.Additional details
- Eprint ID
- 73896
- Resolver ID
- CaltechAUTHORS:20170131-140942712
- Israel Cancer Research Foundation
- Israel Science Foundation
- Created
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2017-02-01Created from EPrint's datestamp field
- Updated
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2021-11-11Created from EPrint's last_modified field