Multidrug transporter MdfA as a target for high-resolution structural studies

• Main Author: O'Grady, Christopher Brian
• Other Authors: Roesler, Bill
• Format: Others
• Language: en
• Published: University of Saskatchewan 2010
• Subjects: nuclear magnetic resonance; protien purification; structural studies; membrane protein purification; Multidrug transporters; membrane protein structural studies; MdfA; multidrug resistance; membrane protein; x-ray crystallography
• Online Access: http://library.usask.ca/theses/available/etd-01202010-093831/

The MdfA is a 410 amino acid-long integral membrane protein, which belongs to the Major Facilitator superfamily of multidrug transporters. It is predicted to consist of 12 transmembrane helices. MdfA uses the energy of the transmembrane proton gradient to pump a variety of toxic compounds out of E. coli cells. No high resolution structure of MdfA is available. The goals of this research project were to develop a practical method for purification of MdfA, to evaluate the feasibility of structure determination by Nuclear Magnetic Resonance (NMR) and X-ray crystallography, and to develop an activity assay for purified MdfA. To this end, MdfA, with a hexa-histidine tag attached to facilitate protein purification, was successfully expressed and incorporated into the cell membrane using an E. coli expression system. MdfA was extracted from the cell membrane with the detergents 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), n-dodecyl-B-D-maltoside (DDM), and 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LMPG) and purified by affinity chromatography on nickel-nitrilotriacetic acid agarose. Pure protein was found to be monodisperse in DHPC, DDM and LMPG micelles. To achieve simple amino acid selective isotope labeling for high-resolution NMR studies, MdfA was expressed in a cell-free translation system. To determine if the purified protein was properly folded, 19F NMR experiments were carried out on 5-fluoro-tryptophan-labeled MdfA while titrating the MdfA substrates ethidium bromide and chloramphenicol into the fluoro-tryptophan-labeled MdfA sample. An activity assay was developed for MdfA incorporated into liposomes using the fluorescent dye 9-amino-6-chloro-2-methoxyacridine (ACMA) to detect proton translocation coupled to substrate transport. Results from both the 19F NMR and the transport activity assay indicated that the purified MdfA was properly folded and functional. NMR experiments with pure MdfA yielded spectra of insufficient quality for high-resolution structure determination but did indicate that structural studies of MdfA by NMR are feasible. Crystallization trials yielded crystals that are likely to contain protein and will serve as a starting point for further optimization of crystallization conditions for X-ray structure determination.