Molecular mechanism of membrane transport in the bacterial multidrug resistance proteins

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The long-term goal of this work is to understand the molecular mechanism of multidrug resistance in bacteria and to advance the methods of investigating the structure of membrane proteins by nuclear magnetic resonance spectroscopy (NMR).  Multidrug resistance refers to the ability of cancer and bacterial cells to expel various chemically different toxic compounds from the cell, thus undermining the effectiveness of chemo-therapy and antibacterial chemicals. Drug extrusion is facilitated by membrane proteins called multidrug transporters. The multidrug resistance protein MdfA from E.coli will be used as a model for studying the molecular mechanism of drug extrusion. We plan to analyze the global fold, or overall architecture, of MdfA, and to map the drug-binding site in the protein, using multi-dimensional NMR, a method uniquely suited for investigating small molecule bind-ing to proteins and for studying protein dynamics in solution.  The MdfA and related transporters use the energy of transmembrane ion gradients to pump drugs out of the cells. We will attempt to pinpoint the ion-binding site in the structure of the protein and to investigate changes in the fold of the protein, which may be involved in drug and ion binding and translocation across the cell membrane. To reach an atomic resolution in the future work on MdfA, we will try to crystallize the protein for solving the structure of MdfA by X-ray crystallography. This work will advance our understanding of the multiple drug resistance phe-nomenon in pathogenic bacteria and cancer cells, and, hopefully, will provide new ideas and contribute to the future development of new approaches to the chemotherapy of can-cer and to the development of new antibacterial treatments. The experimental approaches developed in the course of this project should be of interest to the broad research com-munity in the area of membrane protein biochemistry.