Multidrug-resistance is a situation encountered in cancer patients in which the tumor becomes resistant to a variety of cytotoxic anti-cancer chemotherapeutic agents. It often involves enhanced expression of P- glycoprotein (Pgp), a plasma membrane protein. Involvement of Pgp in resistance to anti-AIDS drugs is also strongly-indicated. Pgp consists of 1280 amino acids, arranged in two repeated halves, each of which contains six predicted transmembrane helices and one ATP-binding site. It acts in an ATP-dependent manner to exclude drugs and a wide range of other hydrophobic compounds from cells, displays substantial drug- stimulated ATPase activity, and is now widely-believed to act as an ATP- driven drug-efflux pump. A catalytic cycle involving alternating catalytic sites and a mechanism for coupling of ATP-hydrolysis to drug-transport, presented by our laboratory, has become widely-adopted as a working model. We recently made a breakthrough, namely the development of a large- scale method for preparation of pure, detergent-soluble, mouse and human Pgp, using Pichia. Not only wild-type but also mutant Pgp may now be obtained in quantity, facilitating a broader range of structural, biophysical and biochemical approaches.
The aim of this proposal is to characterize structure and function of Pgp. Structure will be determined by electron-microscopy and X-ray crystallography. Catalytic mechanism will be studied by specific insertion of fluorescent probes to monitor nucleotide binding parameters and occupancy of catalytic sites, and by mutagenesis of critical catalytic site residues. Coupling of ATP hydrolysis to drug transport will be investigated. The two halves of Pgp will be purified separately and reconstituted, to facilitate understanding of interactions between catalytic sites and membrane domains. Basic knowledge of this kind will be invaluable in devising ways to disable P-glycoprotein and overcome drug-resistance in patients.
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