The molecular process underlying multidrug efflux by P-glycoprotein. (360G-Wellcome-094392_Z_10_Z)

Our overall aim is to describe the molecular mechanism responsible for the ability of P-gp to confer resistance to anti-cancer drugs. Our investigative strategy relies upon electron paramagnetic resonance (EPR) spectroscopic analysis of purified, reconstituted protein with. Key functional regions of P-gp will be subject to site-directed cysteine mutagenesis. This will enable subsequent covalent attachment of the thiol-reactive paramagnetic spectroscopy probe MTSL. The labelled P-gp will be cycle d between different conformation states and EPR spectra obtained for each. EPR spectroscopy will provide information on the relative accessibility, mobility and local environment of each residue addressed. Moreover, measurement at distinct protein states will provide a dynamic understanding of the conformational changes involved in drug translocation. The proposal has been divided into a number of key investigative goals as follows: Expression of mutant P-gp isoforms and their purification in a functional state Description of conformation changes in the transmembrane domains related to drug translocation Generation of a topographical map for TM6 and TM12 Detailing of movements at the nucleotide binding domain interfaces Locating the drug binding sites and description of their reorientation during translocation Using distance measurements to validate and complement the existing structural data for P-gp