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Drug-drug interactions in the binding pocket of P-glycoprotein multidrug resistant transporter

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Title: Drug-drug interactions in the binding pocket of P-glycoprotein multidrug resistant transporter
Author: Vitsupakorn, Danoo
Department: Department of Molecular and Cellular Biology
Program: Biophysics
Advisor: Sharom, Frances
Abstract: The ABC multidrug transporter P-glycoprotein (Pgp, ABCB1) can transport structurally diverse substrates from the lipid bilayer. Pgp binds its substrates inside a large pocket with multiple sub-sites. In the present work, a thiol-reactive agent, (2-pyridyl)dithiobimane, was covalently linked to 5 of the 7 Cys residues in hamster Pgp, with substantial ATPase activity retained. Three of the labelled Cys residues are located in the transmembrane domain, close to the substrate-binding pocket. Binding affinity determination using Trp and bimane fluorescence for tetramethylrosamine (R-site), Hoechst 33342 (H-site) and 6-(dimethylamino)-2-[4-[4-(dimethylamino)phenyl]-1,3-butadienyl]-1-ethyl perchlorate (both sites), showed similar Kd values for native Pgp and Pgp-bimane adducts, suggesting that the binding pocket can accommodate more than one substrate simultaneously. Reconstituted Pgp-bimane proteoliposomes showed the ability to transport all three substrates across the membrane, although with lower Vmax values compared to native Pgp. Thus, the Pgp-bimane adduct can transport substrates when more than one substrate occupies the binding pocket.
Date: 2014-08
Rights: Attribution-NonCommercial-NoDerivs 2.5 Canada
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Attribution-NonCommercial-NoDerivs 2.5 Canada Except where otherwise noted, this item's license is described as Attribution-NonCommercial-NoDerivs 2.5 Canada