New Immobilized Proteoliposome-Based Biosensor System for Investigating Human ATP-Binding Cassette Transporters

Abstract

ATP-binding cassette (ABC) transporters comprise a large family of membrane proteins that transport a variety of organic substrates across cellular membranes. Human ABC transporters are mostly efflux pumps for physiological and xenobiotic substrates related to immune response and cellular detoxification. Nine human ABC transporters play a major role in cellular multi-drug resistance (MDR), thus being called MDR-related proteins (MRPs). So far, the structure of MRPs and human ABC transporters in general is unclear. Sequence analysis and experimental data indicate that functional ABC transporters are composed of two subunits and imply strong positive cooperativity between those entities. To elucidate the transport mechanism and the molecular origin of the cooperativity a solid-supported biosensor system with defined physicochemical properties is needed. One promising approach is the immobilization of MRP-containing proteoliposomes on functionalized surfaces. We report a new system for immobilizing biotin-doped proteoliposomes via the well-known biotin-streptavidin interaction on gold surfaces functionalized with a self assembled monolayer (SAM) of a binary thiol mixture. The SAM composed of a hydroxy-terminated 16-carbon alkanethiol and its biotinylated derivative protects the gold surfaces from unspecific adsorption and allows the immobilization of defined quantities of streptavidin. Proteoliposomes made from natural lipid compositions and doped with a biotinylated anchor lipid can readily be tethered to these surfaces. By thorough biophysical characterization using quartz crystal microbalance (QCM), atomic force microscopy (AFM) and fluorescence techniques all experimental parameters were optimized for application in biosensor systems. We successfully immobilized intact proteoliposomes containing the reconstituted human ABC transporter MRP3 on the described surfaces. Our system allows the investigation of ABC transporters by a variety of surface-enhanced techniques ranging from AFM and QCM to impedance spectroscopy and surface plasmon resonance based methods under well-defined conditions closely mimicking the protein's natural environment.