(204c) Towards an in Vitro Model of Anti-Therapeutic Resistance: Drug Efflux Pump Supported Membranes Tethered on Silica Microstructures
The present study is focused on the implementation of a biomimetic approach to reintroduce the supporting structures of membrane proteins and provide enhanced spacing from the underlying substrate. Central to our approach is to build templated solid supported lipid bilayers that utilize bacteriorhodopsin (bR), a transmembrane protein, as a tethering molecule. We have conjugated bR with biotin-poly(ethylene glycol) (biotin-PEG3400) through amine based coupling for anchoring the tether and also labeled bR with Texas Red to facilitate localization via fluorescence imaging. These structures have been employed as a route to functionally immobilize the yeast drug efflux pump PDR5 (as a GFP fusion), a member of the ABC transporter superfamily. These assemblies are being used to build in vitro models of multidrug resistance for high-throughput screening and drug discovery.
The silica microsphere (5 micron) surface was functionalized with streptavidin in order to make a tethering bridge designed to yield a spacing of 9 - 14 nm from the silica (Silica-PEG3400-biotin (~4.7 nm) ? Streptavidin (~4 nm) ? biotin-PEG3400-bR (~4.7 nm). The objective is to yield supported membranes with a bilayer to substrate spacing that can accommodate membrane proteins with large cytoplasmic domains. The substrates were constructed by initially forming p-aminophenyltrimethoxysilane (APhMS) self-assembled monolayers on silica microspheres followed by subsequent attachment of biotin-PEG3400-NHS, PEG2000-NHS (for added passivation), and finally streptavidin. Bacteriorhodopsin tether supported lipid bilayer membranes were constructed onto these streptavidin-functionalized silica particles (5 micron) using a detergent-based reconstitution method in which the tethered bR conjugates template the assembly of the supported bilayer. Confocal fluorescence microscopy was utilized to analyze the silica surface at different stages of surface modification and examine the passivation of the substrate to the formation of untethered lipid bilayers. Fluidity of the supported membranes was analyzed using fluorescence recovery after photobleaching (FRAP). These lipid diffusion coefficient findings indicated that that native-like fluidity was achieved in the bR tethered supported membranes.
The drug efflux pump PDR5 (as a GFP fusion) has been isolated from S. cervisiae clones from a commercial library (InVitrogen) using chromatography and isoelectric focusing. The mobility of PDR5-GFP has been studied using confocal FRAP in supported membranes (tethered and untethered) and compared with that of the protein in the yeast plasma membrane. Functional assays are being performed using fluorescence based ATPase protocols.