Anodic Aluminum Oxide Nanopores as Substrate for Functional and Structural Studies on G Protein-Coupled Membrane Receptors

Abstract

Functional and structural studies on GPCR are ideally conducted on single, protein-carrying bilayers with unrestricted access for ligands and G-proteins. It is desirable to have a large accessible surface area and protection from a solid support. Reconstitution of GPCR into bilayers supported by porous anodic aluminum oxide (AAO) nanopores meets those requirements. Extrusion of proteoliposomes through the nanopores resulted in formation of tubules of a single lipid bilayer that covers the inner surface of pores. We successfully reconstituted the GPCRs rhodopsin and recombinant peripheral cannabinoid receptor CB2 at functional conditions and high concentration into the cylindrical AAO nanopores with a diameter of 200 nm and a length of 60 μm. One square centimeter of AAO filter yielded 500 cm2 of membrane surface. The lipid tubules are open at both ends such that buffer passes easily through the pores. Detergents used for protein reconstitution are flushed out within minutes. By 2H NMR we demonstrated that neither lipid headgroups nor hydrocarbon chains of fluid bilayers are perturbed by the solid support. Photoactivation of rhodopsin in the pores, monitored by UV-vis spectrophotometry, was indistinguishable from rhodopsin in unsupported liposomes. Metarhodopsin-II in the tubules activated G-protein that was delivered through the pore openings. By NMR diffusion experiments we determined that tubular bilayers are assembled as short pieces with a length of a micrometer or less that adhere to the surface by their edges. The tubules possess undulation with a radius of curvature of 100-400 nm. We have evidence for a layer of water with an average thickness of 3 nm between the bilayers and the pore surface. It explains why neither protein function nor fluid bilayer properties are perturbed by the solid support.