We developed a method for reconstitution of the G protein-coupled membrane receptors (GPCR) into single bilayers using nanoporous anodic aluminum (AAO) filters. The tubular membranes line the inner surface of pores that traverse the filters, permitting easy removal of detergents during sample preparation as well as delivery of ligands and G protein for functional studies. Experiments were conducted with the GPCR bovine rhodopsin. Sample preparation is simple and rapid. Reconstitution of rhodopsin into single, tubular membranes in porous AAO substrates, yielded functional rhodopsin. Neither the lipid matrix properties nor the equilibrium of metarhodopsin I (MI)/metarhodopsin II (MII) photointermediates were different from unsupported, unilamellar liposomes. The oriented bilayers are packed in the substrate at very high density.  One square centimeter of a porous AAO filter (thickness 60 micrometers) yields on the order of 500 cm2 of solid-supported, oriented bilayers, sufficient for conducting NMR studies. The bilayers have a controlled radius of curvature that is determined by pore dimensions. The ability to orient bilayers at a solid interface and to control membrane curvature greatly improved sensitivity and reproducibility of solid-state 2H NMR experiments on deuterated lipids in membranes. Results of (i) a study on the influence of hydrophobic thickness of phosphatidylcholine bilayers on rhodopsin structure and function, and (ii) the influence negative curvature elastic stress in lipid monolayers on the MI/MII equilibrium will be shown. The approach is likely to be applicable to reconstitution of other membrane proteins as well. It appears to be particularly beneficial for studies that require a change of ionic strength, pH, and addition of ligands or soluble proteins during the course of the experiment. Most importantly, it enables structural studies on membrane proteins at functional conditions.

Reconstitution of G protein-coupled membrane receptors into single tubular bilayers using nanoporous substrates

245th National Meeting (2013)

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