Summary Polar lipids must flip-flop rapidly across biological membranes to sustain cellular life [1, 2], but flipping is energetically costly [3] and it’s intrinsic rate is low. To overcome this problem, cells have membrane proteins that function as lipid transporters (flippases) to accelerate flipping to a physiologically relevant rate. Flippases that operate at the plasma membrane of eukaryotes, coupling ATP hydrolysis to unidirectional lipid flipping, have been defined at a molecular level [2]. On the other hand, ATP-independent bidirectional flippases that translocate lipids in biogenic compartments, e.g., the endoplasmic reticulum, and specialized membranes, e.g., photoreceptor discs [4, 5], have not been identified even though their activity has been recognized for more than 30 years [1]. Here we demonstrate that opsin is the ATP-independent phospholipid flippase of photoreceptor discs. We show that reconstitution of opsin into large unilamellar vesicles promotes rapid (τ <10 sec) flipping of phospholipid probes across the vesicle membrane. This is the first molecular identification of an ATP-independent phospholipid flippase in any system. It reveals an unexpected activity for opsin and, in conjunction with recently available structural information on this G-protein coupled receptor [6, 7], significantly advances our understanding of the mechanism of ATP-independent lipid flip-flop.

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The inherent instability of heptahelical G protein-coupled receptors (GPCRs) during purification and reconstitution is a primary impediment to biophysical studies and to obtaining high-resolution crystal structures. New approaches to stabilize receptors during purification and to screen reconstitution procedures are needed. Here we report the development of a novel homogeneous time-resolved fluorescence assay (HTRF) to quantify properly folded CC-chemokine receptor 5 (CCR5). The assay permits high-throughput thermal stability measurements of femtomole quantities of CCR5 in detergent and in engineered nanoscale apolipoprotein bound bilayer (NABB) particles. We show that recombinant expressed CCR5 can be incorporated into NABB particles in high yield, resulting in greater thermal stability compared with CCR5 in detergent solution. We also demonstrate that CCR5 binding to the HIV-1 cellular entry inhibitors maraviroc, AD101, CMPD 167, and vicriviroc dramatically increases receptor stability. The HTRF assay technology reported here is applicable to other membrane proteins and could greatly facilitate structural studies of GPCRs. Keywords GPCR; lipoprotein particle; CCR5; NABB; thermal stability; maraviroc; europium cryptate; timeresolved fluorescence; energy transfer G protein-coupled receptors (GPCRs) constitute a large family of heptahelical membrane proteins that recognize a broad array of extracellular ligands and couple to multiple intracellular signaling pathways.(1,2) Their involvement in the molecular pathophysiology of a number of diseases has made GPCRs a major drug target.(3) Recent high-resolution structures of several GPCRs, including rhodopsin, β 2 -adrenergic receptor, β 1 -adrenergic receptor, A 2A adenosine receptor, and opsin have provided insight into the molecular mechanism of receptor activation, but further advances are necessary to understand their physiological function and to enhance drug discovery. Stabilizing these complex polytopic membrane proteins in purified and/or defined systems remains the most significant present challenge for biochemists and structural biologists. Successful approaches to generating stable GPCRs in detergent solution include truncations or deletions of disordered regions,

Surface-enhanced infrared absorption (SEIRA) difference spectroscopy can probe reactions in a protein monolayer tethered to a nanostructured gold surface. SEIRA studies of membrane proteins, however, remain challenging due to sample stability, effects of the metal surface on function, and the need for a membrane-mimicking environment. Here we demonstrate and characterize a model system for membrane receptor investigations using SEIRA spectroscopy. The system employs nanoscale apolipoprotein bound bilayer (NABB) particles, similar to discoidal high-density lipoprotein particles, as soluble carriers for the G-protein-coupled receptor rhodopsin. The His-tag of the engineered apolipoprotein allows for selective binding of the NABBs to a Ni-NTA modified surface, while the lipid environment of the particle ensures stability and protection of the embedded receptor. Using SEIRA spectroscopy, we followed specific binding of rhodopsin-loaded NABB particles to the surface and formation of a membrane protein monolayer. Functionality of the photoreceptor in the immobilized NABBs was probed by SEIRA difference spectroscopy confirming protein conformational changes associated with photoactivation. Orientation of the immobilized NABB particles was assessed by comparing SEIRA data with polarized attenuated total reflection-Fourier-transform infrared spectroscopy. Thus, SEIRA difference spectroscopy supported by the NABB technology provides a promising approach for further functional studies of transmembrane receptors.

Discoidal lipoproteins are a novel class of nanoparticles for studying membrane proteins (MPs) in a soluble, native lipid environment, using assays that have not been traditionally applied to transmembrane proteins. Here, we report the successful delivery of an ion channel from these particles, called nanoscale apolipoprotein-bound bilayers (NABBs), to a distinct, continuous lipid bilayer that will allow both ensemble assays, made possible by the soluble NABB platform, and single-molecule assays, to be performed from the same biochemical preparation. We optimized the incorporation and verified the homogeneity of NABBs containing a prototypical potassium channel, KcsA. We also evaluated the transfer of KcsA from the NABBs to lipid bilayers using single-channel electrophysiology and found that the functional properties of the channel remained intact. NABBs containing KcsA were stable, homogeneous, and able to spontaneously deliver the channel to black lipid membranes without measurably affecting the electrical properties of the bilayer. Our results are the first to demonstrate the transfer of a MP from NABBs to a different lipid bilayer without involving vesicle fusion.