G protein-coupled receptors (GPCRs) are essential components of cellular signaling pathways. They are the targets of many current pharmaceuticals and are postulated to dimerize or oligomerize in cellular membranes in conjunction with their functional mechanisms. We demonstrate using fluorescence resonance energy transfer how association of rhodopsin occurs by long-range lipid-protein interactions due to geometrical forces, yielding greater receptor crowding. Constitutive association of rhodopsin is promoted by a reduction in membrane thickness (hydrophobic mismatch), but also by an increase in protein/lipid molar ratio, showing the importance of interactions extending well beyond a single annulus of boundary lipids. The fluorescence data correlate with the pK(a) for the MI-to-MII transition of rhodopsin, where deprotonation of the retinylidene Schiff base occurs in conjunction with helical movements leading to activation of the photoreceptor. A more dispersed membrane environment optimizes formation of the MII conformation that results in visual function. A flexible surface model explains both the dispersal and activation of rhodopsin in terms of bilayer curvature deformation (strain) and hydrophobic solvation energy. The bilayer stress is related to the lateral pressure profile in terms of the spontaneous curvature and associated bending rigidity. Transduction of the strain energy (frustration) of the bilayer drives protein oligomerization and conformational changes in a coupled manner. Our findings illuminate the physical principles of membrane protein association due to chemically nonspecific interactions in fluid lipid bilayers. Moreover, they yield a conceptual framework for understanding how the tightly regulated lipid compositions of cellular membranes influence their protein-mediated functions.

Rhodopsin is an important example of a G protein-coupled receptor (GPCR) in which 11-cis-retinal is the ligand and acts as an inverse agonist. Photolysis of rhodopsin leads to formation of the activated meta II state from its precursor meta I. Various mechanisms have been proposed to explain how the membrane composition affects the meta I-meta II conformational equilibrium in the visual process. For rod disk membranes and recombinant membranes containing rhodopsin, the lipid properties have been discussed in terms of elastic deformation of the bilayer. Here we have investigated the relation of nonlamellar-forming lipids, such as dioleoylphosphatidylethanolamine (DOPE), together with dioleoylphosphatidylcholine (DOPC), to the photochemistry of membrane-bound rhodopsin. We conducted flash photolysis experiments for bovine rhodopsin recombined with DOPE/DOPC mixtures (0:100 to 75:25) as a function of pH to explore the dependence of the photochemical activity on the monolayer curvature free energy of the membrane. It is well-known that DOPC forms bilayers, whereas DOPE has a propensity to adopt the nonlamellar, reverse hexagonal (H(II)) phase. In the case of neutral DOPE/DOPC recombinants, calculations of the membrane surface pH confirmed that an increase in DOPE favored the meta II state. Moreover, doubling the PE headgroup content versus the native rod membranes substituted for the polyunsaturated, docosahexaenoic acyl chains (22:6 omega 3), suggesting rhodopsin function is associated with a balance of forces within the bilayer. The data are interpreted by applying a flexible surface model, in which the meta II state is stabilized by lipids tending to form the H(II) phase, with a negative spontaneous curvature. A simple theory, based on principles of surface chemistry, for coupling the energetics of membrane proteins to material properties of the bilayer lipids is described. For rhodopsin, the free energy balance of the receptor and the lipids is altered by photoisomerization of retinal and involves curvature stress/strain of the membrane (frustration). A new biophysical principle is introduced: matching of the spontaneous curvature of the lipid bilayer to the mean curvature of the lipid/water interface adjacent to the protein, which balances the lipid/protein solvation energy. In this manner, the thermodynamic driving force for the meta I-meta II conformational change of rhodopsin is tightly controlled by mixtures of nonlamellar-forming lipids having distinctive material properties.

Rhodopsin is the only member of the pharmacologically important superfamily of G-protein-coupled receptors with a known structure at atomic resolution. A molecular dynamics model of rhodopsin in a POPC phospholipid bilayer was simulated for 15 ns, revealing a conformation significantly different from the recent crystal structures. The structure of the bilayer compared with a protein-free POPC control indicated hydrophobic matching with the nonpolar interface of the receptor, in agreement with deuterium NMR experiments. A new generalized molecular surface method, based on a three-dimensional Voronoi cell construction for atoms with different radii, was developed to quantify cross-sectional area profiles for the protein, lipid acyl chains and headgroups, and water. Thus, it was possible to investigate the bilayer deformation due to curvature of the individual lipid monolayers. Moreover, the generalized molecular surface derived hydrophobic interface allowed benchmarking of the hydropathy sequence analysis, an important structural genomics tool. Five water molecules diffused into internal hydration sites during the simulation, yielding a total of 12 internal waters. The cytoplasmic loops and the C-terminal tail, containing the G-protein recognition and protein sorting sequences, exhibited a high mobility, in marked contrast to the extracellular and transmembrane domains. The proposed functional coupling of the highly conserved ERY motif to the lipid-water interface via the cytoplasmic loops provides insight into lipid effects on G-protein-coupled receptor activation in terms of a flexible surface model, involving the spontaneous monolayer curvature.

Changes in lipid composition have recently been shown to exert appreciable influences on the activities of membrane-bound proteins and peptides. We tested the hypothesis that the conformational states of rhodopsin linked to visual signal transduction are related to biophysical properties of the membrane lipid bilayer. For bovine rhodopsin, the meta I-meta II conformational transition was studied in egg phosphatidylcholine (PC) recombinants versus the native rod outer segment (ROS) membranes by means of flash photolysis. Formation of metarhodopsin II was observed by the change in absorbance at 478 nm after a single actinic flash was delivered to the sample. The meta I/meta II ratio was investigated as a function of both temperature and pH. The data clearly demonstrated thermodynamic reversibility of the transition for both the egg PC recombinants and the native ROS membranes. A significant shift of the apparent pK(a) for the acid-base equilibrium to lower values was evident in the egg PC recombinant, with little meta II produced under physiological conditions. Calculations of the membrane surface pH using a Poisson-Boltzmann model suggested the free energies of the meta I and meta II states were significantly affected by electrostatic properties of the bilayer lipids. In the ROS membranes, phosphatidylserine (PS) is needed for full formation of meta II, in combination with phosphatidylethanolamine (PE) and polyunsaturated docosahexaenoic acid (DHA; 22:6omega3) chains. We propose that the PS surface potential leads to an accumulation of hydronium ions, H(3)O(+), in the electrical double layer, which drive the reaction together with the large negative spontaneous curvature (H(0)) conferred by PE plus DHA chains. The elastic stress/strain of the bilayer arises from an interplay of the approximately zero H(0) from PS and the negative H(0) due to the PE headgroups and polyunsaturated chains. The lipid influences are further explained in terms of matching of the bilayer spontaneous curvature to the curvature at the lipid/rhodopsin interface, as formulated by the Helfrich bending energy. These new findings guide current ideas as to how bilayer properties govern the conformational energetics of integral membrane proteins. Moreover, they yield knowledge of how membrane lipid-protein interactions involving acidic phospholipids such as PS and neutral polyunsaturated DHA chains are implicated in key biological functions such as vision.

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