Curvature and Hydrophobic Forces Drive Oligomerization and Modulate Activity of Rhodopsin in Membranes

Abstract

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.

G protein-coupled receptor-receptor interactions give integrative dynamics to intercellular communication

“…A few experimental studies addressed the relation between membrane thickness and oligomerization. Using FRET experiments, it was shown that the reduction of membrane thickness or the increase of the protein/lipid molar ratio promotes rhodopsin association (Brown, 1994;Botelho et al, 2006).…”

Solid-state 2 H NMR relaxation illuminates functional dynamics of retinal cofactor in membrane activation of rhodopsin

“…Light-induced retinal isomerization destabilizes the inactive receptor conformation (17,18). Activation entails large-scale movements of helices H5 and H6 together with H7 (19,23), exposing transducin (G t ) recognition sites on the C2 and C3 cytoplasmic loops (22). A current picture (17,27,44) is that photon absorption leads to the major reactions (17,44):…”

“…Such changes in local retinal dynamics (Fig. 3) underlie the influences of retinylidene methyl groups on the Meta I-Meta II equilibrium (34) within the pressure field of the lipid bilayer (22). In the currency of protein dynamics, large-scale functional motions involve displacements of many atoms within the protein.…”

“…Release of the inhibitory constraints biases the ensemble in favor of substates that expose recognition elements for transducin (G t ). Variables that reduce the entropy gain-for example, rhodopsin mutants, retinal analogues (1,34,42), or membrane lipids (21,22,49)-yield loss of function through back shifting the Meta I-Meta II equilibrium. Solid-state NMR plays an important role because it is exquisitely sensitive to local order and dynamics.…”

“…However, the lightactivated Meta II state is transient and crystal deformation occurs giving low-resolution data (14). Crystal structures of ligand-free opsin (19) with a bound synthetic G t peptide (20) show an elongation of the protein (21,22) due to helical displacements (19)-but opsin does not contain the activating all-trans-retinal ligand, and its activity does not match rhodopsin (20).…”