Hydration and Membrane Lipids Influence Rhodopsin Activation

Abstract

G-protein–coupled receptors (GPCRs) are integral membrane proteins responsible for signal transduction across cellular membranes and constitute the largest class of medicinal targets. While X-ray crystallographic and cryo-EM (cryogenic electronic microscopy) techniques have revealed the presence of structural water molecules in GPCRs, their exact physiological role in the signaling process remains unclear. Additionally, it is important to note that crystallography and cryo-EM experiments are typically performed under dehydrating conditions, and since bulk-phase water molecules exhibit inherent disorder, relying solely on these techniques may not provide an accurate depiction of the hydration states of a protein molecule. This study employs the visual receptor rhodopsin as a model system to investigate the impact of hydration and membrane properties on the conformational energetics and signaling of GPCRs. Contrary to the conventional biochemical perspective, which overlooks the contribution of biological soft matter such as lipids and water in GPCR activation, our study demonstrates that the activation of rhodopsin within the lipid membrane is intricately linked to alterations in membrane curvature and internal hydration levels. These factors play a pivotal role in modulating signaling efficacy through allosteric mechanisms. We investigated hydration effects on rhodopsin by subjecting it to polymer-induced osmotic stress, which allowed us to regulate the receptor hydration level. Simultaneously, we examined membrane effects by recombining rhodopsin into controlled lipid environments with varying lipid unsaturation, headgroup size, and lipid-to-protein ratios. Reversible shifting of the metarhodopsin equilibrium due to the osmotic stress and lipid environments was probed using UV-visible spectroscopy. We found that rhodopsin activation involves a bulk influx of water and that osmotic dehydration by large, excluded polymers inhibits activation. By contrast, small penetrable osmolytes and their monomers with lower conformational entropy penetrate the receptor core and stabilize the more expanded active state of rhodopsin until reaching a quantifiable saturation point. Notably, we observed an influx of ~80 water molecules into rhodopsin upon activation in both native and POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine) membranes. Therefore, we propose a novel sponge model of GPCR signaling, which emphasizes the regulatory role of water in the binding and unbinding of rhodopsin's G-protein transducin. We explain the lipid effects in terms of coupling of rhodopsin activation to changes in monolayer spontaneous curvature. Negative curvature is associated with the more expanded solvent swollen active Meta II state, while zero intrinsic curvature favors pre-active Meta I. Expansion of the receptor during activation is coupled to a negative monolayer curvature of the membrane, which is energetically favored by small headgroups, unsaturated acyl chains, and more dispersed lipid environments. These findings reinforce the adoption of a flexible surface model of lipid-protein interactions over the outdated fluid mosaic model. In conclusion, our results unveil unexpected yet critical roles of biological soft matter—comprising lipid bilayers and bulk water—in shaping crucial physiological processes.