G-protein-coupled receptors (GPCRs) comprise the largest and most pharmacologically targeted membrane protein family. We used visual rhodopsin as an archetype for understanding how the membrane soft matter (lipids and water) affects the conformational changes involved in GPCR activation. Cellular membranes are highly dynamic and undergo constant remodeling suggesting a biophysical role of fluctuations in mediating protein functions. Our hypothesis was that GPCRs exist as dynamic conformational ensembles with multiple inactive and active conformations biased by the membrane lipids and corresponding hydration state. Shifting of the metarhodopsin equilibrium due to changing the lipid environment was probed under controlled hydration (osmotic stress) using optical (UV-visible) spectroscopy. We considered the balance of curvature free energy with hydrophobic matching and demonstrated how our data support a flexible surface model (FSM) for the coupling between proteins and lipids (Annu.Rev.Biophys. 2017, 46, 379). The FSM is based on the Helfrich formulation of membrane bending energy in which elasticity and curvature strain are induced by lateral pressure imbalances between the lipids that drive key physiological processes at the membrane level. Spontaneous negative monolayer curvature toward water is mediated by (poly)unsaturated, small-headgroup lipids and couples directly to GPCR activation upon light absorption by rhodopsin. We further showed that activation of the archetypical GPCR rhodopsin in the lipid membrane is slaved to bulk water movements into the protein. A universal response of rhodopsin to osmotic stress was observed in the limit of increasing osmolyte size, suggesting the protein adopts a dynamic, solvent-swollen state upon photoactivation (Angew.Chem.Int.Ed. 2021, 60, 2288). The thermal fluctuations are directly accessed by neutron spin echo (NSE) spectroscopy, enabling a deeper understanding of the dynamic synergy between proteins and host membranes on important mesoscopic scales. Our results dramatically showcase how cellular lipids and water function as direct allosteric regulators of membrane protein activity and GPCR signaling.

Steven Fried

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