G-protein–coupled receptors (GPCRs) are integral membrane proteins responsible for signal transduction across cellular membranes and constitute the largest class of medicinal targets. Although three-dimensional structures show the presence of structural water molecules within GPCRs, the functional role of these water molecules has remained unknown. Our previous work has further shown a bulk influx of water stabilizing the active rhodopsin conformation (Angew.Chem.Int.Ed. 2021, 60, 2288). Here we broadened the experimental approach to investigate how the conformational energetics of GPCR activation are modulated by water using a series of hydrophilic polymers including polyethylene glycols (PEGs), dextrans, polyvinylpyrrolidones, and their monomers. Our hypothesis was that the thermodynamic stability of conformational substates of photoactivated rhodopsin is hydration mediated. Accordingly, we changed the osmotic stress on rhodopsin with different concentrations of various polymer solutes and used UV-visible spectroscopy to monitor the shifting of the metarhodopsin equilibrium under controlled hydration. An opposite effect of the large and small osmolytes was observed on the metarhodopsin equilibrium, in which larger osmolytes (e.g., PEG 1500-6000) promoted the inactive metarhodopsin-I (MI) state, while the small osmolytes increased the active metarhodopsin-II (MII) fraction. We propose that these size effects are a result of the degree of penetration of osmolytes into the protein core of rhodopsin. Small polymers penetrate the protein core and favor the open MII state of rhodopsin, while the large polymers behave like ideal osmolytes and dehydrate rhodopsin. Interestingly PEGs and dextrans with extremely large molar masses deviated in their trends from other large polymers, which we attribute to a combined result of crowding and penetration effects. Our results provide us with new insights into the interplay between osmotic stress, penetration, and crowding effects and further our understanding of the vital role of water in the GPCR activation mechanisms.

Osmotic stress and crowding in G-protein-coupled receptor activation

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.