Water is a powerful dynamic allosteric modulator in rhodopsin activation.

Rhodopsin is an archetype for G protein-coupled receptors (GPCRs) which are membrane proteins involved in cellular signal transduction. Although GPCR X-ray structures show the presence of structural water molecules, their physiological role in the signaling process remains uncertain. Our previous work has shown that in lipid membranes rhodopsin activation is coupled to bulk water movements into the protein to stabilize the effector binding conformation [1,2]. Here we extend the experimental approach to explore modulation of the conformational energetics of rhodopsin activation in lipid membranes. We hypothesized that the thermodynamic stability of conformational substates of photoactivated rhodopsin is hydration mediated. Accordingly, we changed the osmotic stress on rhodopsin using different hydrophilic polymers including polyethylene glycols (PEGs), dextrans, polyvinylpyrrolidones, and their monomers. Reversible shifting of the metarhodopsin equilibrium due to the osmotic stress was probed using UV-visible spectroscopy. We observed that the rhodopsin activation is associated with a bulk influx of water due to the osmotic dehydration by large, excluded polymers which inhibit GPCR activation. By contrast small penetrable osmolytes and monomers have lower conformational entropy and showed a nonosmotic trend in rhodopsin activation. Small osmolytes penetrate the receptor core and stabilize the more expanded active state of rhodopsin until reaching a quantifiable saturation point. Both PEGs and dextrans with very high molar masses deviated in their trends from other large polymers due to a crowding effect, whereby extremely large osmolytes induce steric forces on rhodopsin and restrict their volume occupancy. Further studies of hydration effects on binding of the G-protein and receptor catalyzed exchange of GTP for GDP are expected to provide significant clues on the complete mechanism of G-protein binding and release