Hydration And Protonation Effects On Activation Of G-Protein-Coupled Receptors

G-protein-coupled receptors (GPCRs) are a superfamily of pharmaceutically crucial transmembrane receptors that are responsible for numerous physiological signaling pathways. However, the factors influencing the activation of GPCRs remain contested. Here, we use visual rhodopsin as an archetype for assessing effects of hydration changes upon GPCR activation. Using polyethylene glycol (PEG) solutes of varying molecular weights, rhodopsin was subjected to osmotic stress while the response of its metastable equilibrium was quantified using UV-visible spectroscopy [1]. By measuring the ratio of fully active metarhodopsin-II (MII) to inactive metarhodopsin-I (MI), the equilibrium constant (K) was calculated as a function of applied osmotic stress. Analysis using thermodynamic relations revealed an influx of 80-100 water molecules flooding into the interior of rhodopsin during photoactivation. This result was further supported by molecular dynamics (MD) simulations [2]. Solute effects on rhodopsin activation were size-dependent, in which larger polymers caused a back shifting of the equilibrium to the inactive MI state, while smaller solutes induced a forward shift to the active MII state. This size effect is attributed to the osmolyte ability to penetrate into the rhodopsin core: large osmolytes are more excluded and withdraw more water, while penetrating small solutes stabilize the expanded MII conformation for low concentration regimes [1]. Additionally, solutes significantly influence the pKA of Glu134, whose acid-base chemistry is strongly coupled to activation. By changing water availability, osmotic stress perturbs the dielectric constant characterizing the Glu134 environment, and thus the thermodynamics of its protonation. Our results demonstrate the activation of rhodopsin to be contingent upon the rearrangement of water molecules within the protein conformation, dramatically recasting the role of soft matter in biological signal transduction. [1] U. Chawla et al. (2020) Angew. Chem. Int. Ed. https://doi.org/10.1002/anie.202003342. [2] N. Leioatts et al. (2014) Biochemistry 53, 376−385.