Extent of Internal Hydration influence the Activation of GPCR Rhodopsin

Abstract

Rhodopsin is an archetypical GPCR responsible for dim‐light vision, which adopts a dynamic equilibrium between two metastable intermediates, metarhodopsin‐I (MI) and metarhodopsin‐II (MII), upon absorption of light. MI is an inactive conformational state of this GPCR, while MII is a fully active conformational state. We hypothesized that the MI to MII transition following isomerization of the retinal ligand involves a flood of bulk water into the protein core. To observe this change in the hydration level of the receptor during activation that is typically invisible to structural techniques such as X‐ray crystallography or cryo‐EM, we carried out a set of experiments using an osmotic stress approach. In this approach, the hydration level of rhodopsin was changed by placing the protein in native disk membranes in solutions with varying levels of osmotic pressure. Inert hydrophilic polyethylene glycol (PEG) solutes with different molecular weights and concentrations were used as the osmolytes. The effect of osmotic pressure on conformational dynamics was then quantified by measuring the reversible shifting of the metarhodopsin equilibrium using UV‐Visible spectroscopy. Results from these experiments show an influx of 80–100 water molecules into the rhodopsin interior, forming a partially disordered, solvent‐swollen active MII conformational state during photoactivation, a result supported by atomistic molecular dynamics simulations work [1, 2]. Additionally, we observed that the size of the polymer osmolyte played an important role in the modulation of rhodopsin’s activation: large osmolytes (PEG > 600) back shift the equilibrium to inactive MI state, while small osmolytes (PEG < 600) forward‐shift the equilibrium to MII state. We attribute this size dependence to the degree of osmolyte penetration into the rhodopsin core. Large polymers behave similarly to ideal osmolytes and dehydrate rhodopsin, while smaller polymers wriggle into the rhodopsin interior and stabilize the open MII conformation. These results necessitate a new understanding of GPCR activation in which the hydration level of protein is paramount in governing conformational energy landscapes. [1] U. Chawla et al. (2021) Angew. Chem. Int. Ed. 60, 2288–2295. [2] N. Leioatts et al. (2014) Biochemistry 53, 376−385.