Effects of Soft Matter on G-Protein-Coupled Receptor Activation

Abstract

G protein-coupled receptors (GPCRs) constitute both the largest class of medicinal targets and the largest family of human membrane proteins. Current three-dimensional structures have shown the existence of many water molecules immersed inside GPCRs. However, the functional role of these water molecules has remained uncertain. Our previous work has shown that water modulates the activation of visual rhodopsin, a prototypical GPCR [1]. Here we expanded our approach to further investigate how the conformational energetics of GPCR activation are modulated by water using a series of osmolytes that included polyethylene glycols (PEGs), dextrans, polyvinylpyrrolidones (PVPs), and their monomers. We hypothesized that osmotic pressure affects the thermodynamic stability of conformational substates of photoactivated rhodopsin. Accordingly, we changed the osmotic stress on rhodopsin using different concentrations of various polymer solutes. Shifting of the metarhodopsin equilibrium due to the osmotic pressure was probed using UV-visible spectroscopy. The larger osmolytes (e.g., PEG 1500-6000) promote the inactive metarhodopsin-I (MI) state, while the small organic osmolytes such as smaller molar mass PEGs, their monomers, and small sugars increase the active metarhodopsin-II (MII) fraction. 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. Most interestingly, PEGs and dextrans with extremely large molar masses deviated in their trends from other large polymers (PEGs), which we attribute to a combined result of crowding and penetration effects. The results provide us with new insights into the interplay between osmotic stress, penetration, and crowding effects. Our findings further the understanding of the vital role of water in the GPCR activation mechanisms. [1] U. Chawla et al. (2020) Angew.Chem.Int.Ed. doi:10.1002/anie.202003342.