Hydration Mediated G-Protein-Coupled Receptor Activation

Abstract

G-protein-coupled receptors (GPCRs) comprise about 50% of the known drug targets and play crucial roles in numerous of physiological processes. Rhodopsin is a canonical GPCR, which upon photoactivation undergoes a series of conformational changes leading to a chemical equilibrium between closed inactive Meta-I and open active Meta-II states [1,2]. We hypothesize that activation of rhodopsin leads to an increase in hydrated protein volume in the active Meta-II state. Using UV-visible spectroscopy, we tested our hypothesis by evaluating how the Meta-I/Meta-II equilibrium and thermodynamic parameters are influenced by changing the membrane environment. We discovered a surprising bulk water influx (about 75 water molecules) during formation of the active protein. Moreover, our results with high molar mass osmolytes differ from previous osmotic stress studies with small osmolytes. We discovered that osmolytes of varying molar mass affect rhodopsin activation differently. Large osmolytes shift the equilibrium to the inactive Meta-I state, leading to efflux of water. By contrast, small osmolytes lead to an influx of water upon activation. We propose that small osmolytes affect rhodopsin activation similarly to the G-protein transducin, which stabilizes the active Meta-II state. Large osmolytes cannot gain access to the transducin binding site and exert osmotic stress on the protein. Our results are in agreement with molecular dynamics studies showing an influx of water during rhodopsin photoactivation [3]. Hence we propose that rapid high-fidelity signaling by rhodopsin involves cycling of water into and out of the protein core together with activation of transducin. Our studies give important insight in the role of water in activation of GPCRs like rhodopsin. [1] A.V. Struts et al. (2011) PNAS 108, 8263-8268. [2] A. V. Struts et al. (2015) Meth. Mol. Biol. 1271, 133-158. [3] N. Leioatts et al. (2013) Biochemistry 53, 376-385.