Membrane-Lipid Mediated Rhodopsin Signaling Involves an Ensemble of Conformational Substates

Abstract

G-protein-coupled receptors (GPCRs) constitute about 50% of the known drug targets. Using rhodopsin as a model GPCR, we study the role of membrane lipids in the rhodopsin activation. Absorption of light by rhodopsin leads to a series of conformational changes that establishes an equilibrium between inactive Meta-I and an ensemble of activated Meta-II states [1,2]. Using UV-visible and FTIR spectroscopy, we show that the distribution of late conformational substates during rhodopsin activation is lipid-mediated as explained by an ensemble activation mechanism (EAM). Lipid bilayer composition (head groups and acyl chain lengths) and membrane protein-lipid interaction govern the EAM through biasing conformational substates. Rhodopsin reconstituted in DOPC backshifts the equilibrium to the Meta-IIa substate, whereas mixed-chain POPC membranes favor the inactive Meta-I state [3]. The wavenumber-dependent analysis of the FTIR-difference spectra yields a distribution of pKa and alkaline endpoint values, consistent with an ensemble of substates for each lipid bilayer-rhodopsin system. A phenomenological Henderson-Hasselbalch function was fitted to the pH titration curves to derive thermodynamic parameters from the spectral analysis. Our thermodynamic studies show that activation of rhodopsin is accompanied by an entropy gain compensating the unfavorable enthalpy increase, analogous to protein unfolding reactions. The results from the EAM analysis are in agreement with the flexible surface model (FSM), which describes elastic coupling of the membrane lipids to integral membrane proteins through a balance of curvature and hydrophobic forces in lipid-protein interactions [4]. Our study also provides insight into the thermodynamic parameters that govern rhodopsin-like GPCR activation in native membrane lipid environments. [1] A.V. Struts et al. (2011) PNAS 108, 8263-8268. [2] A.V. Struts et al. (2014) Meth. Mol. Biol. (in press). [3] E. Zaitseva et al. (2010) JACS 132, 4815-4821. [4] M.F. Brown (2012) Biochemistry 51, 9782-9795.