Investigation of Lipid Bilayer Effects on Rhodopsin Activation using UV-Visible and Ftir Spectroscopy

Abstract

G-protein-coupled receptors (GPCRs) comprise almost 50% of pharmaceutical drug targets and play crucial roles in signal transduction for a number of physiological processes. Upon photoactivation, rhodopsin undergoes a series of conformational changes leading to visual perception. An ensemble of activated Meta II states is in equilibrium with the inactive precursor Meta I [1]. Lipid bilayer composition and its interaction with membrane proteins govern the ensemble activation mechanism (EAM) as predicted by the flexible surface model (FSM). The FSM describes the balance between intrinsic monolayer curvature and lipid-protein hydrophobic interactions, leading to the elastic coupling of lipids and membrane proteins [2]. Effects of temperature and pH were analyzed for rhodopsin reconstituted in lipid vesicles using UV-visible and FTIR spectroscopy. Thermodynamic properties were derived by fitting phenomenological Henderson-Hasselbalch functions to their respective pH titration curves. Mixed-chain POPC membranes backshift rhodopsin towards Meta I, whereas rhodopsin in DOPC favors the active Meta IIa substate. Analysis of the wavelength-dependent distribution of pKa and alkaline endpoints as estimated from FTIR difference spectra reveals an ensemble of substates for each lipid bilayer-rhodopsin system. The presence of multiple activated conformations is a hallmark of the EAM. Our results are in agreement with the FSM, whereby lipids having a negative monolayer curvature favor the active Meta II state, while lipids with zero spontaneous curvature (POPC) favor the inactive Meta I state [3]. The data give additional insight into the entropy-enthalpy balance which drives the structural conformational changes that occur upon rhodopsin photoactivation. Moreover our work provides fundamental insight into the functionality of other GPCR-related proteins in a natural membrane lipid environment. [1] A.V. Struts (2011) PNAS 108, 8263-8268. [2] M.F. Brown (2012) Biochemistry 51, 9782-9795. [3] E. Zaitseva (2010) JACS 132, 4815-4821.