Flexible Surface Model Explains Time-Resolved Uv-Visible Studies of Rhodopsin Activation in a Membrane Lipid Environment

Abstract

Rhodopsin is the quintessential GPCR--the UV/Visible absorbance of its bound chromophore provides detailed time-resolved information about the GPCR activation steps [1]. At least four species equilibrate on the millisecond time scale after rhodopsin photoexcitation in a membrane environment [2]. The first millisecond-time scale equilibrium is between the protonated Schiff base (PSB) species Meta I480 and the deprotonated SB species Meta IIa and is pH independent. Subsequent equilibria involve spectrally silent transitions of Meta IIa to produce Meta IIb and protonation of Glu134 of the E(R)Y sequence in Meta IIb to give Meta IIbH+ where pKa describes the acid-base equilibrium. We used time-resolved absorbance measurements on the microsecond-to-hundred millisecond time scale to study effects of the membrane lipid environment on the first equilibrium constant, K1, and on the pKa of the final equilibrium. Reconstituted membranes of rhodopsin with POPC, DOPC, or DOPC/DOPE mixtures were studied at 30oC. Results were analyzed by singular value decomposition and globally fit to a sum of exponential terms. We discovered a striking increase in K1 due to either PE head groups or increased acyl chain unsaturation; by contrast pKa changed little. According to the flexible surface model (FSM), there is competition between the curvature elastic energy and the hydrophobic mismatch at the proteolipid boundary that explains the above influences of lipid-protein interactions [3]. The fact that both the lipid acyl chains and polar head groups affect the Meta I-Meta II transition of rhodopsin is a striking illustration of how protein energetics in membranes are governed by chemically nonspecific properties of the lipid bilayer. [1] J. Epps et al. (2006) Photochem. Photobiol.82, 1436-1441. [2] E. Zaitseva et al. (2010) JACS132, 4815-4821. [3] A.V. Botelho et al. (2006) BJ91, 4464-4477.