Lipid Bilayer Influences Rhodopsin Activation Probed by FTIR and UV-Visible Spectroscopy

Abstract

Rhodopsin, the mammalian dim light photoreceptor, is the model protein for studying G protein-coupled receptor (GPCR) structure and function. We postulate that this process can be attributed to an ensemble of activated states - the ensemble activation mechanism (EAM) - and that the EAM is directly affected by membrane protein-lipid bilayer interactions dependent upon cell membrane composition, as predicted by the flexible surface model [1]. Rhodopsin was detergent-solubilized and incorporated into homogenous lipid vesicles (either POPC or DOPC) by dialysis. Samples were prepared at a series of temperature and pH values, bleached, and analyzed by simultaneous Fourier transform infrared (FTIR) and UV-visible spectral acquisition. Activation in mixed-chain POPC bilayers drastically backshifts rhodopsin from the active Meta II photointermediate to the inactive Meta I state. A di-monounsaturated phospholipid like DOPC restores partial activity to rhodopsin, as well as stabilizing the protein in the Meta IIa state. Spectral reduction and analysis via pH titration curves yielded non-Henderson-Hasselbach behavior, indicating more than one activated state exists, thus supporting the concept of an EAM [2]. In addition, temperature changes have a marked effect on rhodopsin activation, decoupling the two protonation switches necessary for full activation at physiological temperatures. By manipulating the lipid environment, we validate the flexible surface model and EAM. Lipids with negative monolayer curvature such as DOPC facilitate rhodopsin activation towards the Meta II state [3]. Thermodynamic parameters showed that free energy changes are related to greater flexibility of rhodopsin in the cell membrane upon activation. Further validation of the flexible surface model is an important contribution to biophysical understanding of GPCR function.[1] A.V. Botelho et al. (2006) BJ 91, 4464-4477. [2] M. Mahalingam et al. (2008) PNAS 105, 17795-17800. [3] E. Zaitseva et al. (2010) JACS 132, 4815-4821.