This project is designed to assess the role of membrane lipid composition, especially polyunsaturated phospholipids, in modulating G protein-coupled receptor (GPCR) signal transduction and to elucidate the mechanism of action of ethanol in these systems. GPCRs are ubiquitous components of signal transduction pathways, including taste, smell, vision, and many neurotransmitter systems. GPCRs are also targets of a great many pharmaceutical drugs. The visual transduction pathway of the retinal rod photoreceptor is the best characterized member of this receptor superfamily and is being used as a model system in these studies. We studied a wide variety of detergents to determine the requirements for optimal preservation of membrane protein structural stability and function, and we investigated the interplay between polyunsaturated lipids and cholesterol in determining protein stability. Purified bovine rhodopsin was reconstituted into vesicles consisting of 1-stearoyl-2-oleoyl phosphatidylcholine or 1-stearoyl-2-docosahexaenoyl phosphatidylcholine with and without 30 mol% cholesterol. Rhodopsin stability was examined using differential scanning calorimetry (DSC). The thermal unfolding transition temperature (Tm) of rhodopsin was scan rate dependent, demonstrating the presence of a rate-limited component of denaturation. Both Tm and Ea varied with bilayer composition. Cholesterol increased Tm both in the presence and absence of DHA acyl chains. In contrast, cholesterol lowered Ea in the absence DHA acyl chains, but raised Ea in the presence of 20 mol% DHA-containing phospholipid. Maximal kinetic stability was found within the range of acyl chain order found in native bovine rod outer segment disk membranes. The results demonstrate that membrane composition has distinct effects on the thermal versus kinetic stabilities of membrane proteins, and suggests that a balance between membrane constituents with opposite effects on acyl chain packing may be required for maximum protein stability. Due to the continuing challenge of reconstituting purified membrane proteins in quantities sufficient for many for many biophysical techniques, there is increasing use of membrane proteins solubilized in detergents. We analyzed the effects of anionic, cationic, zwitterionic and non-ionic detergents on the conformation changes involved in rhodopsin activation and deactivation, which occur over time scales from microseconds to hundreds of seconds. The fastest conformation changes, associated with formation of the G protein-binding conformation, were significantly distorted by all detergents. The slowest conformation changes, associated with release of ligand, proceeded with native-like kinetics in detergents with a high micelle molecular weight, regardless of detergent ionic character. The results will provide membrane biophysicists with useful guidelines for determining when it is appropriate to study solubilized membrane proteins and which detergents may provide reasonably native conditions.
| Niu, Shui-Lin; Doctrow, Brian; Mitchell, Drake C (2009) Rhodopsin activity varies in proteoliposomes prepared by different techniques. Biochemistry 48:156-63 |
