Lipid and Detergent Effects on the Kinetic Stability of the GPCR, Rhodopsin

Abstract

The photoreceptor, rhodopsin is a kinetically stable G-Protein Coupled Receptor that is located in rod outer segment disk membranes. Differential scanning calorimetry (DSC) studies have shown that rhodopsin exhibits an irreversible scan rate dependent endothermic transition (Tm) at approximately 72 oC. The activation energy for thermal denaturation (Eact) calculated from the scan rate dependence of the Tm is sensitive to the integrity of the lipid bilayer. It is also sensitive to proteolytic cleavage of the extramembraneous loops of rhodopsin. Here we investigate the influence of palmitoylation, membrane cholesterol and digitonin solubilization on rhodopsin kinetic stability. DSC experiments were performed using a MicroCal VP-DSC microcalorimeter. Samples were scanned at 15, 30, 60 and 90o/hr. Because the protein transitions are irreversible, a second scan was used to determine the baseline. Rhodopsin palmitoylation at cys 322 and cys 323 anchors the C-terminus to the membrane. The Eact for rhodopsin in disk membranes treated with hydroxylamine to remove these palmitate groups was approximately 35 Kcal/mole less than that of native rhodopsin. The Eact for rhodopsin was also determined at different membrane cholesterol levels. These data indicated a decrease in stability both above and below native membrane cholesterol levels. Finally, unlike solubilization in other detergents, solubilization in digitonin had little effect on rhodopsin kinetic stability.