Effect of Solubilization on Rhodopsin Thermal Denaturation in Rod Outer Segment Disk Membranes

Abstract

The photoreceptor, rhodopsin is a GPCR in rod outer segment disk membranes. Activation by light converts the dark-adapted form, rhodopsin to the bleached form, opsin. Differential scanning calorimetry (DSC) studies showed that rhodopsin and opsin each exhibit an irreversible scan rate dependent endothermic transition (Tm) at approximately 72°C and 55°C respectively as well as a scan rate dependent exothermic transition. Solubilization was used to examine the contribution of the bilayer. Disk membranes were subjected to sub-solubilizing and solubilizing concentrations of octylglucoside (OG) until rhodopsin was completely delipidated. DSC experiments were performed using a MicroCal VP-DSC microcalorimeter. Samples were scanned at 15, 30, 60 and 90°/hr. Because the protein transitions are irreversible, a second scan was used to determine the baseline. As the OG partitioned into the bilayer the endothermic Tm and EACT (activation energy of denaturation) rapidly decreased. Both then remained constant following rhodopsin solubilization. At low detergent concentration the exothermic Tm increased rapidly then remained constant after solubilization. Unlike the endothermic EACT, the degree of solubilization had little effect on the exothermic transition EACT. Digitonin was also used to examine the effect of solubilization. Unlike OG, this detergent binds to cholesterol which is constitutes approximately 10% of the disk lipids (average). The endothermic transition was less affected by digitonin than by OG. The EACT was also determined by thermal bleaching and was in agreement with the DSC data. These results indicate an endothermic transition is observed due to a weakening of the tertiary structure interactions as rhodopsin is heated. This may be accompanied by changes in the packing of the trans-membrane helices as well as changes in protein-lipid interactions. It is likely the exothermic transition results from aggregation.