Abstract
The photoreceptor, rhodopsin is a kinetically stable membrane protein. This G-protein coupled receptor consitiutes greater than 90 percent of the membrane proteins of rod outer segment disk membranes. Therefore biophysical studies of rhodopsin can be carried out in its native membrane. The role of the native bilayer in maintaining the kinetic stability rhodopsin was investigated. The disks were systematically disrupted by the detergent, octyl-β-D-glucopyranoside (OG). Rhodopsin kinetic stability was examined under sub-solubilizing conditions in which the bilayer was partially disrupted as well as fully solubilizing conditions in which rhodopsin was largely delipidated. In differential scanning calorimetry (DSC) studies rhodopsin exhibited an irreversible scan rate dependent endothermic transition and a scan rate dependent exothermic transition at all stages of solubilization. The endothermic Tm decreased and the exothermic Tm increased as the OG partitioned into the bilayer. There was little change once the fully solubilized stage was achieved even though phospholipids were present in the mixed micelles. The activation energy of denaturation (Eact) was calculated at each stage of membrane disruption from the scan rate dependence of the Tm. The endothermic Eact decreased rapidly in the sub-solubilizing phase, but not once the membrane was fully solubilized. The Eact determined from the rate of thermal bleaching was in agreement with the DSC data. The degree of solubilization had no effect on the exothermic transition Eact. The calorimetric enthalpy (ΔHcal) was independent of the extent of solubilization. The thermal transition broadened during the sub-solubilizing phase suggesting increased protein motional freedom. There was no further broadening in the fully solubilized stage. These results indicate that the phospholipids present in detergent micelles do not kinetically stabilize the protein. The membrane bilayer enhances the kinetic stability rhodopsin by increasing the energy barrier to denaturation.
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