Title

Assessment of Bovine Rod Outer Segment (ROS) Disk Membrane Heterogeneity and the Contribution of the Bilayer to Rhodopsin Kinetic Stability

Date of Completion

January 2010

Keywords

Chemistry, Biochemistry|Biophysics, General

Degree

Ph.D.

Abstract

Rod outer segment (ROS) disk membranes undergo modifications in lipid composition as disks are displaced Disks become progressively lower in cholesterol and higher in unsaturated phospholipids. Flow cytometry was used to assess differences amongst disks. First, the scatter properties of beads of similar size (0.1-1.0μm in diameter) to osmotically intact disks, were established. Disk scatter intensity predominantly corresponded to 0.1-0.2μm beads. Flow cytometry was applied to detect caveolin-1, peripherin/rds, and GM1 in disk membranes. Fluorescence labeling experiments indicated that caveolin-1, peripherin/rds, and GM1 were detected in 80%, 70%, and 60% of respective scattering events corresponding to disks. Fluorescence intensity of caveolin-1 and peripherin/rds labeling was directly proportional to scatter intensity, indicating that both caveolin-1 and peripherin/rds are uniform among the disks. Fluorescence intensity of GM1 labeling was independent of side scatter intensity, indicating GM1 is heterogeneously distributed among the disks. Results are consistent with the integral membrane proteins caveolin-1 and peripherin/rds remaining in the bilayer as disks are apically displaced. GM1 distribution may be similar to the spatial distribution of cholesterol in the outer segment.^ The bilayer contribution to rhodopsin kinetic stability was examined using differential scanning calorimetry (DSC). Disk membranes were systematically disrupted with sub-solubilizing and fully solubilizing concentrations of octy1-β-D-glucopyranoside (OG). At all stages of solubilization, rhodopsin exhibited an irreversible endothermic transition followed by an irreversible exothermic transition, both scan rate dependent. The endothermic transition temperature ( Tm) decreased and the exothermic T m increased in the sub-solubilizing stage. However, the T ms changed little as the phospholipid to rhodopsin ratio in the mixed micelles decreased during the fully solubilized stage. The activation energy of denaturation (Eact) was calculated from the scan rate dependence of the Tm. The endothermic Eact decreased in the sub-solubilizing stage, but remained constant in the fully solubilized stage. Eact determined from the rate of thermal bleaching was in agreement with DSC data. The endothermic transition broadened during the sub-solubilizing phase suggesting increased motional freedom. Solubilization had no effect on either transition's exothermic Eact or calorimetric enthalpy (ΔH cal). These results indicate the bilayer enhances the kinetic barrier to rhodopsin denaturation. ^