Structural Stability of Rhodopsin and Opsin Studied by Differential Scanning Calorimetry

The thermal stability of opsin and rhodopsin in disk membranes of bovine retinal rod cell outer segments has been examined by differential scanning calorimetry (DSC). Consistent data is obtained when all parameters of the membrane preparation are carefully reproduced. Calorimetric parameters for opsin and rhodopsin vary as a function of scan rate, membrane concentration and pH. Binding of retinal imparts a stabilization to the protein structure indicated by a temperature of thermal denaturation (Tm) of 71.4oC for rhodopsin as compared to 55.3°C for opsin. Similarly, the ΔHcal for rhodopsin (167 kcal/mole) is 43 kcal/mole higher than that for opsin (124 kcal/mole). By contrast, a much smaller effect is obtained in samples subjected to limited proteolysis indicating that the membrane-embedded portion of the protein lends quantitatively more stability to the protein than does its cytoplasmic surface. DSC confirms that the pH of maximal stability of both opsin and rhodopsin is 6.1. DSC can be used successfully to examine stability of rhodopsin in detergent solution. In membrane suspensions, membrane-membrane interactions are indicated by a dependence of ΔHcal on the membrane concentration. Intermolecular interactions within the disk membrane are indicated by a dependence of opsin’s ΔHcal on the extent of bleaching of the membrane suspension.