ROTATIONAL DIFFUSION OF RHODOPSIN IN THE VISUAL RECEPTOR MEMBRANE - EFFECTS OF TEMPERATURE AND BLEACHING

Rhodopsin present in bovine rod outer segments was covalently labeled with the triplet probe erythrosinyliodoacetamide. The rotational diffusion of the protein in the membrane was studied by measuring the time dependence of the phosphorescence emission anisotropy. In this way it was possible to monotor directly the rotational diffusion of both bleached and unbleached rhodopsin. Nonlinear least-squares analysis of the resulting anisotropy decay curves revealed changes in the rotational dynamics of the protein as a function of temperature and photobleaching. By assuming the coexistence of mobile and immobile protein fractions, it was shown that a decrease in temperature is capable of inducing aggregation of both bleached and unbleached rhodopsin. Differences in the time dependence of the emission anisotropy between bleached and unbleached rhodopsin are interpreted as providing evidence that rhodopsin undergoes a conformational change upon bleaching. Information on the specific binding site of the erythrosin probe indicates that this change is localized in the C-terminal region of the protein. The steady-state fluoresence polarization of 1,6-diphenyl-1,3,5-hexatriene was measured as a function of temperature. A close agreement was found between the activation energy of the fluorescence depolarization and the activation energy of the relaxation of bleached rhodopsin.