Protein-phospholipid-cholesterol interaction in the photolysis of invertebrate rhodopsin.

Three aspects of protein-phospholipid-cholesterol interaction in microvillar membranes of octopus photoreceptor cells were studied: (1) the effect of hydrophobic environment on the kinetics of thermal transformation in the photolysis of rhodopsin; (2) effects of cholesterol in microvillar membranes on protein-lipid interaction; (3) the effect of membrane proteins on the dynamic properties of lipids in membranes. Dynamic properties of hydrophobic environments were observed with electron spin resonance spectroscopy with various lipid spin-labels. The kinetics of thermal transformation of mesorhodopsin to metarhodopsin was followed by flash photolysis experiments. The thermal stability of mesorhodopsin was followed by low-temperature spectrophotometry. The rate of transformation is influenced by the properties of both hydrophobic environments and temperature. The effect of the former is prominent at physiological temperatures, while the A number of functions of proteins are greatly facilitated by the internal fluctuation of protein conformation [for a recent review, see Careri et al. (1979) and Karplus & McCammon (1981)l. Oosawa (1973, 1975) emphasized the importance of the environmental fluctuation of electric potential around proteins with long correlation times and long correlation lengths' in determining the reaction rate of enzymes. This type of spontaneous environmental fluctuation occurs relatively rarely but supplies sufficient free energy, coupled with internal protein fluctuation, to enhance enzymatic activity. The hydrophobic portion of biological membranes is of particular interest in this respect because the spectrum of spontaneous fluctuations is expected to shift to longer correlation times and lengths due to the higher viscosity and ordered structure of hydrophobic loci in membranes. One of the aims of this work is to study the influence of the environment on the function of the protein. The thermal conversion of the photochemical intermediates of rhodopsin (a membrane protein) after photolysis gives an ideal model for the study of the relationship between the function of the protein and the environment, since the transformation of the intermediates (hypso-batho-lumimeso-meta) is strictly thermally driven and the photochemical process is well established (Yoshizawa, 1972; Tsuda et al., 1980; Tsuda, 1982a). The study of the influence of hydrophobic environment on rhodopsin after photolysis is also significant in clarifying how absorption of a photon by rhodopsin leads to visual excitation. From the National Biomedical ESR Center, Department of Radiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 (A.K.), the Department of Physics (M.T. and Y.T.) and the Department of Biochemistry (T.A.), Sapporo Medical College, Sapporo 060, Japan, and the Department of Biophysics, Faculty of Science, Kyoto University, Kyoto 606, Japan (SO.). Received August 17, 1982. This work was supported in part by Grants RR-01008 and GM-22923 from the National Institutes of Health and by grants from the Ministry of Education, Science and Culture, Japan. *Address correspondence to this author at the Department of Biology, Princeton University, Princeton, NJ 08544. 0006-2960/83/0422-1165$01.50/0 latter becomes a key factor at lower temperatures. The effect of cholesterol, which accounts for 19 mol 9% of total lipid in microvillar membranes, on the properties of the membranes was observed by comparing the temperature profiles of the order parameter of spin-labeled stearic acid in liposomes formed of either the total lipid or the polar lipid extracted from microvillar membranes. These experiments demonstrate that cholesterol has a large effect on the dynamic properties of membranes, lowering and broadening the temperature region of the gross phase change in the total lipid liposomes. In contrast, the temperature profile of the order parameter of the fatty acid spin-label in microvillar membranes shows only small differences from that in total-lipid liposomes. The minor effects of proteins on the dynamic properties of the membrane lipid are ascribed to the presence of cholesterol in the microvillar membranes. Intense effort is in progress in many laboratories to gain an understanding of the linkage between the photochemical event in rhodopsin and excitation of plasma membrane of photoreceptor cells. Light-activated enzymes in photoreceptor cells such as GTPase and phosphodiesterase have been proposed to be involved in this process. Apparently, one of the photochemical intermediates is able to activate these enzymes since neither rhodopsin nor its final photoproduct, opsin plus retinal (vertebrate) or metarhodopsin (invertebrate), can (Calhoon et al., 1981). Since rhodopsin is a membrane protein and the light activation of the enzyme cascade seems to be initiated at membranous loci, it is important to understand rhodopsin-lipid interaction during the photolysis of rhodopsin. The influence of lipid environment on rhodopsin has been studied mostly with vertebrate rhodopsin (Applebury et al., 1974; O'Brien et al., 1977; Davoust et al., 1979; Kusumi et al., 1980b, 1982). However, invertebrate photoreceptors have a number of advantages such as the following in studying the mechanism of visual transduction: (1) both rhodopsin and the light-dependent ion channel(s) are located in the same membrane (microvilli); (2) the thermal transformation of invertebrate rhodopsin after photolysis is simpler than that of vertebrate rhodopsin (no complicated transformation after metarhodopsin); (3) metarhodopsin, which is the final photoproduct of rhodopsin in the invertebrate case, is stable at physiological temperatures; (4) invertebrate rhodopsin can be photoregenerated from metarhodopsin. In particular, octopus rhodopsin can be almost completely photoregenerated from metarhodopsin upon irradiation of orange light (Tsuda, 1979a). This offers a good test of the reversibility of photoinduced phenomena and facilitates signal averaging in photolysis experiments. Consequently, invertebrate photoreceptor membranes have become an active subject of investigation in recent ' Correlation time refers to the time correlation of random phenomena while correlation length is a concept that comments on the spatial correlation of random phenomena.