Energy Transfer in Rhodopsin, N -Retinyl-Opsin, and Rod Outer Segments

Abstract

N-retinyl, the chromophore of bleached and reduced rhodopsin, N-retinyl-opsin, was used as a covalently attached fluorescence probe to examine the structure of N-retinyl-opsin and the rod outer segment. The efficiency of energy transfer from the protein part of N-retinyl-opsin to the chromophore is 12 4 5%. It is argued that this implies that the N-retinyl-opsin molecule is asymmetrical. Kropf has estimated the efficiency of energy transfer from the protein to the chromophore in native rhodopsin to be about 50%. This difference of efficiencies seems to imply a large movement of the chromophore away from the tryptophans of the opsin after rhodopsin is bleached.From excitation spectrum measurements, it has been found that light absorbed by the protein of the rod outer segments has more action in sensitizing the fluorescence of the chromophore than does light absorbed by the protein part of pure N-retinyl-opsin. Thus, some other tryptophans or tyrosines in either another N-retinylopsin molecule or another protein must be close enough (about 28 A) to the chromophore to transfer energy to it. Measurements of the polarization of the fluorescence of the chromophore suggest, however, that the chromophores of neighboring N-retinyl-opsin molecules are more than 20 A apart. Moreover, these neighboring chromophores do not transfer energy to each other, tending to rule out any clustering of chromophores of different Nretinyl-opsin molecules and suggesting that rhodopsin chromophores do not transfer energy to each other.The mechanism of visual excitation can not be understood without a fuller understanding of the structure and function of the proteins in the membranes of the photoreceptors, particularly rhodopsin. The significance of rhodopsin goes beyond the immediate problems of visual excitation. Of the best-known membrane proteins and membrane structures, rhodopsin and the rod outer segments (r.o.s.) are among the most suitable for extensive study. Structurally, rhodopsin is quite important since it constitutes about 50% of the protein of the bovine r.o.s. (unpublished data). Functionally, it is important since it is the pigment that absorbs the light used in vision. Rhodopsin can be isolated in fairly large quantities in a pure form, and the purity of rhodopsin and r.o.s. samples can be assayed (1). Much is known already about the photochemistry of rhodopsin; it is almost certain that the first step in visual excitation is the isomerization of the chromophore of rhodopsin, retinal, from the li-cis to the all-trans form (2).Because rhodopsin is arranged in a systematic array in the r.o.s., much work utilizing the techniques of both electron microscopy and x-ray diffraction has been possible (3, 4). Because the rod outer segment is an electrically active membrane (5), a number of electrophysiological findings may Abbreviation: r.o.s., rod outer segments. I would like to present evidence here on the structure of the native rhodopsin molecule, the bleached rhodopsin molecule, and the r.o.s. This evidence is based on...

Light-induced protein conformational changes in the photolysis of octopus rhodopsin

“…Alternatively, the UV difference spectrum is interpreted as being due to alterations of the environment of several aromatic amino acid residues such as tryptophan and tyrosine. As shown in previous studies Pool, 1968, 1969;Ebrey, 1971), the quantum yield of fluorescence of the chromophore in opsin is very low. However, fluorescence emission from the aromatic acids of opsin is rather strong (Ebrey, 1972;Farrens and Khorana, 1995).…”

Fluorescence of crayfish metarhodopsin studied in single rhabdoms

“…Other Fluorescence Sources Located in Photoreceptors Although this paper is the first description of emission from the chromophore site of any rhodopsin-metarhodopsin system (see Busch et al, 1972;Lewis et al, 1976), Stavenga and Franceschini (1981) report that the metarhodopsin of flies also fluoresces in the red. Fluorescence has been observed from aromatic amino acids of vertebrate rhodopsins (e.g., Ebrey, 1972), and from retinol in photoreceptors (Hagins and Jennings, 1959;Eakin and Brandenburger, 1978), as well as from N-retinyl-opsin (Ebrey, 1971).…”

“…The fluorescence properties of retinol are well known (Kahan, 1971), and some stereoisomers of retinal and its Schiff bases are also known to fluoresce (Balke and Becker, 1967;Waddell et al, 1973;Das et al, 1979). Although N-retinyl-opsin formed by reduction of the retinal-opsin Schiff base linkage of rhodopsin (Hall and Bok, 1976) has fluorescence properties similar to retinol itself (Ebrey, 1971), vertebrate rhodopsins emit only weakly if at all from their chromophore sites. Guzzo and Pool (1968) reported a 500-nm stimulated emission from cattle rhodopsin, peaking at 600 nm, but attempts to repeat this work have been unsuccessful (Busch et al, 1972).…”

Fluorescence spectroscopy of rhodopsins: Insights and approaches

“…This approach yields a fluorescent retinal, N-retinyl-opsin, both for bacteriorhodopsin [43] and visual rhodopsin [44, 45]. Reduction of the retinal SB linkage may also be achieved by two-photon excitation [46], where the two-photon photoreduction results in similar photoproducts as for chemical reduction with peaks at 340 nm, 360 nm, and 380 nm.…”

“…This phenomenon was subsequently investigated and established to be due to release of retinal following SB hydrolysis, and an assay for retinal release was established that allows to follow MII decay [77]. Note that the phenomenon of increasing tryptophan fluorescence of rhodopsin after bleaching appears to have been observed in rhodopsin by investigators in the late 1960s and early 1970s [45, 78], although the underlying mechanism was not completely clear at that time. A number of investigations have exploited this phenomenon to monitor the kinetics of retinal release, both in rhodopsin [79–81], and more recently in cone opsins [82, 83].…”