Resonance raman spectroscopy of rhodopsin in retinal disk membranes

Low-temperature resonance Raman spectroscopy has been used to study the conformation and interactions of retinal within its opsin binding site in disk membrane vesicles formed from bovine retinal rod outer segments. At 80° K, laser irradiation within the visible absorption band produces well-defined photostationary states containing only rhodopsin, isorhodopsin, and bathorhodopsin. A double wavelength, pump-probe technique has been devised to distinguish scattering from these three components. In addition to the conventional Raman “probe” laser beam, a “pump” beam at a different wavelength is used to modify the composition of the photostationary state. The fixed wavelength probe holds the resonant enhancement factors constant so that changes in the spectra that are induced by the pump beam are directly related to the composition of the photostationary state. The Raman data demonstrate that, in situ, retinal and opsin are joined by a protonated Schiff base. Deuteration of the Schiff base causes a predictable reduction in the stretching frequency from 1655 to 1630 cm^-1. The C=C stretching frequency of the retinal chain decreases from about 1563 in irans-retinylideneethanolamine to 1551 in isorhodopsin to 1539 cm^-1 in bathorhodopsin, in direct proportion to the magnitude of the bathochromic shift. The spectrum of bathorhodopsin, which contains relatively intense bands at 856, 877, and 920 cm^-1, and weak scattering near 1160 cm^-1, is markedly different from that of all-trans retinylidene solutions The data suggest that the formation of bathorhodopsin from rhodopsin or isorhodopsin may not involve a simple cis-trans isomerization about the 11-cis or 9-cis double bond.