Ultraviolet Chromophore Transitions in the Rhodopsin Spectrum

Abstract

Difference spectra measured at -105°s how two decreases in the ultraviolet absorption spectrum of rhodopsin upon bleaching that cannot be attributed to changes in protein conformation. These absorbancy decreases in rhodopsin are consistent with a cis-trans isomerization of the chromophore.Rhodopsin, the visual pigment, consists of the chromophore 11-ci retinal covalently bound to the protein opsin (1). As seen in Fig. 1, the two principal peaks in the absorption spectrum of rhodopsin are located at 280 and 500 nm. The short-wavelength absorption is due primarily to the aromatic amino acids of opsin; the band at 500 nm corresponds to the lowest r-xr* transition of the chromophore shifted by the protein from 380 nm to the visible region of the spectrum. In addition to the two major bands, there is a weaker peak at 340 nm that is most easily seen in pure preparations of rhodopsin. This band has often been termed the rhodopsin "cis" peak, since it is believed to correspond to the cis peak of the isomers of retinal (2).Retinal isomers have two subsidiary peaks in the ultraviolet, one at 250 and the other at 280 nm (3). There has been a question as to which of these absorption bands is the ci8 peak (4-8). We discuss the assignments of these transitions in a subsequent publication (Honig, B., Kliger, D., Ebrey, T. & Karplus, M., in preparation). In this work, we are concerned only with the identification of two corresponding transitions in the spectrum of rhodopsin. Our approach is to measure changes in the ultraviolet spectrum of rhodopsin that result entirely from the isomerization of the chromophore.Our spectroscopic results enable us to identify early stages in the bleaching sequence of rhodopsin, in which the protein has not undergone measurable conformational changes. Furthermore, they provide another test for Hubbard and Wald's (9) finding that the chromophore of rhodopsin is directly isomerized by light, and that changes in the protein take place at a later time. Finally, they provide a more complete description of the spectroscopic changes that result from the formation of rhodopsin from 11-ci.s retinal and opsin. METHODS AND MATERIALSAll low-temperature spectra presented here were measured with rhodopsin from outer segments of bovine rods, which were prepared by the methods of Ebrey (10), hardened in 4% aluminum potassium sulfate, and solubilized in 2% digitonin.Rhodopsin prepared with digitonin as a detergent is not as free from protein contaminants as rhodopsin that has been solubilized with other detergents, such as Ammonyx LO, and then has been purified by calcium phosphate chro-1897 matography (10). However, since we are only interested in difference spectra, absolute shapes of absorption spectra are not necessary.All of the difference spectra reported here were measured at -105°; other measurements at -80°gave essentially identical results. It is necessary to determine difference spectra at low temperatures for two reasons, (a) at room temperature (240), two of the bleaching products of the rhod...

Effects of Bleaching and Regeneration on the Purple Membrane Structure of Halobacterium halobium

“…The large changes in the near-ultraviolet absorption and CD spectra on bleaching purple membrane could result from (a) a change in the secondary and/or tertiary structure of the bacteriorhodopsin, resulting in changes in the environment of the 7r -w* transitions of the aromatic amino acid side chains (30-32); (b) a loss of possible dipole coupling between the xr -7r* transitions of the retinal and the aromatic amino acids (23,26,28); or (c) a change in contributions from minor irlr* transitions of the chromophore in the near-ultraviolet wavelength region (33).…”

Molecular aspects of photoreceptor function

“…There is considerable evidence indicating that opsin does not undergo significant conformational changes until after the lumirhodopsin stage (see discussion in Ebrey & Honig, 1972). Thus, it appears that the first two spectral shifts observed in the bleaching sequence involve changes in the chromophore rather than changes in the opsin.…”

Multistate Multiconfiguration Quantum Chemical Computation of the Two-Photon Absorption Spectra of Bovine Rhodopsin

“…OPA Properties of the XMCQDPT2/cc-pVTZ//CASSCF/6-31G­(d)/AMBER QM/MM Model of Rh . The absorption spectrum of Rh shows three peaks in the UV–vis region . The two principal peaks are located at 498 and 280 , nm (57.4 and 102.1 kcal/mol, respectively).…”

“…The absorption spectrum of Rh shows three peaks in the UV–vis region . The two principal peaks are located at 498 and 280 , nm (57.4 and 102.1 kcal/mol, respectively). The long-wavelength peak is attributed to the S 0 → S 1 transition and the short-wavelength intense peak is attributed to aromatic amino acids .…”

“…The two principal peaks are located at 498 and 280 , nm (57.4 and 102.1 kcal/mol, respectively). The long-wavelength peak is attributed to the S 0 → S 1 transition and the short-wavelength intense peak is attributed to aromatic amino acids . However, the latter peak is also present in the spectra of rPSB11 in 1,2-dichloroethane solvent with lower intensity.…”