Michael G. Motto

THE ‘OPSIN SHIFT’ IN BACTERIORHODOPSIN: STUDIES WITH ARTIFICIAL BACTERIORHODOPSINS

Abstract— The difference (in cm−1) in absorption maxima between the protonated Schiff base of retinals and the pigment derived therefrom has been defined as the opsin shift. It represents the influence of the opsin binding site on the chromophore. The analysis of the opsin shifts of a series of dihydrobacteriorhodopsins has led to the external point‐charge model, which in addition to a counter anion near the Schiff base ammonium, carries another negative charge in the vicinity of the β‐ionone ring. This is in striking contrast to the external point‐charge model proposed earlier for the bovine visual pigment. The absorption maxima of rhodopsins formed from bromo‐ and phenyl retinals support the two models. A retinal carrying a photoaffinity label has yielded a nonbleachable bacteriorhodopsin.

DOUBLE POINT CHARGE MODEL FOR VISUAL PIGMENTS; EVIDENCE FROM DIHYDRORHODOPSINS*

The visual pigment rhodopsin is known to consist of a chromophore, 1 1 4 s retinal 1 bound to the terminal amino group of a lysine moiety of the apoprotein opsin. It is also generally accepted that the linkage to the protein is through a protonated Schiff base (Shriver et al., 1977).

[21] Heavy atom labeling of retinal in bacteriorhodopsin

The action of light on the pigment causes the 1 1 4 s chromophore to isomerize and become detached from the protein, releasing free allfruns retinal and opsin at the end of the bleaching sequence. A fundamental problem arises when the absorption maximum of the protonated Schiff base formed from simple amines such as butylamine (2) absorb at 440nm in the leveling solvent methanol (Blatz, 1968),‘, whereas when R is protein (3), the maxima range from 430 to 580nm depending on the source of opsin. Typically for bovine rhodopsin the

An external point-charge model for wavelength regulation in visual pigments

Publisher Summary Bacteriorhodopsin—because of its two-dimensional crystal lattice in the membrane—lends itself to study by various diffraction techniques. By the use of diffraction techniques with retinals containing heavy atoms, it might be possible to determine the location of the retinal moiety within the protein. Two compounds are synthesized for this purpose, the 9-demethyl-9- bromoretinal 1 and the 13-demethyl-13-bromoretinal 2, in which either the 9-or the 13-methyl is replaced with a bromine atom. It is thought that a bromine in these positions would minimize any extra steric interactions that might impede the binding of the chromophore to the protein, and also provide a heavy label that could be used in the diffraction studies. For synthesis of 9-demethyl-9-bromoretinal, Ethyl 4- oxocrotonate is treated with bromine in CCl 4 and the resulting aldehyde is condensed—dibal reduction and MnO 2 oxidation give the aldehyde in quantitative yield. For synthesis of 13-demethyl-13-bromoretinal, 2-butyne- l,4-diol is reacted with tert-butyldimethyl silyl chloride to yield monosilyated product; subsequent oxidation with MnO 2 give the aldehyde. Condensation of this aldehyde with Wittig salt give the protected tetraenynol, which was then deprotected and oxidized with MnO 2 to give the tetraenynal, 44% yield, which reacted with aqueous HBr in benzene to give all-trans-13-de methyl-13-bromoretinal, 40% yield.