Valeria Balogh-Nair

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.

Flash photolysis and low temperature photochemistry of bovine rhodopsin with a fixed 11-ene.

Nonbleachable rhodopsins containing retinal moieties with fixed 11-ene structures have been prepared. When the nonbleachable rhodopsin analogue corresponding to the natural pigment was flash-photolysed at 20.8 degrees C, no absorption changes occurred at the monitoring wavelengths of 380, 480, and 580 nm for the time range of 2 microseconds--10 s. This observation is in contrast to that of natural rhodopsin which showed the formation of metarhodopsin I and its decay to meta II. Irradiation of the artificial rhodopsin, 77 K, with light of 460 and 540 nm, also gave no spectral changes; in the case of natural rhodopsin, however, the irradiation leads to formation of the red-shifted intermediate bathorhodopsin. The absence of photochemistry in the artificial pigment shows that an 11-cis to trans photoisomerization of the retinal moiety is a crucial step in inducing the chain of events in te photolysis of rhodopsin.

Structure of ajugarins

The structure of three new ent-clerodanes, which are insect antifeedants, isolated from Ajugaremota, have been determined.

Picosecond kinetic absorption and fluorescence studies of bovine rhodopsin with a fixed 11-ene

A synthetic retinal having a fixed 11-cis geometry has been used to prepare a nonbleachable analogue of bovine rhodopsin. Marked differences in the picosecond absorption and fluorescence behavior of this analogue at room temperature, compared with that of natural rhodopsin, were observed. This not only indicates that the 11-cis to trans isomerization of the retinal moiety is the crucial primary event in the photolysis of rhodopsin, but also it establishes that this isomerization must occur on the picosecond time scale or faster.

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).

[64] Synthetic analogs of retinal, bacteriorhodopsin, and bovine rhodopsin

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

Photoaffinity labeling of bovine rhodopsin

Publisher Summary This chapter discusses the synthetic analogs of retinal, bacteriorhodopsin, and bovine rhodopsin. The synthetic routes leading to analogs of retinal in most cases follow methodology developed for the synthesis of vitamin A and carotenoids. A stereospecific synthesis of vitamin A from 2,2,6-trimethylcyclohexanone used as a key reaction a novel vanadium(V)-catalyzed rearrangement of an ethynyl-substituted 2,2,6-trimethylcyclohexenyl derivative to obtain an 8-oxo compound, which then could be transformed into a 7,8 double bond. A versatile synthesis of retinal and its analogs containing modified rings is accomplished by condensation of key intermediates 3,7-dimethyl-2,4, 6-nonatrien-8-ynoates, i.e., the entire side chain of retinal, to cyclic ketones. Directed aldol condensation coupled with separation of isomers by preparative liquid chromatography is found to be a rapid and efficient procedure for the preparation of pure cis and trans isomers of the methylated analogs of retina.

Chapter 7 The stereochemistry of vision

Abstract Photoaffinity labeled (3-diazoacetoxy)-9- cis -retinal ( 1 ) and (9-methylenediazoacetoxy)-9- cis -retinal ( 20 ) were synthesized and bound to absorption maxima at 465 and 460 nm respectively. Binding studies established that synthetic retinals 1 and 2 bind to the natural binding site and that the integrity of the diazoacetoxy photoaffinity label is preserved in the process. Incorporation of 3-(O 14 COCHN 2 )-labeled 9- cis retinal could be conveniently carried out in high yield using apomembrane solubilized in CHAPS as detergent to afford the pigment analog in a pure form. Photolysis of the diazoacetoxy group within the binding site led to 15–20%, crosslinking of rhodopsin as estimated by using radiocarbon containing labeled retinal 1 thus showing that this synthetic retinal is suitable for photoaffinity labeling of the active site in rhodopsin. Subsequent experiments to establish the site(s) of crosslinking by sequencing studies will then contribute to our knowledge of the structure of rhodopsin.

The primary event in vision investigated by time-resolved fluorescence spectroscopy

Publisher Summary Visual transduction is the process by which rods and cones convert light to a neural signal, which is transmitted to the brain via the optic nerve. The process is initiated by the absorption of light by the visual pigments, and causes a change in the electrical properties of the photoreceptor cell membranes by changing their permeability. The impinging of light on a visual pigment initiates the process of vision. This initial absorption of light is followed by a train of events culminating in the integration, by the brain, of the pattern of perception. The visual pigments are located in the light-sensitive cells, the photoreceptors. There are two basic types, the ciliary cell of the vertebrates and the rhabdomeric cell of the invertebrates, both possessing an inner and an outer segment. It is the outer segment membranes which contain the photoreceptors. In the case of the vertebrate eye, the light entering the eye must travel through the cornea, iris, lens, vitreous humor and various layers of nerve cells in the retina before it reaches the photoreceptors. These receptor cells are of two types, rods which are responsible for scotopic vision (in dim light) and cones which work under bright illumination and are concentrated in the region of the fovea. The human retina has three types of cone cells, with differing absorption maxima at 450, 535, and 560 nm, which provide the basis for color discrimination.

INCORPORATION OF 11,12-DIHYDRORETINAL INTO THE RETINAE OF VITAMIN A DEPRIVED RATS

The picosecond fluorescence kinetics and quantum yield from bovine rhodopsin were measured in the 5-40 degrees K range. The fluorescence rise and decay times are faster than our resolution of 15 ps (full width at half maximum) over this entire temperature range. The size of the observed emission was also temperature independent, and we find that the upper limit of rhodopsins fluorescence quantum yield to be phi f approximately equal to 10(-5). Replacing all of rhodopsins exchangeable protons with deuterons by suspending rhodopsin in D2O had no effect on either the kinetics of the emission or the value of the quantum yield. Our data provide strong confirmation of the idea that the first step in the visual process is an excited-state cis-to-trans isomerization about the C11-C12 double bond of retinal.