Fadila Derguini

Photoisomerization of retinal at 13-ene is important for phototaxis of Chlamydomonas reinhardtii: Simultaneous measurements of phototactic and photophobic responses

A real-time automated method was developed for simultaneous measurements of phototactic orientation (phototaxis) and step-up photophobic response of flagellated microorganisms. Addition of all-trans retinal restored both photoresponses in a carotenoid-deficient mutant strain of Chlamydomonas reinhardtii in a dose-dependent manner. The phototactic orientation was biphasic with respect to both the light intensity and the concentration of retinal. All-trans retinal was more effective than 11-cis retinal to regenerate both photobehavioral responses. Analogs having locked 11-cis configurations and a phenyl ring in the side chain also induced photoresponses, although at concentrations more than two orders of magnitude higher than all-trans retinal. According to the present assay method, the responses were hardly detectable in cells incubated with retinal analogs in which the 13-ene was locked in either its trans or cis configuration. The results strongly suggest that the isomerization of the 13-14 double bond is important for photobehavioral signal transduction and that a single retinal-dependent photoreceptor controls both phototactic and photophobic responses.

Diversion of the sign of phototaxis in a Chlamydomonas reinhardtii mutant incorporated with retinal and its analogs

The blind mutant FN68 of the unicellular flagellate green alga Chlamydomonas reinhardtil is negatively phototactic in the presence of the native chromophore all‐trans retinal. In contrast, analog chromophores such as a ring‐acyclic retinal and those in which trans/cis isomerization about the C11–C12 double bond was blocked induced predominantly positive pho totaxis in the same strain under the same experimental conditions. These observations can be interpreted by assuming that the negative and the positive phototaxis is mediated distinctively by two rhodopsin species which differ in their affinities with the exogenous chromophores. However, a more reasonable explanation, which requires fewer assumptions, is that the sign of phototaxis depends on a delay in intracellular photosignal transduction. This novel view was deduced directly from the widely accepted Hypothesis [1980, Microbiol. Rev. 44, 572–630] on phototaxis mechanisms.

Photoaffinity labeling of rhodopsin and bacteriorhodopsin.

Photoaffinity labeling with bovine rhodopsin using a retinal with a fixed 11-cis-ene cross-linked exclusively to Trp-265/Leu-266 in helix F, showing that the beta-ionone C-3 is close to helix F. Moreover, since these labeled amino acids are in the middle of helix F, while the Schiff-base linkage to Lys-296 at the other terminus of the chromophore is also in the middle of helix G, the chromophore lies horizontally near the center of the lipid bilayer. In bacteriorhodopsin, photoaffinity studies using a retinal with a C-10 tritiated phenylazide appended through a 13 A spacer cross-linked to Arg-175/Asn-176 on the cytoplasmic side of helix F; this indicates that 9-Me points toward the extracellular space. This result agrees with our earlier studies with 9-sulfate analogs but is opposite to that deduced by biophysical measurements.

Chromophore/protein and chromophore/anion interactions in halorhodopsin

Halorhodopsin (HR), the light-driven chloride transport pigment of Halobacterium halobium, was bleached and reconstituted with retinal analogues with the pi electron system interrupted at different locations (dihydroretinals). The absorption maxima of the artificial pigments formed with the dihydroretinals are found to be very similar to those of the corresponding pigments formed by reconstitution of bacteriorhodopsin (BR) and sensory rhodopsin (SR). This strongly suggests that the distribution of charges around the retinal is similar in all three bacterial rhodopsins. Comparison of the primary, and proposed secondary, structures for HR and BR reveal conserved asparagine (asp) and arginine (arg) residues, which are likely candidates for the ionizable amino acids that interact with the retinal. In a second set of experiments absorption shifts due to the binding of anions to Sites I and II in HR, reconstituted with different retinal analogues, were used to estimate the locations of these binding sites relative to the retinal. Site I is localized near the Schiff base, and Site II near the ionone ring. On the basis of these results a structural model for HR is proposed, which accounts for the spectroscopic properties of HR in terms of the three buried arg residues and two of the buried asp residues in the protein.

Cellular electrophysiologic effects of vertebrate digitalis-like substances.

Substances structurally and functionally similar to digitalis glycosides are produced by several vertebrate species. There also is evidence for a digitalis-like substance of human origin. Standard microelectrode techniques were used to study the direct effects on the cellular electrophysiology of canine Purkinje fibers of 1) bufalin, an unconjugated cardiotonic steroid molecule that is produced by the toad Bufo marinus, and 2) an extract of human bile that showed digitalis-like immunoreactivity on radioimmunoassay. The goal of this study was to determine whether these substances have arrhythmogenic effects comparable with those seen with toxic doses of digitalis glycosides. Bufalin, 2 x 10(-8) M, significantly (p less than 0.05) reduced maximal diastolic potential, action potential amplitude and duration and maximal rate of rise of phase 0 (Vmax) within 40 min of onset of exposure. All six fibers developed delayed afterdepolarizations and two developed triggered rhythms. Ouabain was less potent, in that a 2 x 10(-7) M concentration was required to comparably reduce maximal diastolic potential, action potential amplitude and duration and Vmax within 30 min. These Purkinje fibers also developed delayed afterdepolarizations and triggered rhythms. A sample of an extract of human bile that showed digitalis-like immunoreactivity with an antibufalin serum also reduced maximal diastolic potential, action potential amplitude and duration and Vmax, and produced delayed afterdepolarizations and triggered activity. In contrast, immunologically unreactive bile extracts had no appreciable effect on the action potential. In summary, the cardiac toxicity of digitalis substances produced by lower vertebrates is comparable with that induced by the glycosides. Moreover, it appears that humans may produce digitalis-like substances that may be cardiotoxic.

A versatile synthesis of retinoids via condensation of the side-chain to cyclic ketones

Abstract A simple synthesis of ethyl 3,7-dimethyl-2,4,6-nonatriene-8-ynoates and their corresponding alcohols is presented; codensation with various cyclic ketones can lead to retinoids having modified ring structures.

Rhodopsins containing 6- to 9-membered rings. The triggering process of visual transduction

A major unsettled problem in the chemistry of vision is clarification of the triggering mechanism that initiates conformational changes in the rhodopsin structure leading to visual transduction. Although it is established that a & to uans photoisomerization of its chromophore, the protonated Schiff base of 1 1 - &-retinal, occurs, the more subtle aspects of the mechanism in which photon absorption leads to conformational changes are unclear. Theoretical models propose that charge redistribution or bond polarization in the excited state triggers the conformational changes in rhodopsin leading to enzymatic activation. However, experimentally it remained unknown whether or not excited state polarization is involved at all in the visual transduction process and if so to what extent. In order to clarify this crucial but elusive problem, retinal analogs with 8- and 9- membered rings in the side-chain have been synthesized and incorporated into rhodopsin. Flash photolysis, binding, enzymatic assays and other studies of several such rhodopsin analogs and dihydrorhodopsins have shown that excited state polarization is involved but a complete &-- isomerization of the full rigid polyene system is required.

VISUAL PIGMENTS AND BACTERIORHODOPSINS FORMED FROM AROMATIC RETINAL ANALOGS

Abstract— Rhodopsin (Rh) and bacteriorhodopsin (bR) analogs have been prepared from retinals containing various aromatic and heterocyclic nuclei. In the case of Rh, aromatic methyl substituents facilitate the regeneration and stabilize the pigments formed; in bR, however, methyl substituents seem to have little influence. Rhodopsins derived from unsubstituted aromatic retinals show fine structure in the relatively stable “pre‐pigment” intermediate and their maxima are red‐shifted compared to pigments derived from methylated aromatic retinals. This implies that in these aromatic rhodopsins the ring moiety adopts a more planar conformation when unsubstituted. In bR derivatives also the aromatic ring adopts a close‐to‐planar conformation when unsubstituted, but comparison with indene‐derived bR suggests that even the unsubstituted phenyl ring may not be coplanar with the side‐chain.

Fractionation and Proof of Structure of Natural Products

The past few years have witnessed a veritable explosion in the development of new methods for the isolation and structure determination of natural products. This is true in particular for the applications of novel separation technologies and modern spectroscopic techniques for structural analysis. In many instances, the discovery of these new methods was spawned by persistent, formidable obstacles in the preparation of pure substances from complex mixtures or in the microscale determination of complex structures. This is exemplified by the application of the benzoate chirality method for determining linkage sites of sugars in branched glycosides (236, 237) or the development of new countercurrent chromatography techniques for the separation of labile compounds like the tunichromes (53, 54). In other situations, an existing technique of theoretical interest was tailored to solve structural problems, as was the case with the application of two dimensional nuclear magnetic resonance in determining chemical structure.