Retinal Isomerization Research Articles

Solvent isotope effects on retinal cis-trans isomerization in the dark adaptation of bacteriorhodopsin

The solvent isotope effect on the first-order rate constant for dark adaptation of bacteriorhodopsin, near neutral pH (pD), is inverse; k{sub D}/k{sub H} = 1.24. The fit of the variation of isotope effect with the atom fraction of deuterium in the solvent to the Gross-Butler equation leads to the conclusion that the proton(s) in motion is (are) less tightly bound in the reactant than in a reactive intermediate, formed prior to the rate-controlling step of cis-trans isomerization. The near-unity isotope effect on the equilibrium between the bound 13-cis- and all-trans-retinals in the dark-adapted state mixture indicates that the isotope effects on the forward and reverse rate constants for isomerization are approximately equal to each other and equal to the isotope effect on the observed rate constant for dark adaptation. The results support a previously proposed mechanism of rate-controlling Asp-212 nucleophilic catalysis of retinal cis-trans isomerication in the dark adaptation process. 27 refs., 2 figs., 1 tab.

Sensory rhodopsin I: receptor activation and signal relay.

Recent progress is summarized on the mechanism of phototransduction by sensory rhodopsin I (SR-I), a phototaxis receptor in Halobacterium halobium. Two aspects are emphasized: (i) The coupling of retinal isomerization to protein conformational changes. Retinal analogs have been used to probe chromophore-apoprotein interactions during the receptor activation process. One of the most important results is the finding of a steric trigger deriving from the interaction of residues on the protein with a methyl group near the isomerizing bond of the retinal (at carbon 13). Recent work on molecular genetic methods to further probe structure/function includes the synthesis and expression of an SR-I apoprotein gene designed for residue replacements by cassette mutagenesis, and transformation of an H. halobium mutant lacking all retinylidene proteins known in this species to SR-I+ and bacteriorhodopsin (BR)+. (ii) The relay of the SR-I signal to a post-receptor component. A carboxylmethylated protein ("MPP-I") associated with SR-I and found in the H. halobium membrane exhibits homology with the signaling domain of eubacterial chemotaxis transducers (e.g., Escherichia coli Tar, Tsr, and Trg proteins), suggesting a model based on SR-I----MPP-I signal relay.

Photoisomerization of polyenes. 30. Quantum chain processes in photoisomerization of the all-trans, 7-cis, and 11-cis isomers of retinal.

Quantum chain processes in the photoisomerization of retinal isomers (all-trans, 7-cis, and 11-cis) are reflected in the dependence of quantum yield of isomerization on concentration of retinal (to values exceeding unity), the presence of one-photon multiple-bond isomerization, and changes of product distribution on retinal concentration. The processes are believed to take place exclusively from the triplet states. Reaction schemes involving participation of 12-(S)-cis conformers in the quantum chain processes have been advanced to account for all of the results

Consequences of amino acid insertions and/or deletions in transmembrane helix C of bacteriorhodopsin.

Six bacterioopsin mutants containing either single amino acid deletions (delta A84, delta L87), insertions (delta 85A, delta 88A), or both deletions and insertions (delta A84/delta 88A, delta 85A/delta L87) within the first two turns of transmembrane helix C, starting from the extracellular side, have been prepared. The mutant apoproteins refold in phospholipid/detergent micelles and display secondary structures similar to that of the wild type. However, the mutants delta 88A and delta A84/delta 88A do not form a chromophore with retinal. The regenerated chromophore of delta 85A displays absorption maxima and retinal isomer compositions in the dark- and light-adapted states similar to those of the wild type. In delta A84, delta L87, and delta 85A/delta L87 these chromophore properties are altered, and the structures are less stable than that of the wild type, as shown by an enhanced rate of reaction with hydroxylamine in the dark, an increased pKa of the denaturation at acidic pH, and a decreased pKa of Schiff base deprotonation. Proton translocation is abolished in the delta A84 and delta 85A/delta L87 mutants, whereas in delta 85A and delta L87 the activity is reduced to about 25% of the wild-type value at pH 6. The overall properties of the delta 85A, delta 85A/delta L87, and delta L87 mutants indicate that the deletions and/or insertions result in displacement of residues Arg-82, Asp-85, or Asp-96, respectively, which participate in proton translocation. The results are compatible with a helical structure for transmembrane segment C and emphasize the flexibility of intramolecular contacts in bacteriorhodopsin.

All- trans retinal constitutes the functional chromophore in Chlamydomonas rhodopsin

Orientation of the green alga Chlamydomonas in light (phototaxis and stop responses) is controlled by a visual system with a rhodopsin as the functional photoreceptor. Here, we present evidence that in Chlamydomonas wild-type cells all-trans retinal is the predominant isomer and that it is present in amounts similar to that of the rhodopsin itself.The ability of different retinal isomers and analog compounds to restore photosensitivity in blind Chlamydomonas cells (strain CC2359) was tested by means of flash-induced light scattering transients or by measuring phototaxis in a taxigraph. All-trans retinal reconstitutes behavioral light responses within one minute, whereas cis-isomers require at least 50 x longer incubation times, suggesting that the retinal binding site is specific for all-trans retinal. Experiments with 13-demethyl(dm)-retinal and short-chained analogs reveal that only chromophores with a beta-methyl group and at least three double bonds in conjugation with the aldehyde mediate function. Because neither 13-dm-retinal, nor 9,12-phenylretinal restores a functional rhodopsin, a trans/13-cis isomerisation seems to take place in the course of the activation mechanism. We conclude that with respect to its chromophore, Chlamydomonas rhodopsin bears a closer resemblence to bacterial rhodopsins than to visual rhodopsins of higher animals.

Retinal analog restoration of photophobic responses in a blind Chlamydomonas reinhardtii mutant. Evidence for an archaebacterial like chromophore in a eukaryotic rhodopsin

The strain CC-2359 of the unicellular eukaryotic alga Chlamydomonas reinhardtii originally described as a low pigmentation mutant is found to be devoid of photophobic stop responses to photostimuli over a wide range of light intensities. Photophobic responses of the mutant are restored by exogenous addition of all-trans retinal. We have combined computer-based cell-tracking and motion analysis with retinal isomer and retinal analog reconstitution of CC-2359 to investigate properties of the photophobic response receptor. Most rapid and most complete reconstitution is obtained with all-trans retinal compared to 13-cis, 11-cis, and 9-cis retinal. An analog locked by a carbon bridge in a 6-s-trans conformation reconstitutes whereas the corresponding 6-s-cis locked analog does not. Retinal analogs prevented from isomerization around the 13-14 double bond by a five-membered ring in the polyene chain (locked in either the 13-trans or 13-cis configuration) do not restore the response, but enter the chromophore binding pocket as evidenced by their inhibition of all-trans retinal regeneration of the response. Results of competition experiments between all-trans and each of the 13-locked analogs fit a model in which each chromophore exhibits reversible binding to the photoreceptor apoprotein. A competitive inhibition scheme closely fits the data and permits calculation of apparent dissociation constants for the in vivo reconstitution process of 2.5 x 10(-11) M, 5.2 x 10(-10) M, and 5.4 x 10(-9) M, for all-trans, 13-trans-locked and 13-cis-locked analogs, respectively. The chromophore requirement for the trans configuration and 6-s-trans conformation, and the lack of signaling function from analogs locked at the 13 position, are characteristic of archaebacterial rhodopsins, rather than the previously studied eukaryotic rhodopsins (i.e., visual pigments).

CD spectrum of bacteriorhodopsin: Best evidence against exciton model

We summarize the predictions of the exciton model that was originally proposed to explain the observed biphasic band shape of its CD spectrum in the visible region of bacteriorhodopsin (bR). It is shown that to reconcile these predictions with the observed results on the linear dichroism, the retinal isomerization time and, the retinal-retinal distance, the biphasic nature of the observed CD spectrum of bR becomes itself an evidence against the exciton model because of the uncertainty principle.Reduced bR (RbR), which retains its hexagonal structure, shows a monophasic CD spectrum with relatively small rotational strength as compared to bR. This is shown to disagree with predictions made by the exciton model. The results could best be explained in terms of retinal-protein heterogeneity leading to two or more types of bR in which their retinals suffer opposite sense of intramolecular rotational distortion along their retinal long axis. Such a retinal-protein heterogeneity disappears in reduced bR which is known to have a planar (nondistorted) retinal conjugated system, resulting in a monophasic CD with reduced rotational strength, as observed.

8] Clefts and binding sites in protein receptors

Publisher Summary This chapter discusses the methods for determining clefts and binding sites in protein receptors. Direct methods of cleft determination are aimed at the analysis of a receptor whose three-dimensional structure is known. The meaning of the term “protein surface” is discussed in the chapter so that all the methods can be related to a common set of definitions. The different classes of algorithms that have been used to define and display the surface, to look at mappings of topographic features, to quantify surface roughness and curvature, and to search for packing defects and sites are reviewed in the chapter. All these methods require a set of Cartesian coordinates and produce an essentially static picture of the receptor. Biomolecules are dynamic objects and undergo substantial conformational changes; each static picture should, therefore, be regarded as a snapshot. These stills should be combined into a moving trajectory or a partition function; this is implied whenever a new static method is introduced. Indirect methods of receptor mapping are applicable when the receptor structure is unknown, but the structures of a series of ligands that bind to the receptor at a common site are available. The binding site is deduced from the apparent common properties of the ligands—that is, the pharmacophore. The assumptions behind pharmacophore deduction are discussed in the chapter. The algorithms are illustrated in the chapter by case studies: the method of shape mapping is exemplified by a family of retinal isomers.

Electrophysiologic characteristics of human and rat retinas in vitro

To promote studies on the human retina, we investigated the survival of function in postmortem specimens. Visual pigment has been regenerated in normal human retinas, 5 to 58 hours postmortem, by exposure to retinal isomers in the dark. Levels from 0.1 to 0.41 nmol/mg protein were reached. Photoresponses were obtained in 9 of 13 retinas: P III maximum amplitudes ranged from 20-398 microV and thresholds, taking the criterion amplitude as 3 microV, ranged from 8.8-1340 quanta/micros2. In three cases, the b-wave was also seen. The P III amplitude vs. log intensity curves gave values of n between 0.6 and 1.0, and sigma (the stimulus intensity for a half maximal response) between 132-3700 quanta/microns2. Recovery of sensitivity did not always correspond to that of maximum response.

The interaction of Triton X-100 with purple membrane: Effect of light-dark adaptation

The effects of Triton X-100 on purple membrane have been examined for dark- and light-adapted membrane suspensions. Bacteriorhodopsin is more readily solubilized from dark-adapted than from light-adapted preparations, while light-dark adaptation does not influence phospholipid solubilization. Surfactant-induced changes in the absorption spectrum and retinal isomer distribution of bacteriorhodopsin are also illumination-dependent. These results are discussed in the light of structural data.

The interaction of Triton X‐100 with purple membranes

The interaction of the non-ionic surfactant Triton X-100 with Halobacterium purple membranes has been examined at sublytic and lytic surfactant concentrations. These membranes present a number of important peculiarities in their behaviour towards the surfactant. Although solubilization is a very slow process, with a half-time of the order of hours, detergent binding appears to occur at the same fast rate as that found in other membranes. Lipids are solubilized more easily than proteins, so that hardly any protein is solubilized at surfactant concentrations at which about 75% of the lipid is in the form of detergent-mixed micelles; once started, protein solubilization takes place within a narrow range of surfactant concentrations. Retinal provides a built-in probe to monitor detergent-induced conformational changes by spectroscopy in the visible range. No spectral variation is detected at the prelytic stage, i.e. when detergent is incorporated into the membrane in monomeric form. Membrane disruption is accompanied by a blue shift in the absorption maximum, retinal isomerization (from all-trans to 13-cis), and a decrease in specific absorbance (bleaching). Increasing detergent concentrations after solubilization is completed do not produce further shifts in the spectral maximum, but the specific absorbance is progressively decreased. It is shown that Triton X-100 has a complex effect on the retinal chromophore, modifying its configuration and microenvironment (changes in maximum wavelength) and promoting hydrolysis of the retinal-bacteriorhopsin Schiff's base (bleaching).

On retention of chromophore configuration of rhodopsin isomers derived from three dicis retinal isomers

By the chromophore extraction-HPLC method, it has been shown that an isomeric mixture of pigments (9,11- dicis, 9- cis, and 9,13- dicis) was formed from interaction of 9- cis, 11- cis-retinal with bovine opsin. That from 7- cis,9- cis-retinal retained the original geometry and that from 7,13- dicis contained almost completely the isomerized 7- cis pigment.

Comparative study on the chromophore binding sites of rod and red-sensitive cone visual pigments by use of synthetic retinal isomers and analogs

A comparative study on the chromophore (retinal) binding sites of the opsin (R-photopsin) from chicken red-sensitive cone visual pigment (iodopsin) and that scotopsin) from bovine rod pigment (rhodopsin) was made by the aid of geometric isomers of retinal (all-trans, 13-cis, 11-cis, 9-cis, and 7-cis) and retinal analogues including fluorinated (14-F, 12-F, 10-F, and 8-F) and methylated (12-methyl) 11-cis-retinals. The stereoselectivity of R-photopsin for the retinal isomers and analogues was almost identical with that of scotopsin, indicating that the shapes of the chromophore binding sites of both opsins are similar, although the former appears to be somewhat more restricted than the latter. The rates of pigment formation from R-photopsin were considerably greater than those from scotopsin. In addition, all the iodopsin isomers and analogues were more susceptible to hydroxylamine than were the rhodopsin ones. These observations suggest that the retinal binding site of iodopsin is located near the protein surface. On the basis of the spectral properties of fluorinated analogues, a polar group in the chromophore binding site of iodopsin as well as rhodopsin was estimated to be located near the hydrogen atom at the C10 position of the retinylidene chromophore. A large difference in wavelength between the absorption maxima of iodopsin and rhodopsin was significantly reduced in the 9-cis and 7-cis pigments. On the assumption that the retinylidene chromophore is anchored rigidly at the alpha-carbon of the lysine residue and loosely at the cyclohexenyl ring, each of the two isomers would have the Schiff-base nitrogen at a position altered from that of the 11-cis pigments.(ABSTRACT TRUNCATED AT 250 WORDS)

Survival of structure and function in postmortem rat and human retinas: rhodopsin regeneration, cGMP and the ERG

Procedures for regenerating visual pigment and restoring phototransduction have been established with freshly isolated, bleached rat retinas. Phosphatidyl choline liposomes containing a 500 microM mixture of retinal isomers, including the 9-cis and 11-cis forms, were employed and the results compared with dark-adapted retinas, incubated similarly but without retinal. The following were recovered in a 60 min incubation, rhodopsin (plus isorhodopsin) to 91% of the original rhodopsin concentration, 87% of cGMP and 89% of PIII amplitude at saturation. PIII amplitude vs. log intensity curves gave values of n between 0.6 and 1/2.0 and sigma between 85 and 439 quanta/micron 2. Human retinas, ranging from 18 to 58 hours postmortem and treated as above, also produced photoresponses. Of the 7 retinas studied so far, rhodopsin has been regenerated to 0.1-0.35 nmol/mg protein, cGMP to 23.5-49.2 pmol/mg protein, and PIII to 20-50 microV: in some cases a b-wave was also seen. Values of n varied between 0.6 and 1.0, and sigma between 132 and 3700 quanta/micron 2. PIII responses were also seen after retinas, approximately 30 hours postmortem, were incubated for a further 24 hours in fortified medium. After incubation, retinal vacuolation was reduced.

Photocycles of bacteriorhodopsin in light- and dark-adapted purple membrane studied by time-resolved absorption spectroscopy

Nanosecond time-resolved absorption spectra have been measured throughout the photocycle of bacteriorhodopsin in both light-adapted and dark-adapted purple membrane (PM). The data from dark-adapted samples are interpretable as the superposition of two photocycles arising independently from the all-trans and 13-cis retinal isomers that coexist in the dark-adapted state. The presence of a photocycle in dark-adapted PM which is indistinguishable from that observed for light-adapted PM under the same experimental conditions is demonstrated by the observation of the same five relaxation rates associated with essentially identical changes in the photoproduct spectra. This cycle is attributed to the all-trans component. The cycle of the 13-cis component is revealed by scaling the data measured for the light-adapted sample and subtracting it from the data on the dark-adapted mixture. At times less than 1 ms, the resulting difference spectra are nearly time-independent. The peak of the difference spectrum is near 600 nm, although there appears to be a slight (approximately 2 nm) blue-shift in the first few microseconds. Subsequently the amplitude of this spectrum decays and the peak of the difference spectrum shifts in two relaxations. Most of the amplitude of the photoproduct difference spectrum (approximately 80%) decays in a single relaxation having a time constant of approximately 35 ms. The difference spectrum remaining after this relaxation peaks at approximately 590 nm and is indistinguishable from the classical light-dark difference spectrum, which we find, in experiments performed on a much longer time scale, to peak at 588 nm. The decay of this remaining photo-product is not resolvable in the nanosecond kinetic experiments, but dark adaptation of a completely light-adapted sample is found to occur exponentially with a relaxation time of approximately 2,000 s under the conditions of our experiments.

A Model Study of Rhodopsin Regeneration in Visual Cell. Photoisomerization of all-trans Retinal in Phosphatidylcholine Vesicles

Abstract The photochemical behavior has been investigated for all-trans retinal in egg york phosphatidylcholine(EPC) vesicles and dipalmitoylphosphatidylcholine (DPPC) vesicles. In EPC vesicles, 10% of all-trans isomer is isomerized to 11-cis one upon direct irradiation of 15 W-fluorescence light for 180 min. In organic solvents the photoisomerization of all-trans isomer is induced depending on the dielectric constant of solvent. The distributions of retinal isomers in EPC vesicles is similar to those obtained in less polar solvents, such as diethyl ether. Significant difference is not recognized between the distributions of retinal isomers in DPPC vesicles at the above and below Tc. These results indicate that photoisomerization of retinal in PC vesicles is dominated by the electric properties of enviroment of retinal rather than the packing effect of PC membrane.

Activation of Chlamydomonas rhodopsin in vivo does not require isomerization of retinal.

The unicellular eukaryote Chlamydomonas reinhardtii is a phototactic alga that swims toward or away from light, using rhodopsin as the photopigment. The activity of retinal analogues was tested in the mutant FN68, which has high phototactic sensitivity only after incubation with retinal or analogues of retinal. Analogues prevented from isomerizing about the 7-ene, 9-ene, 11-ene, 13-ene, or 15-ene (C = N+H) bonds retained full activity. Also, bleaching, protonation of the N, and a stable geometrically altered chromophore are not required for full activity. An attractive hypothesis is that charge redistribution in the excited state of retinal directly triggers the activity of rhodopsin.

Retinal isomer ratio in dark-adapted purple membrane and bacteriorhodopsin monomers.

On the basis of data obtained by spectroscopic analysis and chromatography of retinal extracts, a consensus has been adopted that dark-adapted purple membrane (pm) contains 13-cis- and all-trans-retinal in equal amounts, whereas the light-adapted membrane contains all-trans-retinal only. We have developed an improved extraction technique which extracts up to 70% of the retinal in pm within 4 min. In the extracts from dark-adapted pm at room temperature, we consistently find 66-67% 13-cis-retinal and 33-34% all-trans-retinal, and more than 98.5% all-trans isomer in light-adapted samples. The spectrum obtained by reconstitution of bacterioopsin with 13-cis-retinal at 2 degrees C (to minimize isomerization) shows an absorbance maximum at 554 nm and agrees well with the spectrum for the 13-cis component calculated from the dark-adapted and light-adapted bR spectra with our extraction data. The ratio of 13-cis:all-trans isomer in dark-adapted pm is 2:1 and nearly constant between 0 and 38 degrees C but begins to decrease distinctly above 40 degrees C, and more rapidly near 70 degrees C, reaching 0.75 at 90 degrees C. The van't Hoff plot of the isomer ratio shows a nonlinear temperature dependence above 40 degrees C, indicating a more complex system than a simple thermal 13-cis/all-trans isomer equilibrium. We attribute the broadening, absorbance decrease, and blut shift of the visible absorption band with increasing temperature to the appearance of at least one and possibly two or three new chromophores which contain, mainly or exclusively, the all-trans isomer.(ABSTRACT TRUNCATED AT 250 WORDS)

Photoisomerization of all-trans-retinal in organic solvents and organized media

Abstract The photochemistry of all-trans -retinal (ATR) in n -hexane, ethanol, aqueous sodium dodecylsulphate, n -butyl stearate and phosphatidylcholine—H 2 O media under direct irradiation (λ ⩾ 380 nm) has been investigated. Photostationary state compositions of retinal isomers have been determined. Results are discussed in terms of the effects of solvent polarity on the relative position of the n,π* and π,π* states of the excited polyene, the π electron density and the spatial requirements vis-a-vis the solvent-induced barrier for isomerization of different double bonds. Characteristics of UV absorption bands of ATR in different media are also discussed.

All- cis-retinal and 7- cis,9- cis,11- cis-retinal 1

A modified 15 + 5 route led to 7- cis,9- cis,11- cis-retinonitrile in high selectivity and also a lesser amount of all- cis-retinonitrile. DIBAL reduction gave the corresponding retinal isomers. The absorption maximum 287nm of all- cis-retinal reveals the distorted nature of the chromophore.