Two processes lead to a stable all-trans and 13-cis isomer equilibrium in dark-adapted bacteriorhodopsin; effect of high pressure on bacteriorhodopsin,bacteriorhodopsin mutant D96N and fluoro-bacteriorhodopsin analogues

The combination of absorption spectroscopy and extraction techniques was applied to study the effect of high pressure on the dark-adapted state of bacteriorhodopsin, 14-(12-,10-)fluoro-bacteriorhodopsin, a D96N bacteriorhodopsin mutant, and 14-(12-,10-)fluoro-D96N. Evidence is presented that, at high pressure, the isomers' equilibrium is shifted from all- trans isomers towards the 13-cis isomers. Two groups of values for calculated molar volume changes indicate that there are at least two different processes leading to a stable all-trans and 13-cis isomers' equilibrium called the dark-adapted bacteriorhodopsin. The first process may be attributed to changes in the distances and rearrangement of functionally important residues and a retinal Schiff base. It is suggested that the moved residues (probably Asp-212 with the contribution of Tyr-185 and/or Asp-85) closer to the chromophore could catalyse its trans-cis isomerization. These changes require smaller pressure changes and induce larger volume changes (large-volume-change process). The second process may be attributed to the formation of the three hydrogen bonds that additionally decrease the volume and strengthen further stabilization of the 13-cis isomer. To induce these changes, larger changes of pressure are required and the final molar volume changes are smaller (small-volume-change process). The total molar volume change between all-trans bacteriorhodopsin and 13-cis bacteriorhodopsin in the dark-adapted state of native bacteriorhodopsin was found to be about -28 mL/mol, which is much higher than the value of about -7 mL/mol obtained previously (Tsuda and Ebrey 1980, Schulte and Bradley 1995). The data provide a novel insight into factors leading to stable isomer equilibrium in dark-adapted bacteriorhodopsin.     

Met-145 is a key residue in the dark adaptation of bacteriorhodopsin homologs.

Composition of retinal isomers in three proton pumps (bacteriorhodopsin, archaerhodopsin-1, and archaerhodopsin-2) was determined by high performance liquid chromatography in their light-adapted and dark-adapted states. In the light-adapted state, more than 95% of the retinal in all three proton pumps were in the all-trans configuration. In the dark-adapted state, there were only two retinal isomers, all-trans and 13-cis, in the ratio of all-trans: 13-cis = 1:2 for bacteriorhodopsin, 1:1 for archaerhodopsin-1, and 3:1 for archaerhodopsin-2. The difference in the final isomer ratios in the dark-adapted bacteriorhodopsin and archaerhodopsin-2 was ascribed to the methionine-145 in bacteriorhodopsin. This is the only amino acid in the retinal pocket that is substituted by phenylalanine in archaerhodopsin-2. The bacteriorhodopsin point-mutated at this position to phenylalanine dramatically altered the final isomer ratio from 1:2 to 3:1 in the dark-adapted state. This point mutation also caused a 10 nm blue-shift of the adsorption spectrum, which is similar to the shift of archaerhodopsin-2 relative to the spectra of bacteriorhodopsin and archaerhodopsin-1.     

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)     

Characterization of the chromophore of the third rhodopsin-like pigment of Halobacterium halobium and its photoproduct.

Halobacterium halobium contains at least three retinal-containing pigments: bacteriorhodopsin, halorhodopsin, and a third rhodopsin-like pigment (tR) absorbing at approximately 590 nm, tR590. Illumination of tR590 gives rise to a very long-lived blue absorbing photoproduct, tR370. Using high-performance liquid chromatography we show that the chromophore of tR590 is primarily all-trans retinal and its conversion by light to tR370 causes the chromophore to isomerize primarily to the 13-cis conformation. Irradiation of the tR370 gives rise to a transient photoproduct absorbing at approximately 520 nm that decays back to the initial pigment tR590. In addition to all-trans retinal, the apomembrane of tR can also combine with 13-cis retinal but not with the 9- or 11-cis isomers.     

Effect of high pressure on the absorption spectrum and isomeric composition of bacteriorhodopsin.

The effects of high pressure upon the absorption spectra and isomeric composition of the dark (bRD) and light adapted (bRL) forms of bacteriorhodopsin were examined. Pressure favors the 13-cis form of bacteriorhodopsin (bR13-cis). The equilibrium isomeric composition and absorption spectra of bacteriorhodopsin samples at a given pressure were the same starting from either light or dark adapted bacteriorhodopsin. From the effect of pressure on the equilibrium constant between bRall-trans in equilibrium bR13-cis in the dark, the molar volume change between bRall-trans and bR13-cis was found to be -7.8 +/- 3.2 ml/mol. This volume change suggests a difference in conformation between dark- and light-adapted bacteriorhodopsin, but the magnitude of the change is small, involving only a small number of the protein residues.     

High-pressure near-infrared Raman spectroscopy of bacteriorhodopsin light to dark adaptation.

Near-infrared (NIR) Raman spectroscopy is employed as an in situ probe of the chromophore conformation to study the light to dark-adaptation process in bacteriorhodopsin (bR) at variable pressure and temperature in the absence of undesired photoreactions. In dark-adapted bR deconvolution of the ethylenic mode into bands assigned to the all-trans (1526 cm-1) and 13-cis (1534 cm-1) isomers yields a 13-cis to all-trans ratio equal to 1 at ambient pressure (Schulte et al., 1995, Appl. Spectrosc. 49:80-83). Detailed spectroscopic evidence is presented that at high pressure the equilibrium is shifted toward the 13-cis isomers and that the light to dark adaptation kinetics is accelerated. The change in isomeric composition with temperature and pressure as well as the kinetics support a two-state model activation volumes of -16 ml/mol for the transition of 13-cis to all-trans and -22 ml/mol for the reverse process. These compare with a conformational volume difference of 6.6 ml/mol, which may be attributed to the ionization of one or two residues or the formation of three hydrogen bonds.     

An energy-based approach to packing the 7-helix bundle of bacteriorhodopsin.

Based on the heavy-atom coordinates determined by the electron microscopy for the seven main helical regions of bacteriorhodopsin with the all-trans retinal isomer, energy optimizations were carried out for helix bundles containing the all-trans retinal and 13-cis retinal chromophores, respectively. A combination of simulated annealing and energy minimization was utilized during the process of energy optimization. It was found that the 7-helix bundle containing the all-trans isomer is about 10 kcal/mol lower in conformational energy than that containing the 13-cis isomer. An energetic analysis indicates that such a difference in energy is consistent with the observation that absorption of a 570-nm proton is required for the conversion of a bacteriorhodopsin from its all-trans to 13-cis form. It was also found that the above conversion process is accompanied by a significant conformational perturbation around the chromophore, as reflected by the fact that the beta-ionone ring of retinal moves about 5.6 A along the direction perpendicular to the membrane plane. This is consistent with the observation by Fodor et al. (Fodor, S.P.A., Ames, J.B., Gebhard, R., van der Berg, E.M.M., Stoeckenius, W., Lugtenburg, J., & Mathies, R.A., 1988, Biochemistry 27, 7097-7101). Furthermore, it is interesting to observe that although the retinal chromophore undergoes a significant change in its spatial position, the orientation of its transition dipole changes only slightly, in accord with experimental observations. In other words, even though orientation of the retinal transition dipole is very restricted, there is sufficient room, and degrees of freedom, for the retinal chromophore to readjust its position considerably. This finding provides new insight into the subtle change of the retinal microenvironment, which may be important for revealing the proton-pumping mechanism of bacteriorhodopsin. The importance of electrostatic and nonbonded interactions in stabilizing the 7-helix bundle structure has also been analyzed. Electrostatic interactions favor an antiparallel arrangement among adjacent helices. Nonbonded interactions, however, drive most of the closely packed helices into an arrangement in which the packing angles lie around -160 degrees, a value very near the -154 degrees value computed earlier as the most favorable packing arrangement of two poly(Ala) alpha-helices (Chou, K.-C., Némethy, G., & Scheraga, H.A., 1983, J. Phys. Chem. 87, 2869-2881). The structural features of the 7-helix bundle and their relationship to those found in typical 4-helix bundle proteins are also discussed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cis astaxanthin and especially 9-cis astaxanthin exhibits a higher antioxidant activity in vitro compared to the all-trans isomer

In recent years, a number of studies have implicated the potent antioxidant property of astaxanthin in various experimental systems; however, these studies employed only the all-trans isomer. On the other hand, it has been reported that all-trans natural astaxanthin is readily isomerized to cis-trans, especially 9-cis and 13-cis isomers, under certain conditions by chemical analysis; however, the biological activities of the cis isomers of astaxanthin are little known. In the present study, we investigated the antioxidant activity of 9-cis and 13-cis astaxanthin compared to the all-trans isomer in vitro. In a stable radical DPPH scavenging activity test and in rat microsome and rabbit erythrocyte ghost membrane lipid peroxidation systems induced by AAPH and t-BuOOH, respectively, the results apparently showed that cis-astaxanthin, especially 9-cis astaxanthin, exhibited a higher antioxidant effect than the all-trans isomer. In addition, during polyunsaturated fatty acid (PUFA) oxidation, both DHA and linoleic acid hydroperoxides formation were markedly inhibited by astaxanthin isomers addition in the order 9-cis >13-cis >all-trans. Furthermore, 9-cis also exhibited the most effective inhibition of the generation of ROS induced by 6-hydroxydopamine (6-OHDA) in human neuroblastoma SH-SY5Y cells among the astaxanthin isomers, as well as on the degradation of collagen type II induced by DHA and linoleic acid hydroperoxides. The above-mentioned results suggest, for the first time, that cis isomer astaxanthin, especially 9-cis astaxanthin, has a much higher antioxidant potency than that of the all-trans isomer.     

The effect of protein conformation change from alpha(II) to alpha(I) on the bacteriorhodopsin photocycle

The bacteriorhodopsin (bR) photocycle was followed by use of time-resolved Fourier-transform infrared (FTIR) spectroscopy as a function of temperature (15-85 degrees C) as the alpha(II) --> alpha(I) conformational transition occurs. The photocycle rate increases with increasing temperature, but its efficiency is found to be drastically reduced as the transition takes place. A large shift is observed in the all-trans left arrow over right arrow 13-cis equilibrium due to the increased stability of the 13-cis isomer in alpha(I) form. This, together with the increase in the rate of dark adaptation as the temperature increases, leads to a large increase in the 13-cis isomer concentration in bR in the alpha(I) form. The fact that 13-cis retinal has a much-reduced absorption cross-section and its inability to pump protons leads to an observed large reduction in the concentration of the observed photocycle intermediates, as well as the proton gradient at a given light intensity. These results suggest that nature might have selected the alpha(II) rather than the alpha(I) form as the helical conformation in bR to stabilize the all-trans retinal isomer that is a better light absorber and is capable of pumping protons.     

On the photocycle and light adaptation of dark-adapted bacteriorhodopsin.

Pulsed Nd laser (25 ns, 530 nm) photolysis experiments were carried out at room temperature in aqueous suspensions of dark- and light-adapted fragments of the purple membrane of Halobacterium halobium. It is shown that the (50%) 13-cis isomeric component (BR13-cis) of dark-adapted bacteriorhodopsin (BRDA) undergoes a photocycle involving a characteristic transient absorbing in the neighborhood of 610 nm. At relatively high excitation intensities BR13-cis is converted to the same 410 nm (M) transient that characterized the photocycle of the all-trans isomer (BRtrans) of light-adapted bacteriorhodopsin (BRLA). This process, which competes with the generation of the "610" species, is attributed to the photo-induced conversion, during the pulse, of BR13-cis (or of its primary photoproduct "X") to a species in the BRtrans photocyte. The relationship between these observations and the mechanism of BRDA hv leads to BRLA adaptation at low excitation intensities (for which a quantum yield limit, 0 less than or equal to (3.5 +/- 0.7) X 10(-2) , is established) is discussed.     

Molecular dynamics study of the 13-cis form (bR548) of bacteriorhodopsin and its photocycle.

The structure and the photocycle of bacteriorhodopsin (bR) containing 13-cis,15-syn retinal, so-called bR548, has been studied by means of molecular dynamics simulations performed on the complete protein. The simulated structure of bR548 was obtained through isomerization of in situ retinal around both its C13-C14 and its C15-N bond starting from the simulated structure of bR568 described previously, containing all-trans,15-anti retinal. After a 50-ps equilibration, the resulting structure of bR548 was examined by replacing retinal by analogues with modified beta-ionone rings and comparing with respective observations. The photocycle of bR548 was simulated by inducing a rapid 13-cis,15-anti-->all-trans,15-syn isomerization through a 1-ps application of a potential that destabilizes the 13-cis isomer. The simulation resulted in structures consistent with the J, K, and L intermediates observed in the photocycle of bR548. The results offer an explanation of why an unprotonated retinal Schiff base intermediate, i.e., an M state, is not formed in the bR548 photocycle. The Schiff base nitrogen after photoisomerization of bR548 points to the intracellular rather than to the extracellular site. The simulations suggest also that leakage from the bR548 to the bR568 cycle arises due to an initial 13-cis,15-anti-->all-trans,15-anti photoisomerization.     

Regeneration of rhodopsin and bacteriorhodopsin. The role of retinal analogues as inhibitors

The rate of regeneration of rhodopsin, from 11-cis-retinal and opsin, and bacteriorhodopsin from all-trans-retinal and bacterio-opsin, in the presence or absence of compounds whose structures partially resemble retinal were measured. Some of these compounds severely slowed down the regeneration process, but did not influence the extent of regeneration. In the case of compounds with a carbonyl functional group they were not joined to the active site of the apo-protein via a Schiff's base linkage since after treatment with NaBH4 an active apo-protein remained. The most effective inhibitors of rhodopsin regeneration were molecules whose structure could be superimposed on 9-cis or 11-cis retinal up to carbon atom 11. These C13 and C15 molecules were not distinguished between aldehyde, ketone or alcohol functional groups. The regeneration of bacteriorhodopsin was not inhibited by retinal analogues with short side chains. The most effective inhibitors were the all-trans C17-aldehyde (beta-ionylideneacetaldehyde) or C18-ketone (beta-ionylidenepent-3-ene-2-one) which, compared to retinal, lack two or three carbon atoms from the end of the poylene chain. The inhibition was very dependent upon the presence of the all-trans isomer and required aldehyde or ketone as functional group nitriles and alcohols were less effective. However, similarly to retinol, the all-trans C17 and C18 alcohols underwent a bathochromic shift and showed fine-structured spectra when mixed with bacterio-opsin.     

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.     

Effects of light adaptation on the purple membrane structure of Halobacterium halobium.

Absorption, circular dichroism and optical rotatory dispersion of the bacteriorhodopsin containing purple membrane form Halobacterium halobium were studied in regard to the structural stability of this membrane during the photoisomerization of the retinal of the bacteriorhodopsin from the 13-cis to the all-trans configuration. The following conclusions were reached: (a) the macromolecular structure (protein-protein interaction which may result in the possible exciton interaction of the retinal pi-pi* (NV1) transition moments and protein-lipid interaction) are not significantly altered, (b) possibilities of delocalized conformation changes of the apoprotein involving secondary and/or tertiary structure can be ruled out, (c) localized secondary structure conformation changes of the apoprotein must be limited to the involvement of no more than one or two amino acid residues and localized tertiary structure conformation changes of the apoprotein must be limited to a very short segment of the protein chain containing only a few aromatic amino acid residues, and (d) the interaction between the apoprotein and retinal seems to be relatively more pronounced when the retinal is in the all-trans form than the 13-cis from and also the apoprotein seems to impose a more pronounced dissymmetric constraint on the retinal in the all-trans form than in the 13-cis form.     

A nonbleachable rhodopsin analogue with a slow photocycle

Archaeal rhodopsins, e.g. bacteriorhodopsin, all have cyclic photoreactions. Such cycles are achieved by a light-induced isomerization step of their retinal chromophores, which thermally re-isomerize in the dark. Visual pigment rhodopsins, which contain in the dark state an 11-cis retinal Schiff base, do not share such a mechanism, and following light absorption, they experience a bleaching process and a subsequent release of the photo-isomerized all-trans chromophore from the binding pocket. The pigment is eventually regenerated by the rebinding of a new 11-cis retinal. In the artificial visual pigment, Rh(6.10), in which the retinal chromophore is locked in an 11-cis geometry by the introduction of a six-member ring structure, an activated receptor may be formed by light-induced isomerization around other double bonds. We have examined this activation of Rh(6.10) by UV-visible and FTIR spectroscopy and have revealed that Rh(6.10) is a nonbleachable pigment. We could further show that the activated receptor consists of two different subspecies corresponding to 9-trans and 9-cis isomers of the chromophore. Both subspecies relax in the dark via separate pathways back to their respective inactive states by thermal isomerization presumably around the C(13)=C(14) double bond. This nonbleachable pigment can be repeatedly photolyzed to undergo identical activation-relaxation cycles. The rate constants of these photocycles are pH-dependent, and the half-times vary between several hours at acidic pH and about 1.5 min at neutral to alkaline pH, which is several orders of magnitude longer than for bacteriorhodopsin.     

Selectivity of retinal photoisomerization in proteorhodopsin is controlled by aspartic acid 227

Similarly to bacteriorhodopsin, proteorhodopsin that normally contains all-trans and 13-cis retinal is transformed at low pH to a species containing 9-cis retinal under continuous illumination at lambda > 530 nm. This species, absorbing around 430 nm, returns thermally in tens of minutes to initial pigment and can be reconverted also with blue-light illumination. The yield of the 9-cis species is negligibly small at neutral pH but increases manyfold (>100) at acid pH with a pK(a) of 2.6. This indicates that protonation of acidic group(s) alters the photoreaction pathway that leads normally to all-trans --> 13-cis isomerization. In the D97N mutant, in which one of the two acidic groups in the vicinity of the retinal Schiff base is not ionizable, the yield of 9-cis species at low pH shows a pH dependence similar to that in the wild-type but with a somewhat increased pK(a) of 3.3. In contrast to this relatively minor effect, replacement of the other acidic group, Asp227, with Asn results in a remarkable, more than 50-fold, increase in the yield of the light-induced formation of 9-cis species in the pH range 4-6. It appears that protonation of Asp227 at low pH is what causes the dramatic increase in the yield of the 9-cis species in wild-type proteorhodopsin. We conclude that the photoisomerization pathways in proteorhodopsin to 13-cis or 9-cis photoproducts are controlled by the charge state of Asp227.     

Storage of retinal in the eggs of the ascidian,Halocynthia roretzi

Retinoids in the eggs of the solitary ascidian, Halocynthia roretzi, were analyzed by high performance liquid chromatography. Retinal was the almost exclusive retinoid (>99%), and the concentration of retinal was 25.9-40.1 (30.6 on average) ng/mg of protein. The egg retinal consisted of four isomers: all-trans (50.9%), 9-cis (6.8%), 11-cis (20.4%) and 13-cis (21.9%). The presence of retinal in the eggs of this ascidian is a characteristic shared with the wide range of oviparous vertebrates, although the isomer composition differs between ascidian eggs and vertebrate eggs; in vertebrate eggs, almost all the retinal is in the all-trans form. The egg retinal was bound to a protein complex via a Schiff base linkage. The electrophoretic characteristics of the protein complex were similar to that of egg yolk proteins of oviparous vertebrates. The results presented in this study strongly suggest that, as is found with oviparous vertebrates, retinal in the ascidian eggs is the essential mode of retinoid storage, and is the precursor of photoreceptive pigment chromophores and retinoic acid during development.     

Light- and dark-adapted bacteriorhodopsin, a time-resolved neutron diffraction study

Recently, neutron diffraction experiments have revealed well-resolved and reversible changes in the protein conformation of bacteriorhodopsin (BR) between the light-adapted ground state and the M-intermediate of the proton pumping photocycle (Dencher, Dresselhaus, Zaccai and Büldt (1989) Proc. Natl. Acad. Sci. USA 86, 7876-7879). These changes are triggered by the light-induced isomerization of the chromophore retinal from the all-trans to the 13-cis configuration. Dark-adapted purple membranes contain a mixture of two pigment species with either the all-trans- or 13-cis-retinal isomer as chromophore. Employing a time-resolved neutron diffraction technique, no changes in protein conformation in the resolution regime of up to 7 A are observed during the transition between the two ground-state species 13-cis-BR and all-trans-BR. This is in line with the fact that the conversion of all-trans BR to 13-cis-BR involves an additional isomerization about the C15 = N Schiff's base bond, which in contrast to M formation minimizes retinal displacement and keeps the Schiff's base in the original protein environment. Furthermore, there is no indication for large-scale redistribution of water molecules in the purple membrane during light-dark adaptation.     

Crystal structure of the 13-cis isomer of bacteriorhodopsin in the dark-adapted state

The atomic structure of the trans isomer of bacteriorhodopsin was determined previously by using a 3D crystal belonging to the space group P622. Here, a structure is reported for another isomer with the 13-cis, 15-syn retinal in a dark-adapted crystal. Structural comparison of the two isomers indicates that retinal isomerization around the C13[double bond]C14 and the C15[double bond]N bonds is accompanied by noticeable displacements of a few residues in the vicinity of the retinal Schiff base and small re-arrangement of the hydrogen-bonding network in the proton release channel. On the other hand, aromatic residues surrounding the retinal polyene chain were found to scarcely move during the dark/light adaptation. This result suggests that variation in the structural rigidity within the retinal-binding pocket is one of the important factors ensuring the stereospecific isomerization of retinal.