Isomeric composition of retinal chromophore in dark-adapted bacteriorhodopsin

1. Retinal isomers extracted from the acid-hydrolysate of cetyltrimethylammonium bromide-treated dark-adapted bacteriorhodopsin (bRD) were analyzed in a high performance liquid chromatograph (HPLC) system. The extract from bRD contains almost equal molar amounts of both 13-cis retinal and all-trans retinal isomers. The extent of isomerization and the yield of both isomers during the isolation process were investigated by the application of the same extraction procedure to artificial bacteriorhodopsin reconstituted with 13-cis retinal isomer (13-cis bacteriorhodopsin) and also to light-adapted bacteriorhodopsin (bRL) which has been shown to contain only the all-trans isomer (all-trans bacteriorhodopsin). 2. A reconstituted bacteriorhodopsin, which had been prepared from apo-bacteriorhodopsin and an equimolar mixture of both 13-cis retinal and all-trans retinal isomers, showed an absorption spectrum having the same maximum wavelength as that of bRD even at the beginning of the reconstitution process. 3. Analysis of the photosteady states of bRD at -190 degrees C revealed that it was composed of two different species, one having 13-cis retinal and the other having all-trans retinal isomers in approximately equal molar amounts. These two also gave their respective photoproducts. 4. From these results it can be concluded that bRD contains both 13-cis retinal and all-trans retinal isomers in nearly equal molar amounts as its chromophore.     

A molecular piston mechanism of pumping protons by bacteriorhodopsin

Summary In this review the proton-pumping mechanism proposed recently for bacteriorhodopsin [Chou, K. C. (1993) Journal of Protein Chemistry, 12: 337–350] is illustrated in terms of a phenomenological model. According to the model, the-ionone of the retinal chromophore in bacteriorhodopsin can be phenomenologically imagined as a molecular piston. The photon capture by bacteriorhodopsin would pull it up while the spontaneous decrease in potential energy would push it down so that it would be up and down alternately during the photocycle process. When it is pulled up, the gate of pore is open and the water channel for the proton translocation is through; when it is pushed down, the gate of pore is closed and the water channel is shut up. Such a model not only is quite consistent with experimental observations, but also provides useful insights and a different view to elucidate the protonpumping mechanism of bacteriorhodopsin. The essence of the model might be useful in investigating the mechanism of ion-channels of other membrane proteins.Abbreviations bR bacteriorhodopsin - All-trans bR bacteriorhodopsin with all-trans retinal chromophore - 13-cis bR bacteriorhodopsin with 13-cis retinal chromophore - All-trans bundle the 7-helix bundle in the all-trans bR - 13-cis bundle the 7-helix bundle in the 13-cis bR - rms root-mean-square     

Why 11-cis-Retinal?

The C₂₀ diterpenoid compound retinal is the chromophore of thevisual pigments the rhodopsins, and the pigments present inHalobacterium halobium, namely, bacteriorhodopsin (proton pump),halorhodopsin (chloride pump), and the sensory rhodopsins (phototaxisreceptor). In all cases, they are bound covalently to the receptorprotein by a protonated Schiff base. However, in rhodopsins,the retinal is the 11-cis isomer, whereas in H. halobium pigmentsit is the all-trans isomer. Why did Nature choose retinal asthe chromophore, and why 11-cis in some cases and all-transin other cases? Also why is the chromophore a protonated Schiffbase? These points are addressed after giving an outline ofthe current status of the various photoreceptor pigments     

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.     

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.     

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.     

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.     

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.     

Studies on cephalopod rhodopsin: photoisomerization of the chromophore.

Photoisomerization of the chromophore of squid rhodopsin is dependent upon the irradiation temperature. Above 0 degrees C, only 11-cis in equilibrium all-trans reaction proceeds and the all-trans leads to 9-cis reaction is limited to extremely low efficiency. At liquid nitrogen temperature, 11 cis in equilibrium all-trans in equilibrium 9-cis reaction takes place. At intermediary low temperatures (-80 degrees C to -15 degrees C) another isomer of retinal may be produced by the irradiation, which forms a pigment having an absorbance maximum at 465 nm (P-465). The formation of P-465 decreases remarkably in the narrow temperature range from -30 degrees C to 0 degrees C where mesorhodopsin converts to metarhodopsin. Medsorhodopsin is quite different from metarhodopsin in the photoisomerization of the chromophore because P-465 is produced from the former but not from the latter. No P-465 is produced both at liquid nitrogen temperature and above 0 degrees C. P-465 is more labile than any of the other photoproducts so far known, that is isorhodopsin, alkaline and acid metarhodopsins. P-465 is converted to metarhodopsin by irradiation.     

Chromophore configuration of pharaonis phoborhodopsin and its isomerization on photon absorption.

The configuration of the retinylidene chromophore in pharaonis phoborhodopsin (ppR) and its changes during the photoreaction cycle were investigated by means of a chromophore extraction method followed by HPLC analysis. The ppR has an all-trans chromophore, and unlike bacteriorhodopsin, it exhibits no dark isomerization of the chromophore. Irradiation of a ppR sample in the presence of 10 mM hydroxylamine, at which concentration a negligible amount of ppR was bleached, caused the formation of 90% 13-cis- and 10% all-trans-retinal oximes. Because the ppR sample under the continuous irradiation was a mixture containing original ppR, ppRM, and a small amount of ppRO, the above results showed that the chromophores of ppRM and ppRO are in a 13-cis form and an all-trans form, respectively. Therefore, the all-trans chromophore of ppR is isomerized to the 13-cis form on photon absorption, and it is thermally reisomerized to the all-trans form on the conversion process from ppRM to ppRO. The extracted retinal oximes from ppR and ppRO were mainly the 15-syn form, while that from ppRM was mainly the 15-anti form. This fact indicated that the attack of hydroxylamine on the chromophore is stereoselective owing to the unique structure of the chromophore binding site near the Schiff base region of the chromophore.     

Interaction of aromatic retinal analogues with apopurple membranes of Halobacterium halobium

Absorption spectral properties of aromatic analogues of retinal with apopurple membrane of Halobacterium halobium were studied. The spectra of the all-trans forms were composed of two or more absorption bands. During incubation at 20 degrees C, an absorption band above 500 nm increased in intensity gradually at the expense of an absorption band in the shorter wavelength region with no isomerization of the chromophore. The longer wavelength species was shown to be the protonated form of the shorter wavelength species by changing the pH of the medium. Upon irradiation with blue light, the bandwidth of the spectrum became smaller with isomerization of the chromophore to its 13-cis form. Irreversible binding of protons on the membrane occurred during this process. The rate of the increase in the longer wavelength absorption band was especially low in the reaction with the all-trans form of retinal analogues having a bulky substituent at the para or meta positions of the phenyl ring. In contrast, the 13-cis isomer of aromatic retinal analogues gave a single absorption peak. The extent of the spectral shift upon binding to apopurple membranes was compared over a series of aromatic retinals, and the results were explained in terms of steric interactions of the chromophore with the protein.     

Trans/13-cis isomerization is essential for both the photocycle and proton pumping of bacteriorhodopsin.

We studied an analogue of bacteriorhodopsin whose chromophore is based on all-trans retinal. A five-membered ring was built around the 13-14 double bond so as to prohibit trans to 13-cis isomerization. No light-induced photochemical changes were seen, other than those due to a small amount (approximately 5%) of unbleached bacteriorhodopsin remaining in the apomembrane used for regeneration. The techniques used included flash photolysis at room and liquid nitrogen temperatures and Fourier-transform infrared difference spectroscopy. When the trans-fixed pigment was incorporated into phospholipid vesicles, no evidence of light-initiated proton pumping could be found. The results indicate that trans to 13-cis isomerization is essential for the photochemical transformation and function of bacteriorhodopsin.     

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).     

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.     

The 3, 4-didehydroretinal chromophore of goldfish porphyropsin

The isomeric configuration of the 3,4-didehydroretinal chromophore of goldfish porphyropsin was determined by high performance liquid chromatography (HPLC) and by the regeneration of this visual pigment with authentic isomers of 3,4-didehydroretinal. A nonisomerizing, quantitative method using hydroxylamine and methylene chloride was employed to extract the 3,4-didehyroretinal chromophore from the rod outer segment membrane (containing the porphyropsin). When this extracted chromophore was injected into the HPLC, only a single major peak was observed and this peak coeluted with the authentic 11-cis 3,4-didehydroretinyl oxime. This suggests that the chromophore of goldfish porphyropsin is 11-cis 3,4-didehydroretinal. When the bleached rod outer segments (containing the opsin) were incubated with different 3,4-didehydroretinal isomers (13-cis, 11-cis, 9-cis, and all-trans), only the 11-cis isomer resulted in the degeneration of porphyropsin. This also suggests that the porphyropsin chromophore exists in the 11-cis configuration.     

Conformational change in bacterio-opsin on binding to retinal.

The detailed mechanism of retinal binding to bacterio-opsin is important to understanding retinal pigment formation as well as to the process of membrane protein folding. We have measured the temperature dependence of bacteriorhodopsin formation from bacterio-opsin and all-trans retinal. An Arrhenius plot of the apparent second-order rate constants gives an activation energy of 11.6 +/- 0.7 kcal/mol and an activation entropy of -4 +/- 2 cal/mol deg. Comparison of the activation entropy to model compound reactions suggests that chromophore formation in bacteriorhodopsin involves a substantial protein conformational change. Cleavage of the polypeptide chain between residues 71 and 72 has little effect on the activation energy or entropy, indicating that the connecting loop between helices B and C is not involved in this conformational change.     

FTIR study of the photoisomerization processes in the 13-cis and all-trans forms of Anabaena sensory rhodopsin at 77 K

Archaeal-type rhodopsins can accommodate either all-trans- or 13-cis,15-syn-retinal in their chromophore binding site in the dark, but only the former isomer is functionally important. In contrast, Anabaena sensory rhodopsin (ASR), an archaeal-type rhodopsin found in eubacteria, exhibits a photochromic interconversion of both forms, suggesting that ASR functions as a photosensor which interacts with its 14 kDa soluble transducer differently in the all-trans and 13-cis,15-syn forms. In this study, we applied low-temperature Fourier transform infrared (FTIR) spectroscopy to the 13-cis,15-syn form of ASR (13C-ASR) at 77 K and compared the local structure around the chromophore and its structural changes upon retinal photoisomerization with those of the all-trans form (AT-ASR) [Furutani, Y., Kawanabe, A., Jung, K. H., and Kandori, H. (2005) Biochemistry 44, 12287-12296]. By use of [zeta-15N]lysine-labeled ASR, we identified the N-D stretching vibrations of the Schiff base (in D2O) at 2165 cm(-1) for 13C-ASR and at 2163 and 2125 cm(-1) for AT-ASR. The frequencies indicate strong hydrogen bonds of the Schiff base with a water molecule for both 13C-ASR and AT-ASR. In contrast, the N-D stretching vibration appears at 2351 cm(-1) and at 2483 cm(-1) for the K states of 13C-ASR (13C-ASR(K)) and AT-ASR (AT-ASR(K)), respectively, indicating that the Schiff base still forms a hydrogen bond in 13C-ASR(K). Rotational motion of the Schiff base upon retinal isomerization is probably smaller for 13C-ASR than for AT-ASR, the latter altering hydrogen bonding of the Schiff base similar to bacteriorhodopsin (BR), a light-driven proton pump. Appearance of several hydrogen-out-of-plane vibrations and amide I vibrations in 13C-ASR(K), but not in AT-ASR(K), suggests that structural changes are distributed widely along the polyene chain for 13C-ASR. On the other hand, retinal photoisomerization in AT-ASR breaks the hydrogen bond of the Schiff base, and localized structural changes in the Schiff base region are induced.     

Photoinduced transformations in bacteriorhodopsin membrane monitored with optical microcavities

Photoinduced molecular transformations in a self-assembled bacteriorhodopsin (bR) monolayer are monitored by observing shifts in the near-infrared resonant wavelengths of linearly polarized modes circulating in a microsphere cavity. We quantify the molecular polarizability change upon all-trans to 13-cis isomerization and deprotonation of the chromophore retinal ( approximately -57 A(3)) and determine its orientation relative to the bR membrane ( approximately 61 degrees ). Our observations establish optical microcavities as a sensitive off-resonant spectroscopic tool for probing conformations and orientations of molecular self-assemblies and for measuring changes of molecular polarizability at optical frequencies. We provide a general estimate of the sensitivity of the technique and discuss possible applications.     

Photochromism of halorhodopsin. cis/trans isomerization of the retinal around the 13-14 double bond

The isomeric composition of retinal in membrane-bound and in purified but detergent-free, dark-adapted halorhodopsin was found to be about 70% 13-cis and 30% all-trans. Any illumination increased the all-trans content relative to the dark-adapted state, but blue illumination shifted the isomeric composition more toward all-trans while red illumination of blue-adapted samples shifted it more toward 13-cis. In the presence of chloride this photoisomerization caused the kind of photochromic behavior reported earlier in Smith, S. O., Marvin, M. J., Bogomolni, R. A., and Mathies, R. A. (1984) J. Biol. Chem. 259, 12326-12329, i.e. blue light caused the absorption maximum to move toward longer wavelengths and red light reversed the shift. Only the all-trans chromophore exhibited the complete photocycle described earlier in detergent-solubilized halorhodopsin, and this was the form that could be associated with light-driven chloride transport activity in cell envelope vesicles. In the absence of chloride the spectroscopic changes caused by illumination were much smaller. Reconstitution of bleached preparations with 13-cis- and all-trans-retinal, in the presence and absence of chloride, confirmed that the difference between the absorption maxima of the two isomeric forms of the chromophore is affected by chloride: 13-cis-halorhodopsin absorbs at about 567-568 nm with and without chloride, and the all-trans pigment absorbs near 568 nm in the absence of chloride, but at 578 nm in its presence. The simplest explanation of this finding is that most of the red-shift which accompanies the 13-cis----all-trans transition originates from electrostatic interaction of the retinal with chloride bound in its vicinity.     

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.