Structural change of bacteriorhodopsin in the purple membrane above pH 10 decreases heterogeneity of the irreversible photobleaching components

Kinetic investigations of irreversible photobleaching of bacteriorhodopsin (bR) in purple membrane (PM) at high temperature have previously shown two kinds of bR species upon light illumination. The bR species consist of kinetically fast- and slow-denatured components, whose proportions were dependent upon structural changes in dark, as shown by CD. In order to elucidate electrostatic contribution on the heterogeneous stability and the bR structure in PM, photobleaching behaviour and structural changes over a wide pH range were investigated by kinetics as well as various spectroscopic techniques. Kinetics revealed that photobleaching below pH 9 obeyed double-exponential functions, whereas measurements above pH 10 were characterized by a single-decay component. FT-IR deconvoluted spectra showed a alpha(II)-to-alpha(I) transition in the transmembrane helices around pH 10. Near-IR Raman scattering spectra demonstrated the equilibrium shift of retinal isomers from all trans to 13-cis form. Near-UV CD spectra suggested configurational changes in the aromatic residues around the retinal pocket. An exciton-to-positive transition in visible CD spectrum was observed. This indicates disorganization in the 2D-crystalline lattice of PM, which occurred concomitantly with the changes above pH 10. A model for the changes in kinetic behaviour and molecular structure around pH 10 is discussed, focusing on changes in charge distribution upon alkalinization.     

Photoelectric properties of a detector based on dried bacteriorhodopsin film

The photoelectric response of a detector using dried bacteriorhodopsin (bR) film as the light sensing material is mathematically modeled and experimentally verified in this paper. The photocycle and proton transfer kinetics of dried bR film differ dramatically from the more commonly studied aqueous bR material because of the dehydration process. The photoelectric response of the dried film is generated by charge displacement and recombination instead of transferring a proton from the cytoplasmic side to the extracellular side of the cell membrane. In this work, the wild-type bR samples are electrophoretically deposited onto an indium tin oxide (ITO) electrode to construct a simple multiple layered photo-detector with high sensitivity to small changes in incident illumination. The light absorption characteristics of the thin bR film are mathematically represented using the kinetics of the bR photocycle and the charge displacement theorem. An electrically equivalent RC circuit is used to describe the intrinsic photoelectric properties of the film and external measurement circuitry to analyze the detector's response characteristics. Simulated studies and experimental results show that the resistance of the dried bR film is in the order of 10(11) Omega. When the input impedance of the measurement circuitry is one order of magnitude smaller than the dried film, the detector exhibits a strong differential response to the original time-varying light signal. An analytical solution of the equivalent circuit also reveals that the resistance and capacitance values exhibited by the dried bR film, in the absence of incident light, are almost twice as large as the values obtained while the material is under direct illumination. Experimental observations and a predictive model both support the notion that dried bR film can be used in simple highly sensitive photo-detector designs.     

X-ray diffraction and flash photolysis studies of M intermediate of lattice-contracted purple membrane

The effects of cross-linking and lattice contraction of purple membrane (PM) on the photodynamics of bacteriorhodopsin (bR) and on the tertiary structure were studied by flash photolysis and X-ray diffraction. To get a contracted lattice form of PM, native PM, and/or PM cross-linked by glutaraldehyde were treated with deoxycholate or Triton X-100. Part of the Triton-treated cross-linked PM was further incubated with Bio-Beads SM-2 to remove Triton X-100. In the modified PM, several long-lived components of the M intermediate appeared, the features of which were related to the environment of bR. Also, X-ray diffraction studies using synchrotron radiation were performed on the modified PM under intense light irradiation (lambda greater than 500 nm) in which 40-80% of bR was photoconverted to the M state. In the Triton-treated cross-linked PM dispersed in 0.25% Triton X-100, the unit cell of membrane crystalline lattice was enlarged from 58.8 to 59.8 A and the crystalline order decreased with irradiation. The analysis of X-ray diffraction patterns suggests that light-induced conformational changes of bR correlated with the Triton content of the environment and an increase of substitution disorder was caused by these changes, but the average location of bR was unchanged. However, the other modified PM showed no significant changes of diffraction, upon light irradiation.     

Transient absorption and photovoltage study of' self-assembled bacteriorhodopsin/polycation multilayer films

A series of organized (PDAC/PM)(n) (poly(diallyldimethylammonium chloride)/purple membrane) multilayer films were prepared by alternate adsorptions of positively charged PDAC polyelectrolyte and negatively charged purple membrane (PM). The kinetics of the photocycle of bacteriorhodopsin (bR) in PM was studied by flash photolysis and transient photovoltage methods. Although the orientation of the adsorbed bR depends on the pH of the PM suspension, the kinetics of the photo-induced reaction cycle in dehydrated films is independent of the deposition pH. In dry (PDAC/PM)(n) films the decay of the M intermediate to the initial bR state is multiexponential and delayed to several minutes for both orientations. A simultaneous two-exponential decay in millisecond time domain was observed at red wavelengths. The source of the red-shifted absorption is suggested to be the C(610) intermediate of the cis photocycle of bR.     

Influence of an electrical potential on the charge transfer kinetics of bacteriorhodopsin

The adsorption of bacteriorhodopsin(bR)-containing purple membranes (PM) to black lipid membranes (BLM) was used to study the charge translocation kinetics of bR upon flash excitation.

The discharge of the PM-BLM system after charging upon illumination is found to proceed quite slowly (discharge time up to several minutes) but is considerably accelerated by addition of the protonophore FCCP.

Therefore, the dependence of the proton transfer kinetics in bR on electrical potentials generated by preceding flashes of varying repetition rate and intensity was investigated. The kinetics are slowed down with increasing flash intensity as well as repetition rate. This effect is partly abolished by small amounts of FCCP.

A new model is introduced which takes into account the instantaneous feedback of the electrical potential on the kinetics of the pump current. It explains the observed deviations from first-order kinetics and renders an approach with “distributed kinetics” unnecessary.

     

Photovoltage kinetics of the acid-blue and acid-purple forms of bacteriorhodopsin: evidence for no net charge transfer.

Time-resolved photovoltage measurements were performed with the acid-blue (bR605A) and acid-purple (bR565A) forms of bacteriorhodopsin (bR) in the time range from 25 ns to 100 s. The bR605A and bR565A pigments were formed by titration with H2SO4 in the absence and presence of 150 mM KCI, respectively. Qualitatively the kinetics of the charge displacement in these two states are similar and consist of two fast phases in one direction (100 ns bandwidth limited and approximately 1 microsecond) followed by a decay in the opposite direction via one component for bR605A (4.4 +/- 0.6 ms) or two components for bR565A (33 +/- 8 microseconds and 3.6 +/- 0.5 ms). The transient photovoltage signal returns exactly to the initial value after several milliseconds, well before the passive discharge of the electrical measuring system at 2 s. We conclude that no net charge transfer occurs in either bR605A or bR565A. The direction of the fast components is opposite that of net proton translocation in bR at pH 7. So, if the charge that moves back and forth is due to a proton, it moves first in the direction of the cytoplasmic side of the membrane (< 1 microsecond) and returns to its initial position via the 4.4 ms (bR605A) or the 33 microseconds and 3.6 ms (bR565A) decay components. The amplitude of the charge motion in both low pH forms is too large to be due to isomerization alone and is comparable to one of the major components in bR at pH 7.2     

Effect of partial delipidation of purple membrane on the photodynamics of bacteriorhodopsin

The effect of lipid-protein interaction on the photodynamics of bacteriorhodopsin (bR) was investigated by using partially delipidated purple membrane (pm). When pm was incubated with a mild detergent, Tween 20, the two major lipid components of pm, phospholipids and glycolipids, were released in different ways: the amount of phospholipids released was proportional to the logarithm of the incubation time; the release of glycolipids became noticeable after the release of approximately 2 phospholipids/bR, but soon leveled off at approximately 50% of the initial content. It was found that the thermal decay of the photocycle intermediate N560 was inhibited by the removal of less than 2 phospholipids per bR. This inhibition was partly explained by an increase in the local pH near the membrane surface. More significant changes in the bR photoreactions were observed when greater than 2 phospholipids/bR were removed: (1) the extent of light adaptation became much smaller, and this reduction correlated with the release of glycolipids; (2) N560 became difficult to detect; (3) the M412 intermediate, which is characterized by a pH-insensitive lifetime, was replaced by a long-lived M-like photoproduct with a pH-sensitive lifetime. The heavy delipidation apparently altered the mechanism by which the deprotonated Schiff base receives a proton. An important conformational change in the protein moiety is suggested to take place during the M412 state, this conformational change being inhibited in the rigid lipid environment.     

Revisiting the folding kinetics of bacteriorhodopsin

The elucidation of the physical principles that govern the folding and stability of membrane proteins is one of the greatest challenges in protein science. Several insights into the folding of α-helical membrane proteins have come from the investigation of the conformational equilibrium of H. halobium bacteriorhodopsin (bR) in mixed micelles using SDS as a denaturant. In an effort to confirm that folded bR and SDS-denatured bR reach the same conformational equilibrium, we found that bR folding is significantly slower than has been previously known. Interrogation of the effect of the experimental variables on folding kinetics reveals that the rate of folding is dependent not only on the mole fraction of SDS but also on the molar concentrations of mixed micelle components, a variable that was not controlled in the previous study of bR folding kinetics. Moreover, when the molar concentrations of mixed micelle components are fixed at the concentrations commonly employed for bR equilibrium studies, conformational relaxation in the transition zone is slower than hydrolysis of the retinal Schiff base. As a result, the conformational equilibrium between folded bR and SDS-denatured bR cannot be achieved under the conventional condition. Our finding suggests that the molar concentrations of mixed micelle components are important experimental variables in the investigation of the kinetics and thermodynamics of bR folding and should be accounted for to ensure the accurate assessment of the conformational equilibrium of bR without the interference of retinal hydrolysis.     

Heterogeneity in regeneration of bacteriorhodopsin from bacterio-opsin and all-trans retinal at high temperatures: implications for dynamic structural fluctuations

Measurements of regeneration kinetics were performed in order to investigate the regeneration mechanisms of bacteriorhodopsin (bR) from thermally unfolded bacterio-opsin (bO) and all-trans retinal. Regeneration kinetics data were successfully fitted to a single exponential function when regeneration was performed at 25 degrees C after incubation at high temperatures. Conversely, the process of regeneration after the addition of retinal to bO at high temperatures occurred at two different rate constants. These findings strongly suggest that the slower regeneration of bR at high temperatures occurs as a result of dynamic structural fluctuation of bO, whereas the faster process corresponds to regeneration from bO, which retains a native structure capable of retinal binding.     

The photoactivated PYP domain of Rhodospirillum centenum Ppr accelerates the recovery of the bacteriophytochrome domain after white light illumination

Ppr from the purple phototrophic bacterium, Rhodospirillum centenum (also known as Rhodocista centenaria), is a hybrid of photoactive yellow protein (PYP), bacteriophytochrome (Bph), and histidine kinase (HK) domains. The holo-Ppr (containing both chromophores) exhibits characteristic absorption maxima at 435 nm due to the PYP domain and at 400, 642, and 701 nm due to the Bph domain. Illumination of the Ppr with white light causes a bleach of both PYP and Bph absorbance; weak blue light primarily bleaches the PYP, and red light activates only the Bph. When excited by blue light, the PYP domain in Ppr recovers with biphasic kinetics at 445 nm (32% with a lifetime of 3.8 min and the remainder with a lifetime of 46 min); white light primarily results in fast recovery, whereas the 130-residue PYP construct shows only the faster kinetics in both blue and white light. Furthermore, there is a slight red shift of the ground state Bph when the PYP is activated; thus, both spectroscopy and kinetics suggest interdomain communication. When Ppr is illuminated with red light, the recovery of the Bph domain to the dark state is significantly slower than that of PYP and is biphasic (57% of the 701 nm decay has a lifetime of 17 min and the remainder a lifetime of 50 min). However, when illuminated with white light or red followed by blue light, the Bph domain in Ppr recovers to the dark-adapted state in a triphasic fashion, where the fastest phase is similar to that of the fast phase of the PYP domain (in white light, 25% of the 701 nm recovery has a lifetime of approximately 1 min) and the slower phases are like the recovery after red light alone. Apo-holo-Ppr (with the biliverdin chromophore only) recovers with biphasic kinetics similar to those of the slower phases of holo-Ppr when activated by either red or white light. We conclude that the photoactivated PYP domain in Ppr accelerates recovery of the activated Bph domain. Phytochromes can be reversibly switched between Pr and Pfr forms by red and far-red light, but the consequence of a bleaching phytochrome is that it cannot be photoreversed by far-red light. We thus postulate that the function of the PYP domain in Ppr is to act as a blue light switch to reverse the effects of red light on the Bph.     

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.     

A study of light-induced conductivity in bacteriorhodopsin

Measurements have been made of light-induced conductivity changes and the associated kinetics of the relaxation processes in aqueous suspensions and sonicated liposomes containing bacteriorhodopsin (bR). Aqueous suspensions exhibit a single relaxation time of 1 to 2 ms. The addition of D₂O to the aqueous suspension slows down the relaxation time, fourfold. Similar behaviour is seen in sonicated liposomes with a relaxation time of 2 to 3 ms. Activation energies of approximately 14 and 6 kJM^-1 are obtained for the effect in sonicated liposomes and aqueous suspension containing bR, respectively. These relaxation processes with lifetime of 1 to 2 ms suggest conformational changes in the protein moiety of bR which most probably may be associated with protonation-deprotonation processes or less likely the release and binding of small ions.     

Heterogeneity in Regeneration of Bacteriorhodopsin from Bacterio-Opsin and All-trans Retinal at High Temperatures: Implications for Dynamic Structural Fluctuations

Measurements of regeneration kinetics were performed in order to investigate the regeneration mechanisms of bacteriorhodopsin (bR) from thermally unfolded bacterio-opsin (bO) and all-trans retinal. Regeneration kinetics data were successfully fitted to a single exponential function when regeneration was performed at 25 °C after incubation at high temperatures. Conversely, the process of regeneration after the addition of retinal to bO at high temperatures occurred at two different rate constants. These findings strongly suggest that the slower regeneration of bR at high temperatures occurs as a result of dynamic structural fluctuation of bO, whereas the faster process corresponds to regeneration from bO, which retains a native structure capable of retinal binding.     

Sensitive detection of protein-lipid interaction change on bacteriorhodopsin using dodecyl β-D-maltoside

A light-driven proton pump bacteriorhodopsin (bR) forms a two-dimensional hexagonal lattice with about 10 archaeal lipids per monomer bR on purple membrane (PM) of Halobacterium salinarum. In this study, we found that the weakening of the bR-lipid interaction on PM by addition of alcohol can be detected as the significant increase of protein solubility in a nonionic detergent, dodecyl β-D-maltoside (DDM). The protein solubility in DDM was also increased by bR-lipid interaction change accompanied by structural change of the apoprotein after retinal removal and was about 7 times higher in the case of completely bleached membrane than that of intact PM. Interestingly, the cyclic and milliseconds order of structural change of bR under light irradiation also led to increasing the protein solubility and had a characteristic light intensity dependence with a phase transition. These results indicate that there is a photointermediate in which bR-lipid interaction has been changed by its dynamic structural change. Because partial delipidation of PM by CHAPS gave minor influence for the change of the protein solubility compared to intact PM in both dark and light conditions, it is suggested that specific interactions of bR with some lipids which remain on PM even after delipidation treatment have a key role for the change of solubility in DDM induced by alcohol binding, ligand release, and photon absorption on bR.     

Dramatic in situ conformational dynamics of the transmembrane protein bacteriorhodopsin.

The conformational dynamic capabilities of the in situ bacteriorhodopsin (bR) can be studied by determination of the changes of the bR net helical segmental tilt angle (the angle between the polypeptide segments and the membrane normal) induced by various perturbations of the purple membrane (PM). The analysis of the far-UV oriented circular dichroism (CD) of the PM provides one means of achieving this. Previous CD studies have indicated that the tilt angle can change from approximately 10 degrees to 39 degrees depending on the perturbants used with no changes in the secondary structure of the bR. A recent study has indicated that the bleaching-induced tilt angle can be enhanced from approximately 24 degrees to 39 degrees by cross-linkage and papain-digestion perturbations which by themselves do not alter the tilt angle. To add further credence, this study has been repeated using midinfrared (IR) linear dichroic spectral analysis. In contrast to the CD method, analysis by the IR method depends on the orientation of the amide plane of the helix assumed. Excellent consistency is achieved between the two methods only when it is assumed that the structural characteristics of the alpha-helices of the bR are equally alpha I and alpha II in nature. Furthermore, the analysis of the IR data becomes essentially independent of the three amide transitions utilized. The net tilt angle of segments completely randomized relative to the incident light must be 54.736 in view of helix symmetry. A value of 54.735 degrees +/- 0.001 degree was achieved by the IR method for the ethanol-treated PM film, establishing this kind of film as an ideal random state standard and demonstrating the accuracy potential of the IR method.     

Photoconversion from the light-adapted to the dark-adapted state of bacteriorhodopsin.

Dark and light adaptation of bacteriorhodopsin in purple membrane multilayers at less than 100% relative humidity differs from that seen in suspensions. Equilibrium between the two bacteriorhodopsin isomers (bR cis 550 and bR trans 570) in the light-adapted state becomes dependent on the wavelength of actinic light. Excitation at the red edge of the visible absorption band causes dark adaptation in a light-adapted sample. Using polarized actinic and measuring light, we show that acceleration of the dark adaptation through heating by actinic light cannot explain this observation. A light-driven bR trans 570 to bR cis 550 reaction that competes with the well-known 13 cis-to-all-trans light adaptation reaction must exist under our experimental conditions. Trans-to-cis conversion is a one-photon process distinct from the two photon process observed by others in purple membrane suspensions (Sperling, W., C. N. Rafferty, K. D. Kohl, and N. A. Dencher, 1978, FEBS (Fed. Eur. Biochem. Soc.) Lett. 97:129-132). Its quantum efficiency increases monotonously on reducing the hydration level, and is paralleled by an increase in the lifetime of the M410 intermediate of the trans photocycle. We suggest that at this point a branch leads from the all-trans into the 13-cis photocycle. It is probably the same reaction that causes the reduced light adaptation in monomeric bacteriorhodopsin (Casadio, R., H. Gutowitz, P. Mowery, M. Taylor, and W. Stoeckenius, 1980, Biochim. Biophys. Acta. 590:13-23; Casadio, R., and W. Stoeckenius, 1980, Biochemistry. 19:3374-3381).     

Roles of functional lipids in bacteriorhodopsin photocycle in various delipidated purple membranes

Purple membrane (PM) is composed of several native lipids and the transmembrane protein bacteriorhodopsin (bR) in trimeric configuration. The delipidated PM (dPM) samples can be prepared by treating PM with CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) to partially remove native lipids while maintaining bR in the trimeric configuration. By correlating the photocycle kinetics of bR and the exact lipid compositions of the various dPM samples, one can reveal the roles of native PM lipids. However, it is challenging to compare the lipid compositions of the various dPM samples quantitatively. Here, we utilize the absorbances of extracted retinal at 382 nm to normalize the concentrations of the remaining lipids in each dPM sample, which were then quantified by mass spectrometry, allowing us to compare the lipid compositions of different samples in a quantitative manner. The corresponding photocycle kinetics of bR were probed by transient difference absorption spectroscopy. We found that the removal rate of the polar lipids follows the order of BPG ≈ GlyC < S-TGD-1 ≈ PG < PGP-Me ≈ PGS. Since BPG and GlyC have more nonpolar phytanyl groups than other lipids at the hydrophobic tail, causing a higher affinity with the hydrophobic surface of bR, the corresponding removal rates are slowest. In addition, as the reaction period of PM and CHAPS increases, the residual amounts of PGS and PGP-Me significantly decrease, in concomitance with the decelerated rates of the recovery of ground state and the decay of intermediate M, and the reduced transient population of intermediate O. PGS and PGP-Me are the lipids with the highest correlation to the photocycle activity among the six polar lipids of PM. From a practical viewpoint, combining optical spectroscopy and mass spectrometry appears a promising approach to simultaneously track the functions and the concomitant active components in a given biological system.     

Effect of protein-protein interaction on light adaptation of bacteriorhodopsin

Triton X-100 solubilized monomers of bacteiorhodopsin (bR) show a decrease in the extent of light adaptation; the red shift and the absorbance increase of the visible absorption band are reduced no less than half the values observed in purple membrane (p.m.) with a corresponding reduction in the isomerization of 13-cis- to all-trans-retinal. Cross-linking of bR with glutaraldehyde before exposure to Triton prevents dissociation of the lattice and reduction in light adaptation. Experiments with cross-linked and lipid-extracted p.m. show that Triton effectively substitutes for the native membrane lipids and that the lattice structure apparently stabilizes the light-adapted state of bR under illumination. In lipid vesicles at molar lipid protein ratios greater than or equal to 80, bR exists as monomers above the lipid-phase transition and aggregates below the phase transition. Above the lipid-phase transition and aggregates below the phase transition. Above the lipid-phase transition light adaptation in the monomers, measured as either the red shift of the visible absorbance maximum or the isomerizaiton o 13-cis- to all-trans-retinal, is also reduced to less than half of the extent observed in intact purple membrane or in the bR aggregates formed in lipid vesicles below the plhase transition. At very high lipid to protein ratios, bR molecules cannot aggregate when the temperature is decreased below the phase transition, and these monomers in a solid lipid phase show the same reduced extent of light adaptation as monomers above the phase transition, thus confirming that this effect is mainly due to the absence of protein-protein interaction and not to the state of the lipid. The extent of the red shift upon light adaptation may be used as a convenient indicator to distinguish the aggregated and monomeric states of bR.     

Photosensory Adaptation in Plants

Photosensory adaptation (range adjustment) is of great importance in plants (including fungi and various microorganisms) that operate in large intensity ranges. The adaptation mechanisms of plants have some features in common with those of vertebrates and invertebrates. As with those systems, plants have biphasic exponential dark-adaptation kinetics, that are much slower than the corresponding light adaptation kinetics. One needs to distinguish between sensor adaptation, which regulates range adjustment, and effector adaptation (habituation), which regulates the motor apparatus of the organism (flagellar movement or cell wall growth). In Phycomyces, and perhaps Stentor, sensor adaptation is mediated by the photoreceptor system. In contrast to vertebrates and invertebrates, dark adaptation can be controlled in some plants by special photoreceptors. In Phycomyces, these can be either photo-products of the actinic photoreceptor(s) or not yet identified receptor pigments. In higher plants phytochrome can alter the state of adaptation.     

Effect of acid pH on the absorption spectra and photoreactions of bacteriorhodopsin.

Purple membranes (PM) from Halobacterium halobium were incorporated into 7.5% polyacrylamide gels to prevent aggregation which occurs in suspensions at low pH. At pH 7.0, the circular dichroism (CD) spectra and visible absorption spectra of light- and dark-adapted bacteriorhodopsin (bR558, respectively) and the flash photolysis cycle of bR568 in gels were essentially the same as those in PM suspensions. Titration of the gels with hydrochloric acid showed the transition to a form absorbing at 605 nm (bR605 acid) with pK = 2.9 and to a second form absorbing at 565 nm (bR565 acid) with pK = 0.5. Isosbestic points were seen for each transition in both light- and dark-adapted gels. In addition, a third isosbestic point was evident between pH 3.5 and 7. Visible CD spectra of bR568, bR605 acid, and bR565 acid all showed the bilobed pattern typical of bR568 in suspensions of PM. Flash kinetic spectrophotometry (with 40-microseconds time resolution) of bR605 acid and bR565 acid showed transient absorbance changes with at least one transiently blue-shifted form for each and an early red-shifted intermediate for bR565 acid. Chromophore extraction from membrane suspensions yielded all-trans-retinal for bR565 acid and a mixture of 13-cis and trans isomers for bR605 acid.