Photocycle of 13-cis-retinal in bacteriorhodopsin caused by destruction to the cooperative effect of purple membrane

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LIGHT INDUCED ISOMERISATION, AT ACIDIC pH, INITIATES HYDROLYSIS OF BACTERIORHODOPSIN TO BACTERIO-OPSIN AND 9-CIS-RETINAL

Abstract— Bacteriorhodopsin (BR) from the purple membrane of Haiobacterium halobium contains covalently bound retinal in the 13- cis and all- trans configurations. Several forms of bacteriorhodopsin are known, with different absorption maxima which are designated as BRλ_max (nm). At acidic pH, BR605 is formed from BR560. The following sequence of reactions was found, which is initiated by irradiation of BR605 with red light:

An all- trans /13- cis to 9- cis isomerisation occurs in the light induced reaction BR605 ∼ BR500. BR500 seems to contain covalently bound retinal, whereas BR390 contains free retinal. By irradiation with light, BR500, BR450 and BR390 can be reconverted to BR560.     

BIOSYNTHESIS AND ASSEMBLY OF PURPLE MEMBRANE OF HALOBACTERIUM HALOBIUM S9: LIGHT STIMULATION OF BACTERIORHODOPSIN FORMATION

Abstract— The effect of light on purple membrane biogenesis in Halobacterium halobium S9 strain was investigated. When bacteria were grown in the dark, the 570nm absorption due to bacteriorhodopsin increased more slowly than under illumination, but eventually after longer incubation, reached the same level as that seen in the illuminated culture.
Analysis of membrane fractions by sucrose density gradient centrifugation revealed that two different membrane fractions, containing purple and brown membrane could be detected in the exponential growth phase. Another fraction whose density was higher than that of purple membrane, disappeared concomitantly with the increase in purple membrane and brown membrane, indicating that it may be related to purple membrane formation.
HPLC analysis of membrane proteins showed that there was no significant difference in de novo synthesis of bacterio–opsin between dark and illuminated cultures. This led us to conclude that light stimulated retinal binding to bacterio–opsin and/or retinal biosynthesis rather than bacterio–opsin synthesis. Bacteriorhodopsin seemed to form the brown membrane fraction first, which then spontaneously reorganized into purple membrane.
When incorporated in liposomes, bacteriorhodopsin in brown membrane was found to have rather higher proton pump activity than that in purple membrane. The H⁺ pumping activity was quite heat labile. This and the CD spectra indicated that bacteriorhodopsin in brown membrane might exist without forming normal timer unit.     

BLEACHING OF BACTERIORHODOPSIN IN PURPLE MEMBRANE, BROWN HOLO-MEMBRANE ANDL–1690-SOLUBILIZED MONOMERS WITH HYDROXYLAMINE IN LIGHT AND THE DARK

Abstract— The reactions of hydroxylamine with bacteriorhodopsin in the states of the purple membrane, the brown holo-membrane andL–1690-solubilized monomers were studied in light and the dark. The bleaching rate was strongly dependent on the state of the bacteriorhodopsin and largely enhanced by light. Analysis of the isomeric composition of the resultant retinaloximes by high performance liquid chromatography (HPLC) showed that more all- trans retinaloximes were present in the dark than 13- cis retinaloximes. However, under illumination, the relative amounts of 13- cis retinaloximes increased greatly, indicating the occurrence of trans to cis isomerization during the photochemical cycle of bacteriorhodopsin. Molar extinction coefficients of 13- cis retinaloximes were determined to estimate the isomeric composition of retinaloximes by HPLC analysis.     

Femtosecond spectroscopy of the first events of the photochemical cycle in bacteriorhodopsin

The first steps in the photochemistry of bacteriorhodopsin (BR) are investigated with light pulses of 160 fs duration. Four samples are studied: (i) the purple membrane, (ii) deuterated purple membrane, (iii) BR trimers and (iv) BR monomers. In all samples the first intermediate J is formed within 430±50 fs. No isotope effect is observed in the formation of J upon deuteration, in contrast to previous reports with much higher excitation energies. Thus proton movement to or from the retinal Schiff's base is not relevant during the first step. Comparing the data for trimeric and monomeric BR suggests an upper limit of 50 fs for the transfer of excitation energy from the excitonically coupled trimer to a single retinal chromophore.     

The light-activated proton pump Bop I of the archaeon Haloquadratum walsbyi

We have isolated and characterized the light-driven proton pump Bop I from the ultrathin square archaeon Haloquadratum walsbyi, the most abundant component of the dense microbial community inhabiting hypersaline environments. The disruption of cells by hypo-osmotic shock yielded Bop I retinal protein highly enriched membranes, which contain one main 27 kDa protein band together with a high content of the carotenoid bacterioruberin. Light-induced pH changes were observed in suspensions of Bop I retinal protein-enriched membranes under sustained illumination. Solubilization of H. walsbyi cells with Triton X-100, followed by phenyl-Sepharose chromatography, resulted in isolation of two purified Bop I retinal protein bands; mass spectrometry analysis revealed that the Bop I was present as only protein in both the bands. The study of light/dark adaptations, M-decay kinetics, responses to titration with alkali in the dark and endogenous lipid compositions of the two Bop I retinal protein bands showed functional differences that could be attributed to different protein aggregation states. Proton-pumping activity of Bop I during the photocycle was observed in liposomes constituted of archaeal lipids. Similarities and differences of Bop I with other archaeal proton-pumping retinal proteins will be discussed.     

pH-dependent bending in and out of purple membranes comprising BR-D85T

The light-driven proton pump bacteriorhodopsin (BR) embedded in a purple membrane (PM) from Halobacterium salinarum undergoes a series of conformational changes while transporting a proton from the cytoplasmic to the extracellular side over the course of the so-called photocycle. Wild-type BR variant D85T, where aspartic acid 85 is replaced by threonine, allows for the study of structural intermediates of this photocycle that are formed in a light-dependent manner in the wild-type and in thermal equilibrium by tuning the pH of the D85T purple membrane suspension. Especially the last and least studied O-intermediate of the photocycle of bacteriorhodopsin has caught recent attention. First AFM images of D85T under acidic conditions resembling wild-type BR under physiological conditions in the O-photocycle-intermediate are presented. Bacteriorhodopsins embedded in the strongly bent purple membranes were analyzed by single molecule force spectroscopy (SMFS) providing the first single molecule force spectra of BR in the O-intermediate. SMFS was further employed to determine the absolute sign of membrane curvature. Complementary electrostatic force microscopy (EFM) was performed to support PM side discrimination and determination of the bending direction. Bending of PM-D85T was analyzed in more detail providing further insight into the structure-function relationship of the bacteriorhodopsin proton pump as well as PM behaviour at the solid-liquid junction. Findings reported here are of general interest to the field of chemomechanical transducers.     

THE PHOTOREACTION OF THE DEIONIZED FORM OF THE PURPLE MEMBRANE INVESTIGATED BY FTIR DIFFERENCE SPECTROSCOPY

Abstract— Deionization of the purple membrane of Halobacterium halobium shifts the visible absorption maximum from 570 to 605 nm and inhibits proton transport. FTIR-difference-spectra of this blue membrane at 280 K reveal that the retinal chromophore adopts a 13 -cis and all -trans geometry in a light dependent ratio. In contrast to purple membrane the 13-cu isomer forms much faster in the dark. The all- trans component produces an L-intermediate which can be stabilized at 170 K. Spectral characteristics are similar to normal L. including comparable changes of internal aspartic acids of the opsin. However, stronger changes in the amide-I absorption are observed. IR bands of the chromo-protein states are assigned to retinal normal modes by the use of bacteriorhodopsin regenerated with'C-labeled retinals.     

THE WHITE MEMBRANE OF CRYSTALLINE BACTERIOOPSIN IN HALOBACTERIUM HALOBIUM STRAIN R1mW AND ITS CONVERSION INTO PURPLE MEMBRANE BY EXOGENOUS RETINAL

Abstract— A new strain isolated from Halobacterium halobium designated R₁mW, contained negligible amounts of isoprenoid pigments, had a yellowish white color due to respiratory pigments and showed no proton movement in response to light. However, addition of all-trans-retinal converted R₁mW into purple cells. Formation of both halorhodopsin and bacteriorhodopsin was indicated by induction of light-dependent proton uptake and release, respectively. Both haloopsin and bacterioopsin were thus postulated to be present in R₁mW. Electron micrographs of freeze-fractured cytoplasmic membranes revealed patches in a hexagonal array of trimeric particles, comparable to the purple membrane structure. These white membrane patches were isolated by procedures similar to those for the purple membrane. The white membrane's buoyant density was about 1.18 g/m/, and its main component migrated on sodium dodecylsulfate polyacrylamide gels at the same rate as bacteriorhodopsin. The white membrane showed only a small absorption peak at ～410nm due to contaminating respiratory pigments and a strong absorption at around 275 nm and shorter wavelengths. The white membrane was thus considered to be mainly composed of bacterioopsin, which was readily converted into bacteriorhodopsin by an addition of all-trans-retinal. The absorption and CD spectra of the white membrane were measured before and after addition of retinal. The molar extinction coefficient of dark-adapted bacteriorhodopsin formed was determined to be 53000M^?1 cm^?1 at 560 nm from retinal binding studies. The CD spectrum of the white membrane was negligible in the visible region but showed several bands assigned to aromatic and backbone structures in the UV region. Retinal addition caused considerable changes in the spectrum, yielding the CD spectrum of crystalline purple membrane bacteriorhodopsin. The white membrane thus appears to be a preparation suitable for structure-function studies of bacteriorhodopsin.     

Photobleaching of bacteriorhodopsin solubilized with triton X-100

In the current studies, we examined the effects of hexagonal lattice formation with lipid membranes on the structural stability of native bacteriorhodopsin (bR). Denaturation kinetic measurements for bR solubilized with the mild nonionic detergent Triton X-100 (TX100) were performed in the dark and under illumination by visible light. The solubilized bR was stable in the dark over a wide concentration range of TX100 (1 to 200 mM). In purple membranes, a bilobed band was observed in visible circular dichroism spectra due to interactions between neighboring chromophores. At all concentrations of TX100, this was replaced by a single positive band. Upon illumination with visible light, TX100-solubilized bR clearly showed photobleaching to bacterioopsin. These experimental results suggest that photobleaching is due to a lack of intermolecular interactions inside the purple membrane lattice. Extensive kinetic measurements further revealed that the rate constant of photobleaching is strongly dependent on the detergent concentration, although the activation energy for photobleaching does not significantly change with the TX100 concentration. The mechanism of photobleaching for the solubilized bR is discussed with respect to detergent micelle properties.     

STRUCTURAL STUDIES OF BROWN MEMBRANE BY X-RAY DIFFRACTION, CIRCULAR DICHROISM AND ABSORPTION SPECTRA

Abstract— Time course of formation and crystallization of bacteriorhodopsin upon the addition of retinal to brown apo-membrane has been studied by X-ray diffraction, circular dichroism (CD) spectra and absorption spectra. The rate of formation of bacteriorhodopsin decreases markedly at low pH (4.8) and low temperature (5°C). Furthermore, the formation of bacteriorhodopsin does not proceed in fully dried membranes. The half-time of the increase of exciton CD band is about 70s at 17°C and pH 7.0, and is comparable to that of the formation of bacteriorhodopsins (∼48s). The crystallization of bacteriorhodopsin proceeds to a large extent within 30 min at pH 7.0 and 5°C.
The bilobed CD band of the brown holo-membrane attributed to exciton coupling of bacteriorhodopsin molecules becomes negligibly small at pH 4.8, even though X-ray diffraction pattern indicates the lattice structure to be similar to that of the native purple membrane.     

D96N mutant bacteriorhodopsin immobilized in sol-gel glass characterization

The D96N mutant form of bacteriorhodopsin (BR) purple membrane fragments isolated from the bacteriumHalobacterium salinarium has been immobilized by entrapment in sol-gel glass. The protein was characterized for M state decay rate at different temperatures and pH values. Bleaching efficiency and absorbance maxima vs pH were also determined. The kinetic effects of triethanolamine and diethanolamine were also examined. Results indicated that the immobilized BR was affected in a manner similar to the mutant BR in aqueous suspension. Addition of guanidine, however, caused the immobilized BR to show kinetic parameters more closely related to the wild-type protein than the D96N mutant control. Samples of the aqueous suspension were characterized for particle size and particle size distribution. Dried samples of the immobilized BR were analyzed by field emission microscopy and BET to characterize both the purple membrane fragments and the sol-gel pore characteristics.     

Thermochromism of bacteriorhodopsin and its pH dependence

Purple membranes (PMs), which consist of the photochromic membrane protein bacteriorhodopsin (BR) and lipids only, show complex thermochromic properties. Three different types of reversible temperature-dependent spectral transitions were found, involving spectral states absorbing at 460, 519, and 630 nm. These thermochromic absorption changes were analyzed in the range from 10 to 80 degrees C. In dependence on the bulk pH value, hypsochromic or bathochromic shifts in the BR absorption spectra are observed in BR gels as well as in BR films. The thermochromic changes between both purple and blue or purple and red were quantified in the CIE color system. The molecular changes causing these effects are discussed, and a model is presented in terms of intramolecular protonation equilibriums. The thermochromic properties of BR may be of interest in applications like security tags, as this feature may complement the well-known photochromic properties of BR.     

Photocycle of 13-cis-retinal in bacteriorhodopsin caused by destruction to the cooperative effect of purple membrane

The steady absorption and kinetic changes of M₄₁₂ intermediate of the light- and dark-adapted bacteriorhodopsin (BR) solubilized by different concentrated Triton X-100 were investigated. The results indicated that the cooperative effect existing within the trimeric BR of native purple membrane (PM) was damaged in the system containing the surfactant since the component and structure of the bilayer lipid membrane in PM varied due to the solubilization of partial PM lipids by Triton X-100. The destruction to the cooperative effect of BR ultimately caused 13-cis-retinal of the dark-adapted BR to take part in BR photocycle and also to generate the deprotonated M₄₁₂ intermediate. Project supported by the key and major projects from the Chinese Academy of Sciences (Grant Nos. Kj951-A1-501-05 and Kj952-S1-03)     

Optical properties of Triton X-100-treated purple membranes embedded in gelatin films

The purple membrane (PM) of the microorganism Halobacterium salinarium contains a hexagonally packed monolayer of the light-sensitive protein, bacteriorhodopsin (BR). The optical characteristics of gelatin-immobilized PMs depend strongly on the chemical environment of the PMs in the matrix. Here we present photoinduced absorptive and holographic characteristics of gelatin-embedded PMs solubilized with the non-ionic detergent, Triton X-100. The BR/detergent interaction was shown to slow the M-to-initial state transition of the photocycle and to increase the photosensitivity of the BR films. The lifetime of the holographic grating in Triton X-100-treated BR films was 2–3 times greater, when compared to the unmodified sample. Holographic grating growth times in BR films were shown to change depending on the extent of solubilization. The measured holographic sensitivity appeared to maximize in the range of Triton X-100/BR molar ratios from 15:1 to 25:1. The possible advantages of solubilized PM films as they are applied to optoelectronic devices are discussed.     

Redox potential of bacteriorhodopsin in oriented purple membrane films

The redox potentials of bacteriorhodopsin (BR) in oriented purple membrane (PM) films formed by the electrophoretic sedimentation (EPS) technique and the Langmuir–Blodgett (LB) technique were measured. The PM LB film and PM EPS film formed at 0.4 V versus SCE electrode potential exhibited two redox potentials at −0.48 and −0.76 V for the cis- and trans-isomers of the retinal Schiff base in the BR, respectively. The reduction current of BR in the PM LB film was greater than that in the PM EPS film and the reduction current of BR in the PM cast film was ca. 70% smaller values of the PM LB film. The results indicate that the electron transfer was affected by a difference in the orientation of PM patches in the films.     

The laser-induced blue state of bacteriorhodopsin: mechanistic and color regulatory roles of protein-protein interactions, protein-lipid interactions, and metal ions

In this paper we characterize the mechanistic roles of the crystalline purple membrane (PM) lattice, the earliest bacteriorhodopsin (BR) photocycle intermediates, and divalent cations in the conversion of PM to laser-induced blue membrane (LIBM; lambda(max)= 605 nm) upon irradiation with intense 532 nm pulses by contrasting the photoconversion of PM with that of monomeric BR solubilized in reduced Triton X-100 detergent. Monomeric BR forms a previously unreported colorless monomer photoproduct which lacks a chromophore band in the visible region but manifests a new band centered near 360 nm similar to the 360 nm band in LIBM. The 360 nm band in both LIBM and colorless monomer originates from a Schiff base-reduced retinyl chromophore which remains covalently linked to bacterioopsin. Both the PM-->LIBM and monomer-->colorless monomer photoconversions are mediated by similar biphotonic mechanisms, indicating that the photochemistry is localized within single BR monomers and is not influenced by BR-BR interactions. The excessively large two-photon absorptivities (> or =10(6) cm(4) s molecule(-1) photon(-1)) of these photoconversions, the temporal and spectral characteristics of pulses which generate LIBM in high yield, and an action spectrum for the PM-->LIBM photoconversion all indicate that the PM-->LIBM and Mon-->CMon photoconversions are both mediated by a sequential biphotonic mechanism in which is the intermediate which absorbs the second photon. The purple-->blue color change results from subsequent conformational perturbations of the PM lattice which induce the removal of Ca(2+) and Mg(2+) ions from the PM surface.     

Effect of external electric fields on membrane proteins: The bacteriorhodopsin

One of the possible mechanisms of the effect of external electric fields on biological systems is through their membrane proteins. At present, contradicting reports exist about the effect of external electric fields on the behaviour of the bacteriorhodopsin, the only protein molecule in the purple membrane fragments. Complex electro-optic studies (electric light scattering; electric dichroism and electric birefringence) and detailed investigations of the electric field strength dependence of the electro-optic effects on different fractions of purple membranes provide no evidence of alterations in the behaviour of bacteriorhodopsin. The effects observed are explained well by the orientations of the whole purple membrane fragments. The faster process detected in the electric light scattering decay curve could be attributed to changes in the form and/or the volume of purple membrane aggregates. The importance of these studies in understanding the dependence of the membrane properties on the variation of the transmembrane potential, which are essential for membrane pumping and channel behaviour, is discussed. Further possibilities of the application of the methods described in the study of other membrane proteins and other membrane problems are also discussed.     

Asymmetric distribution of biotin labeling on the purple membrane

This work examined the biotin modification of bacteriorhodopsin (BR) in the purple membrane (PM). The results of flash kinetic absorption measurements showed that photocycle was maintained in biotinylated BR. Biotinylated BR also maintained its photoelectric activity, as indicated by the photoelectric response of the bilayer lipid membrane (BLM). Atomic force microscopy (AFM) of stretavidiin-bound biotin revealed that biotin molecules covered both surfaces of the, but the amount of biotinylated BR on the extracellular (EC) surface was markedly higher than on the cytoplasmic (CP) surface. Further studies showed that, after reaction with fluorescamine (FL), biotin labeling occurred only on the CP surface. These results are informative for future work on bioconjugation of BR as well as work on oriented assembly and the design of BR-based photoelectric devices.     

pH-dependent transitions in xanthorhodopsin

Xanthorhodopsin (XR), the light-driven proton pump of the halophilic eubacterium Salinibacter ruber, exhibits substantial homology to bacteriorhodopsin (BR) of archaea and proteorhodopsin (PR) of marine bacteria, but unlike them contains a light-harvesting carotenoid antenna, salinixanthin, as well as retinal. We report here the pH-dependent properties of XR. The pKa of the retinal Schiff base is as high as in BR, i.e. > or =12.4. Deprotonation of the Schiff base and the ensuing alkaline denaturation cause large changes in the absorption bands of the carotenoid antenna, which lose intensity and become broader, making the spectrum similar to that of salinixanthin not bound to XR. A small redshift of the retinal chromophore band and increase of its extinction, as well as the pH-dependent amplitude of the M intermediate indicate that in detergent-solubilized XR the pKa of the Schiff base counterion and proton acceptor is about 6 (compared to 2.6 in BR, and 7.5 in PR). The protonation of the counterion is accompanied by a small blueshift of the carotenoid absorption bands. The pigment is stable in the dark upon acidification to pH 2. At pH < 2 a transition to a blueshifted species absorbing around 440 nm occurs, accompanied by loss of resolution of the carotenoid absorption bands. At pH < 3 illumination of XR with continuous light causes accumulation of long-lived photoproduct(s) with an absorption maximum around 400 nm. The photocycle of XR was examined between pH 4 and 10 in solubilized samples. The pH dependence of recovery of the initial state slows at both acid and alkaline pH, with pKas of 6.0 and 9.3. The decrease in the rates with pKa 6.0 is apparently caused by protonation of the counterion and proton acceptor, and that at high pH reflects the pKa of the internal proton donor, Glu94, at the times in the photocycle when this group equilibrates with the bulk.