New intermediates in the photochemical transformation of rhodopsin

The conditions of preferential accumulation of intermediates of the photochemical reaction cycle of bacteriorhodopsin (BR) P550 and P419 at low temperature are found. Upon illumination P550 and P419 undergo photochemical conversions into the light-adapted form of BR (P570), forming during this conversions a number of new intermediates: P550 leads to P560-- -- -- leads to P570; P419 leads to P421-- -- -- leads to P565-- -- -- leads to P585-- -- -- leads to P570; P419 leads to P470-- -- -- leads to P570. All intermediates are photoactive. All light reactions are photoreversible and give formation to the products with absorption maximum shifted to the red as compared to the initial state. The absorption spectra of intermediates are complex and include several bands which are more pronounced in the spectrum of P419 (maxima at 442, 419, 398 nm, a shoulder at 375 nm) and P421, less in the spectrum of P570 (maximum at 578 nm, shoulders at 540 and 608 nm) and others.     

Mechanism of Nonlinear Photoinduced Anisotropy in Bacteriorhodopsin and its Derivatives

Polymer films made with photosensitive chromophore protein bacteriorhodopsin (BR) from the extreme halophile Halobacterium salinarium as well as films made with BR derivatives exhibit a nonlinear photoinduced anisotropy. Two different methods can be used to induce anisotropy in polymer BR films. The first method is based on the anisotropic properties of the initial form of the photocycle, BR570 (B-type anisotropy). Another method is based on the anisotropic properties of the longest-lived photocycle intermediate M412 (M-type anisotropy). CW gas lasers were employed to induce a reversible anisotropy in polymer BR films. Nonlinear photoinduced anisotropy is discussed in the context of a model for the anisotropic photoselection of BR molecules under linearly polarized light. A comparison of the experimental dependencies of nonlinear photoinduced anisotropy on laser intensity with similar calculated dependencies enables one to determine the molecular dichroism of BR and its derivatives not only for the initial form of the photocycle, B but also for the longest-lived intermediate M. Here we present the data showing the correlation between the laser induced nonlinear anisotropic properties and chromophore/protein interactions in BR. The effect of polymer binder on the nonlinear photoanisotropic properties of polymer BR films is also described.     

EPR and low temperature spectrophotometric study of the phototransformation products of bacteriorhodopsin]

It is shown that BR and intermediate products of its phototransformation P600, P550 and P415 (the maximum at -196 degrees C at 419 nm) are not paramagnetic. Illumination of samples containing P415 (P419) at -- 196 degrees C with light in the region of 360-480 nm results in the formation of paramagnetic centres with a sunglet spectrum deltaH=18 Oe and g=2.002 (R1). In parallel formation of a new photoproduct P421 in the absorption spectrum is observed. During subsequent heating at -140 degrees C formation of an asymmetric signal with deltaH=45 Oe and g=2.006 and g=2.03 was observed. In the absorption spectra a dark transition. P421-P565 was observed under the same conditions. P565 differs from initial BR P570 as to its photochemical properties. R1 is identified as retinal radical, R2 as a peroxide radical of the BR-complex lipids. Paramagnetic, spectral, and photochemical properties of some products of BR transformation are compared. A scheme of oxidative-phosphorylation processes with participation of Mn ions in BR phototransformation.     

菌紫质D96N样品的光暗适应型及其转化机制

通过对菌紫质D96N基因突变形成的薄膜样品在光照下动态光谱的测定和分析,研究了此样品的光适应型、暗适应型特性及其之间的转化机制。实验证实,样品受光照激发后很快从暗适应型（D态）光适应型（B态）进入光循环,经过一系列的光化学中间体到边较稳定的M态。M态经过约1小时热驰豫完全回到光适应型B态,B态再经过24小时缓慢热驰豫完全回到暗适应型D态。B态受光则进入光循环,不受光则转化为D态,从而认为B态是菌紫质光循环路径中一个重要的分支节点。据此,提出了该样品在光照下B,D和M态之间转化的模型。     

A study on the fabrication of chemically modified bacteriorhodopsin film and its photochemical properties

Chemically modified bacteriorhodopsin (BR) films embedded in gelatin matrix were fabricated. It was found that chemically modified BR/gelatin film remained the original conformation of BR and possessed homogeneity. Then, the photochemical conversion from all-trans O state to 9-cis P state in the chemically modified BR film/gelatin was investigated. For comparison purpose, the same conversion in the wild-type BR (BRwt) film was also presented here. To our know, it is the first report to show that the absorption changes of the P state in the chemically modified BR film were larger than in the BRwt film upon illumination with red light at the low actinic power density.     

Transient photovoltages in purple membrane multilayers. Charge displacement in bacteriorhodopsin and its photointermediates

The photovoltaic properties of bacteriorhodopsin molecules and their photochemical intermediates have been investigated in an experimental cell consisting of multilayered films of highly oriented, dry fragments of purple membrane and lipid sandwiched between two metal (Pd) electrodes. The electrical time constant of these sandwich cells containing between 5 and 30 layers is less than 10(-5) S. Bright illumination of these cells with actinic flashes of approximately 1 ms duration generates transient photovoltages. These photovoltages, which make the extracellular surface of purple membrane positive with respect to the intracellular surface, follow the time course of the flash with no detectable latency. The amplitude of the photovoltages increases linearly with light intensity and their action spectrum matches the absorption spectrum of the light-adapted state of bacteriorhodopsin, BR570. In these dry multilayer cells, the slow photointermediates of bacteriorhodopsin, M412, N520 and O640 are long lived. Illumination of the sandwich cells with long duration (200 ms) pulses of light results, therefore, in the formation of photomixtures containing all these slow photointermediates. Flash illumination of the sandwich cells immediately following the conditioning pulse produces photovoltages whose action spectra match the absorption spectra of the M412 and N520 photointermediates. The M412 photovoltages, like the BR570 photovoltages, follow the time course of the actinic flash with no detectable latency and increase in amplitude linearly with light intensity. But, unlike the BR570 photovoltage, the M412, N520 and O640 photovoltages make the extracellular surface of purple membrane negative with respect to the intracellular surface. Through the of their specific photovoltaic signals, M412 and N520 are shown to be kinetically distinct photointermediates of bacteriorhodopsin. Detection of fast photovoltages with these characteristics in the absence of any ionic solution, and in parallel with spectrophotometric changes, suggest that they arise from charge displacements in the bacteriorhodopsin molecules and their photointermediates as they undergo photochemical conversion in response to the absorption of photons.     

Photochemical conversion of the O-intermediate to 9-cis-retinal-containing products in bacteriorhodopsin films.

The photochemical activity of the O-state was investigated in bacteriorhodopsin (BR) films containing wildtype BR at pH 6.5 in the presence of glycerol. The formation of a photoproduct of O with an absorption maximum at 490 nm and 9-cis-retinal configuration was found. This 490-nm product was named P and shows a slow thermal reaction into a compound with a maximal absorption at 380 nm which was named Q and contains free 9-cis-retinal in the proteins binding site. The photoproducts of O, i.e., P and Q, are very similar, or even identical, to those previously observed in blue membranes. Common to the O-state and blue membrane forms of bacteriorhodopsin is a protonated aspartic acid 85, and we suggest that it is the reduced negative charge around the Schiff base which is responsible for the 9-cis photoisomerization. The release of a proton from aspartic acid 85 is linked to the conversion of the O-state back to the initial state of BR. Therefore the conditions of low proton mobility in BR films containing glycerol favor the accumulation of the O-state. For optical and holographic applications such BR films are very attractive. It is possible to create photoproducts with red light which are thermally stable at room temperature and that can be photochemically erased. Dependent on the light composition both properties can be realized in the same sample material. This feature may bridge the gap between information processing and short-term and long-term storage of information with BR.     

Picosecond time-resolved fluorescence spectroscopy of K-590 in the bacteriorhodopsin photocycle.

The fluorescence spectrum of a distinct isometric and conformational intermediate formed on the 10(-11) s time scale during the bacteriorhodopsin (BR) photocycle is observed at room temperature using a two laser, pump-probe technique with picosecond time resolution. The BR photocycle is initiated by pulsed (8 ps) excitation at 565 nm, whereas the fluorescence is generated by 4-ps laser pulses at 590 nm. The unstructured fluorescence extends from 650 to 880 nm and appears in the same general spectral region as the fluorescence spectrum assigned to BR-570. The transient fluorescence spectrum can be distinguished from that assigned to BR-570 by a larger emission quantum yield (approximately twice that of BR-570) and by a maximum intensity near 731 nm (shifted 17 nm to higher energy from the maximum of the BR-570 fluorescence spectrum). The fluorescence spectrum of BR-570 only is measured with low energy, picosecond pulsed excitation at 590 nm and is in good agreement with recent data in the literature. The assignment of the transient fluorescence spectrum to the K-590 intermediate is based on its appearance at time delays longer than 40 ps. The K-590 fluorescence spectrum remains unchanged over the entire 40-100-ps interval. The relevance of these fluorescence data with respect to the molecular mechanism used to model the primary processes in the BR photocycle also is discussed.     

Absorption spectrum of allophycocyanin isolated from Anabaena cylindrica: variation of the absorption spectrum induced by changes of the physico-chemical environment

The absorption spectrum of allophycocyanin of Anabaena cylindrica was studied. The extinctions of the main absorption bands (650 and 620 nm) varied depending on the protein concentration, ionic strength, and pH. At higher protein concentrations or higher ionic strength, the 650 nm band became stronger and the 620 nm band became weaker. At pH values lower than 6.0, reverse changes occurred in association with protein dissociation into monomer. Similar spectral variation was also induced by sugars and polyols. Glucose, sucrose, or glycerol (1-5 M) induced an increase in the 650 nm band and a decrease in the 620 nm band without causing any changes in protein conformation. Propylene glycol and ethylene glycol showed a reverse effect and caused protein dissociation into monomer. The difference spectra of all spectral changes were identical, consisting of a sharp and strong peak at 650 nm and a broad and weak one in the reverse direction at a wavelength below 620 nm. The spectral variation probably results from shifts of the electronic state of phycocyanobilin. We postulated that a protein field favorable to the state producing the 650 nm band is established around phycocyanobilin when the protein takes a "tight state" through protein association or by the action of sugar in aqueous environment; in a "relaxed state" in the monomer, the state of phycocyanobilin similar to that in phycocyanin becomes dominant.     

Ultrafast infrared spectroscopy of bacteriorhodopsin.

Picosecond infrared spectroscopy is developed and used for the first time to study the dynamics of photoexcited bacteriorhodopsin (BR). Both spectral and time-resolved data are obtained. The results open an entirely new approach to investigations of the BR photocycle. The infrared difference spectrum (K minus BR570) recorded at ambient temperature between 1,560 and 1,700 cm-1 is not identical with the spectrum reported for a frozen sample. Three bands of the K state at 1,622, 1,610, and 1,580 cm-1 and the bleaching at 1,637 cm-1 (C = NH stretch) are seen. These new spectral lines appear in less than 10 ps.     

Reversible inhibition of proton release activity and the anesthetic-induced acid-base equilibrium between the 480 and 570 nm forms of bacteriorhodopsin.

In purple membrane added with general anesthetics, there exists an acid-base equilibrium between two spectral forms of the pigment: bR570 and bR480 (apparent pKa = 7.3). As the purple 570 nm bacteriorhodopsin is reversibly transformed into its red 480 nm form, the proton pumping capability of the pigment reversibly decreases, as indicated by transient proton release measurements and proton translocation action spectra of mixture of both spectral forms. It happens in spite of a complete photochemical activity in bR480 that is mostly characterized by fast deprotonation and slow reprotonation steps and which, under continuous illumination, bleaches with a yield comparable to that of bR570. This modified photochemical activity has a correlated specific photoelectrical counterpart: a faster proton extrusion current and a slower reprotonation current. The relative areas of all photocurrent phases are reduced in bR480, most likely because its photochemistry is accompanied by charge movements for shorter distances than in the native pigment, reflecting a reversible inhibition of the pumping activity.     

Bacteriorhodopsin wildtype and variant aspartate-96 → aspargine as reversible holographic media

Air dried films of purple membranes (PM) from Halobacterium halobium containing the photochromic protein bacteriorhodopsin (BR) were prepared and the BR-photocycle of this material analyzed. The absorption maxima of the initial state B (λ_max = 570 nm) and the photochemical intermediate M (λ_max = 412 nm), which is the longest living intermediate in suspension (τ ≈ 10 ms), were spectrally well separated. Light-induced population gratings between B and M were used for reversible holographic recording in these dry PM films. The resolution (>5,000 lines/mm) of PM films was comparable to the corresponding values of conventional photochromic recording materials. The longterm stability toward photochemical degradation of PM films is excellent (> 100.000 recording cycles). The spectral bandwidth (400-680 nm) of such films covers nearly the whole visible spectrum. Both the photochemical transition from B → M with wavelengths in the green-red range and from M → B with blue light were utilized for holographic recording. The latter possibility (M → B) seems to be advantageous for several applications because the holographic grating is only formed during reconstruction. Higher reading intensities lead to higher population of the M-state and result in an increase of the fringe contrast instead of decreasing it. New possibilities for the further development of holographic media based on bacteriorhodopsin are raised by the availability of PM variants with modified optical properties. By the use of the variant BR-326, which differs from the wildtype PM by a single amino acid exchange (aspartate-96 → asparagine), the sensitivity of PM films is increased by ~50% from 12 cm²/J to 19 cm²/J for recording with 568 nm. The sensitivity for recording with 413 nm (33 cm²/J) is not influenced by the amino acid exchange. The observed diffraction efficiency η of PM films with BR-326 is twice that of BR-wildtype (BR-WT) films and is in the range of conventional organic photochromics (≈ 1%). In dried films of both BR-WT and BR-326 the M-decay was shown to be at least biexponential.     

Kinetic isotope effects in the photochemical cycle of bacteriorhodopsin.

Kinetics were determined for the four transients K590, L540, M410, O660 of the photochemical cycle of bacteriorhodopsin (BR570) both in 1H2O and in 2H2O over a wide temperature range. Breaks in the Arrhenius plots, observed at 25 degrees-32 degrees for the longest-lived transients coincide with a transition point in the microviscosity of the membrane as measured by depolarization of an added fluorescent probe. The earliest isotope effect occurs in the decay of L540, and is present in the subsequent formation and decay of M410 and O660. Thus in the light-driven proton pump of BR570, proton ejection from the Schiff base correlates with decay of L540 and reprotonation occurs with the decay of both M410 and O660 back to BR570.     

Luminescence of bacteriorhodopsin in purple membranes from Halobacterium haolbium cells]

Red luminescence of purple membranes from Halobacterium halobium cells was found out, and its emission, excitation and polarization spectra were investigated. Simultaneous parallel measurements of absorption and luminescence changes in one sample brought about by the action of light were also carried out. The bands in the spectra can be attributed to a number of bacteriorhodpsin (BR) forms: BR(595,520), BR(650,575),BR(600-620), BR(700,625), BR(730,660) BR(780,695), where the number above is the position of the luminescence maxima, below--that of absorption. Proceding from the quantum yield of the luminescence (10(-3)) and of photoreaction (10(-1)) of BR, the photoisomerization rate constant of the latter was estimated (10(11) sec(-1). The temperature dependence of the luminescence quantum yield points to the existence of two or three quenching processes with different activation energies. BR phosphorescence was not observed in the region 500-1100 nm. High degree (36%) os luminescence polarization shows that there is no homogeneous energy transfer between BR molecules, or there is regularity in orientation of their dipoles. Energy migration from the bulk of carotenoids to BR was not found. However limited heterogeneous transfer between the different BR forms cannot be ruled out. The absence (or limitation) of migration indicated that there is a spatial separation of the chromophores. Data on possible participation of triplet states in the BR photoconversions are discussed.     

菌紫质稳态异构体光致变色反应动力学初探

实验证实,在适当的酸度调节下暗适应菌紫质（BR）的光致变色反应由B→蓝膜→P→Q→B的循环转换构成。在无光照下,B、Q态在中性介质中,蓝膜、P态在酸性介质中均呈高化学稳定性;蓝膜→P和Q→B的态转换须分别用650nm和400nm可见光激励,用紫外－可见光谱对两个光化学过程的动力学特性进行监测,证实它们均为一级反应。菌紫质的四个稳态在可见光区具有不同的特征吸收波长,在信息记录方面可望有一定应用前景。     

Photoelectric response of polarization sensitive bacteriorhodopsin films

Polarization sensitivity is introduced into oriented bacteriorhodopsin (BR) films through a photochemical bleaching process, which chemically modifies the structure of the purple membrane by breaking the intrinsic symmetry of the membrane-bound BR trimers. The resulting photovoltage generated in an indium-tin oxide (ITO)/BR/ITO detector is found to be anisotropic with respect to cross-polarized probe beams. A model, based on the polarization dependent photoselection of the BR molecules qualitatively explains the photochemical bleaching process and the observed anisotropic response. The effect reported here can be used to construct a polarization sensitive BR-based bio-photoreceiver.     

The N intermediate of bacteriorhodopsin at low temperatures: stabilization and photoconversion

In aqueous suspensions of purple membranes (pH 10.2, 0.4 M KCl) an intermediate having an absorption maximum at 570-575 nm (at -196 degrees C) was produced by first heating the M intermediate up to -30 degrees C and then stabilizing it by subsequent cooling to -60 degrees C. We suggest that this species is the intermediate N (or P or R) found and characterized earlier near room temperature. Upon illumination at -196 degrees C N is transformed into a bathochromically absorbing species KN which has an absorption maximum near 605 nm and an extinction 1.35 times that of N. This light reaction is photoreversible. The quantum yield ratio for the forward and back reaction is 0.18 +/- 0.02. The maximum photo steady state concentration of KN is about 0.24. The N intermediate was also trapped in water suspensions of purple membranes at neutral pH and low salt concentration by illumination at lambda greater than 620 nm during cooling. In addition to N another intermediate absorbing in the red (maximum at 610-620 nm) was accumulated in smaller amounts. It is not photoactive at -196 degrees C and apparently is the O intermediate or a photoproduct of N.     

Proton transfers in the photochemical reaction cycle of proteorhodopsin

The spectral and photochemical properties of proteorhodopsin (PR) were determined to compare its proton transport steps to those of bacteriorhodopsin (BR). Static and time-resolved measurements on wild-type PR and several mutants were done in the visible and infrared (FTIR and FT-Raman). Assignment of the observed C=O stretch bands indicated that Asp-97 and Glu-108 serve as the proton acceptor and donor, respectively, to the retinal Schiff base, as do the residues at corresponding positions in BR, but there are numerous spectral and kinetic differences between the two proteins. There is no detectable dark-adaptation in PR, and the chromophore contains nearly entirely all-trans retinal. Because the pK(a) of Asp-97 is relatively high (7.1), the proton-transporting photocycle is produced only at alkaline pH. It contains at least seven transient states with decay times in the range from 10 micros to 200 ms, but the analysis reveals only three distinct spectral forms. The first is a red-shifted K-like state. Proton release does not occur during the very slow (several milliseconds) rise of the second, M-like, intermediate, consistent with lack of the residues facilitating extracellular proton release in BR. Proton uptake from the bulk, presumably on the cytoplasmic side, takes place prior to release (tau approximately 2 ms), and coincident with reprotonation of the retinal Schiff base. The intermediate produced by this process contains 13-cis retinal as does the N state of BR, but its absorption maximum is red-shifted relative to PR (like the O state of BR). The decay of this N-like state is coupled to reisomerization of the retinal to all-trans, and produces a state that is O-like in its C-C stretch bands, but has an absorption maximum apparently close to that of unphotolyzed PR.     

Fluorescence spectral properties of troponin C mutant F22W with one-, two-, and three-photon excitation.

We report the first measurements of protein fluorescence with three-photon excitation, using a mutant of troponin C (TnC) that contains a single tryptophan residue F22W. From the emission intensity dependence on laser power we determine that TnC F22W displays one-, two-, and three-photon excitation at 285, 570, and 855 nm, respectively. The emission spectra and intensity decays are identical for one-, two-, or three-photon excitation. The steady-state and time 0 anisotropies are distinct for each mode of excitation, but the correlation times were the same, suggesting that three-photon excitation of proteins can be accomplished without significant effects of the locally intense illumination. The excitation anisotropy spectrum from 830 to 900 nm displays only negative values, suggesting dominant excitation via the 1Lb state of tryptophan from 830 to 900 nm.