Light-induced currents from oriented purple membrane: II. Proton and cation contributions to the photocurrent

The sign of B2, the micro-second component of the photocurrent from oriented purple membrane, is that of positive charge moving away from the purple membrane in the direction of proton release. B2 could be due to internal dipole or proton movement, proton release, or metal cation release. We found that the waveform of B2 is virtually insensitive to changes in the salt concentration as long as it is >40 mM KCl, >5 mM CaCl₂, or >0.5 mM LaCl₃. However, below these limits, B2's apparent rate of decay increases as the salt concentration decreases without any change in the initial amplitude. This salt dependence suggests that B2 is due to a positive charge, either a metal cation or a proton, moving from the membrane into the solution. That the positive charge is not a metal cation is suggested by the waveform of B2 remaining unchanged upon replacing the cations both in solution and in the binding sites of the purple membrane. Direct evidence that the positive charge movement is due to protons was obtained by examining the correlation of B2 with the proton dependent processes of bacteriorhodopsin in buffers and dyes. Based on these observations, we suggest that most, if not all, of the intrinsic B2 component of the photocurrent at moderate salt concentration is due to proton release.

The photocurrents from purple membranes whose surface potential has been reduced by delipidation or chemical modification of carboxyl groups with methyl esters were found to be only modestly changed. This suggests that the salt effect is not through its modulation of the surface potential. Rather, we propose that in low salt B2 represents the sum of a proton release from the surface of the purple membrane and a second current component, due to cations moving back towards the membrane, which is only important in low salt. The cation counter current is induced by proton release which creates a transient uncompensated negative charge on the membrane.

     

Effect of pH buffer molecules on the light-induced currents from oriented purple membrane

The effect of pH buffers on the microsecond photocurrent component, B2, of oriented purple membranes has been studied. We found that under low salt conditions (less than 10 mM monovalent cationic salt) pH buffers can dramatically alter the waveform of the B2 component. The effect is induced by the protonation process of the buffer molecules by protons expelled from the membrane. These effects can be classified according to the charge transition upon protonation of the buffer. Buffers that carry two positive charges in their protonated form add a negative current component (N component) to B2. Almost all of the other buffers add a positive current component (P component) to B2, which is essentially a mirror image of the N component. Buffers with a pK less than 5.5 have only a small positive buffer component. The pH dependence of the buffer effect is closely related to the pK of the buffer; it requires that the buffer be in its unprotonated form. The rise time of the buffer component increases with the concentration of the buffer molecules. All the buffer effects can be inhibited by the addition of 5 mM of a divalent cation such as Ca2+. Reducing the surface potential slows down the N component but accelerates the P component without affecting the amplitude of the buffer effect significantly. Many of the buffer effects can be explained if we assume that upon protonation of the buffer by a proton expelled from the membrane by light, the buffer molecules move toward the membrane. This backward movement of buffer molecules forms a counter current very similar to that due to cations discussed in Liu, S. Y., R. Govindjee, and T. G. Ebrey. (1990. Biophys. J. 57:951-963).     

Kinetics of the fast electric signal from oriented purple membrane

The photoinduced electric response of oriented purple membranes associated with processes before the K-intermediate decay of bacteriorhodopsin was measured in the 180-300 K temperature range. These response signals consist of two kinetically distinct components (both temperature dependent). The experimental data show a correlation between the time constants of the rise of the signal and solution resistance. A model is proposed to assign these components to two diffusion-limited processes of charge displacement in the solution. The displacement is caused by the electric field of the photoinduced transient dipole which is formed in the primary act of the bacteriorhodopsin photocycle. The two processes are assigned as: (a) the conduction of electrical current through H-bonds (time resolved only in the temperature range 180-200 K) and (b) the diffusion of charges through the interfacial layer.     

The effect of antibiotics on the photocycle and protoncycle of purple membrane suspensions.

The interrelation was studied between the phototransient absorbing maximally at 412 nm (M412) and light-induced proton release under steady-state conditions in aqueous suspensions of 'purple membrane' derived from Halobacterium halobium. The decay of M412 was slowed down by the simultaneous application of the ionophoric antibiotics valinomycin and beauvericin. The former had only slight activity alone and the latter was effective only in conjunction with valinomycin. The steady-state concentration of M412 which was formed on illumination was a direct function of the concentration of valinomycin. Maximum stabilization of M412 was obtained when the valinomycin was approximately equimolar with the bacteriorhodopsin. Addition of salts to the medium increased the number of protons released per molecule of M412 without affecting the level of M412 which was produced by continuous illumination. The effectiveness of the salts in this respect depended on the nature of the cation. Ca2+ and their antagonists La3+ and ruthenium red were found to have especially high affinity for the system. The extent of light-induced acidification could not be enhanced by increasing the pH of the medium from 6.5 to 7.8. The possible mechanism of action of the ionophores and of the cations on the photocycle and on the proton cycle is discussed.     

Structural transition of bacteriorhodopsin is preceded by deprotonation of Schiff base: microsecond time-resolved x-ray diffraction study of purple membrane

The structural changes in the photoreaction cycle of bacteriorhodopsin, a light-driven proton pump, was investigated at a resolution of 7 angstroms by a time-resolved x-ray diffraction experiment utilizing synchrotron x rays from an undulator of SPring-8. The x-ray diffraction measurement system, used in coupling with a pulsed YAG laser, enabled us to record a diffraction pattern from purple membrane film at a time-resolution of 6 micros over the time domain of 5 micros to 500 ms. In the time domain, the functionally most important M-intermediate appears. A series of time-resolved x-ray diffraction data after photo-excitation showed clear intensity changes caused by the conformational changes of helix G in the M-intermediate. The population of the reaction intermediate was prominently observed at approximately 5 ms after a photo-stimulus. In contrast, absorption measurement indicated the deprotonation of the Schiff base predominantly occurred at approximately 300 micros after a photo-stimulus. These results showed that the conformational changes characterizing structurally the M-intermediate predominantly occur at a later stage of the deprotonation of the Schiff base. Thus, the M-intermediate can be divided into two metastable stages with different physical characteristics.     

Effects of pressure and temperature on the M412 intermediate of the bacteriorhodopsin photocycle. Implications for the phase transition of the purple membrane

The effects of pressure and temperature on the decay kinetics of the M412 (M) intermediate in the photocycle of bacteriorhodopsin were studied to provide information about the phase transitions of the purple membrane lipids. The activation volume (delta V++) for the decay of M is expected to be different below and above a phase transition. However, no abrupt change in delta V++ was found from 3.5 degrees to 60 degrees C. But a sharp break was observed in a plot of the logarithm of the rate of M decay vs. pressure. Extrapolation of this break point to standard atmospheric pressure gives a temperature of -42 degrees C, which probably corresponds to the phase transition of the purple membrane lipids. This conclusion is supported by studies of the effect of pressure on the M kinetics of bacteriorhodopsin incorporated into dimyristoylphosphatidylcholine vesicles, whose phase transition has previously been characterized.     

Photocurrent measurements of the purple membrane oriented in a polyacrylamide gel.

When illuminated, oriented purple membranes isolated from Halobacterium halobium give a photoelectric effect. The frequency response of a photocurrent measuring system for purple membranes oriented and immobilized in a polyacrylamide gel is analyzed from DC to 100 MHz. The waveform of the photocurrent can depend on both the sample conditions (including bathing solution) and the measuring system (electrode and ammeter) at both the low and high frequency ends. In the DC-1 kHz range (millisecond signals), the apparent lifetime of the photocurrent component is distorted if the electrode is not platinized and if the conductivity of the bathing solution is not low. In the 1 kHz to 1 MHz range (microsecond signals), the frequency response is flat under most conditions. In the MHz range (nanosecond signals), the apparent lifetime of the photocurrent component will be distorted if the conductivity of the bathing solution is not high and if the input impedance of the ammeter is not low and constant throughout the frequency range. With our optimized apparatus, we could measure the photocurrent components from oriented purple membrane with lifetimes from 70 ms to 32 ns without distortion by the measuring system.     

Effects of the crystalline structure of purple membrane on the kinetics and energetics of the bacteriorhodopsin photocycle.

Time-resolved difference spectra were measured for Triton X-100 solubilized bacteriorhodopsin monomers between 100 ns and 100 ms after photoexcitation. The results are consistent with the general scheme K in equilibrium L in equilibrium M1 in equilibrium M2 in equilibrium N in equilibrium O----BR proposed previously for purple membranes [Váró, G., & Lanyi, J.K. (1990) Biochemistry 29, 2241-2250]. The rate constants which involve proton release or uptake, i.e., kLM1, kNO, and kON, were significantly higher in the monomeric protein than in purple membrane; the other steps were less affected. Analysis of the temperature dependencies of the rate constants between 5 and 30 degrees C yielded the enthalpies and entropies of activation for all steps except the two absent back-reactions. Comparison of these with data for purple membranes [Váró, G., & Lanyi, J.K. (1991) Biochemistry 30, 5016-5022] shows that the crystalline structure affects the energetics of the photocycle. In bacteriorhodopsin immobilized by the lattice of the purple membrane, the entropy changes leading to all transition states are more positive. Thus, the forward reactions proceed with less conformational hindrance. However, the thermal (enthalpic) barriers are higher. These effects are particularly pronounced for the M1----M2 and O----BR reactions. Large changes of the enthalpy and entropy levels of intermediates in the M2----BR reaction segment, but not in the K----M1 segment, upon solubilization of the protein are consistent with our earlier proposal that major protein conformational changes occur in the photocycle and they begin with the M1----M2 reaction.     

Interpretation of the absorption and circular dichroic spectra of oriented purple membrane films.

The absorption and circular dichroic (CD) spectra of purple membrane films in which the plane of the membranes is oriented perpendicular to the incident beam are compared with the solution spectra. This enables one to relate structural features of the purple membrane to a coordinate system as defined by a normal to the membrane plane and two mutually perpendicular in-plane axes. The film and solution absorption spectra were similar except for a relative depression in the 200 - 225-nm region of the film spectrum. However, the CD spectra showed significant differences in the visible region, where the biphasic band in the solution spectrum was replaced by a single positive band at 555 nm in the film spectrum and in the far ultraviolet region, where the 208-nm band was deleted from the film spectra of the native and regenerated membranes. Moreover, a small shoulder occurred at 208 nm in the film spectrum of the bleached membrane. The near ultraviolet spectra also showed differences, whereas the 317-nm band remained essentially the same for both spectra. Based on excitonic interpretations of the visible and far ultraviolet spectra the following conclusions were reached: (a) a relatively strong in-plane monomeric interaction occurs between te retinyl chromophore and apoprotein; (b) the helical axes of the native and regenerated membrane proteins are oriented primarily normal to the membrane plane; and (c) the helical axes of the bleached membrane proteins are tilted more in-plane than the axes of the native or regenerated membrane. Additional conclusions were that an interaction occurs between an in-plane magnetic dipole moment of the retinyl chromophore and probably an in-plane electric dipole moment of a nearby aromatic amino acid(s), and that although the membrane is anisotropic with respect to coupling between electric and magnetic moments of the aromatic amino acids, the transition dipole moments of the aromatic amino acids are not preferentially oriented in either direction.     

Reaction of the purple membrane with a carbodiimide.

Purple membrane was reacted with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at pH 4.5 and 8.0. At pH 4.5, the reaction yields cross-linked bacteriorhodopsin. The cross-linking is inhibited by pretreatment of the membrane with papain, or by the presence of carbohydrazide or glycine ethyl ester in the reaction mixture. The product of the pH 8.0 reaction is not cross-linked, but it displays altered properties. Two measures of photochemical activity (light-induced change in proton binding (delta h) and decay of photointermediate M) show changes indicative of slowed proton uptake. The delta h is increased by ethyl dimethylaminopropylcarbodiimide. This increase is unaffected by pretreatment of the membrane with papain, and it is not reversed by NH2OH. However, the reaction is inhibited by millimolar concentrations of CaCl2. The altered delta h is not apparent in detergent-solubilized membranes. Ethyl dimethylaminopropylcarbodiimide does not appear to cause a large alteration in the membrane surface charge, as measured by Ca2+ binding. We conclude that (1) at acid pH, ethyl dimethylaminopropylcarbodiimide can be used for cross-linking or for attachment of specific probes to the C-terminal region of bacteriorhodopsin, and hence to the cytoplasmic side of the purple membrane, and (2) at alkaline pH, ethyl dimethylaminopropylcarbodiimide reacts at a diffent type of site and appears to inhibit the proton pump.     

The fluorescence from the chromophore of the purple membrane protein.

The fluorescence from the purple membrane protein (PM) of Halobacterium halobium and its relation to the primary photochemical events have been studied. The emission spectrum at 77 degrees K has structure, with peaks at 680, 710-715, and 730-735 nm. The excitation spectrum shows a single peak centered at 580 nm. This and a comparison of the fluorescence intensity at 77 degrees K under a variety of conditions with the amounts of the bathoproduct (or K, the only photoproduct seen at this temperature) formed suggest that the source of the fluorescence is the purple membrane itself, not the photoproduct. From the difference in several of their properties, we suggest that the fluorescing state of the pigment is different from the excited state which leads to photoconversion.     

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.     

Light-induced FTIR difference spectroscopy of the S(2)-to-S(3) state transition of the oxygen-evolving complex in Photosystem II

We have applied flash-induced FTIR spectroscopy to study structural changes upon the S(2)-to-S(3) state transition of the oxygen-evolving complex (OEC) in Photosystem II (PSII). We found that several modes in the difference IR spectrum are associated with bond rearrangements induced by the second laser flash. Most of these IR modes are absent in spectra of S(2)/S(1), of the acceptor-side non-heme ion, of Yradical(D)/Y(D) and of S(3)'/S(2)' from Ca-depleted PSII preparations. Our results suggest that these IR modes most likely originate from structural changes in the oxygen-evolving complex itself upon the S(2)-to-S(3) state transition in PSII.     

Signal-induced degradation of I(kappa)B(alpha): association with NF-kappaB and the PEST sequence in I(kappa)B(alpha) are not required.

Signal-induced degradation of I(kappa)B(alpha) via the ubiquitin-proteasome pathway requires phosphorylation on residues serine 32 and serine 36 followed by ubiquitination on lysines 21 and 22. We investigated the role of other regions of I(kappa)B(alpha) which may be involved in its degradation. Here we report that the carboxy-terminal PEST sequence is not required for I(kappa)B(alpha) signal-induced degradation. However, removal of the PEST sequence stabilizes free I(kappa)B(alpha) in unstimulated cells. We further report that a PEST deletion mutant does not associate well with NF-(kappa)B proteins but is degraded in response to signal. Therefore, we conclude that both association with NF-(kappa)B and a PEST sequence are not required for signal-induced I(kappa)B(alpha) degradation. Additionally, the PEST sequence may be required for constitutive turnover of free I(kappa)B(alpha).     

复苏植物旋蒴苣苔早期光诱导蛋白的特征分析

随着全球气候变暖,旱胁迫越来越成为粮食生产的严重威胁。探索复苏植物耐受极端旱胁迫的机理,积累抗旱相关基因资源,对于植物适应逆境的基础研究,和农业可持续发展,均有重要的作用。旋蒴苣苔(B. hygrometrica)广泛分布于我国山区,是复苏植物的代表性物种,而且有高质量的全基因组序列可以分析利用。本研究对旋蒴苣苔(B. hygrometrica)基因组中的早期光诱导蛋白(ELIPs)基因家族进行挖掘和分析。旋蒴苣苔(B. hygrometrica)基因组中早期光诱导蛋白(ELIPs)基因的数目远高于其他植物(拟南芥,水稻,辣椒,番茄)。系统发育分析显示,早期光诱导蛋白(ELIPs)基因序列具有很高的植物特异性。这说明,早期光诱导蛋白(ELIPs)基因对不同植物进化和适应各自的生长环境具有重要的作用。对处于不同程度干旱状态的旋蒴苣苔(B. hygrometrica)叶片的基因表达分析,表明一部分早期光诱导蛋白(ELIPs)基因表现出很强的对旱处理的响应。不同的早期光诱导蛋白(ELIPs)基因表现出三种不同的响应模式。这些结果表明,旋蒴苣苔(B. hygrometrica)早期光诱导蛋白(ELIPs)基因参与植株应对旱胁迫,而且有精细的调控机制控制不同早期光诱导蛋白(ELIPs)基因的表达量。     

PsaC subunit of photosystem I is oriented with iron-sulfur cluster F(B) as the immediate electron donor to ferredoxin and flavodoxin.

The PsaC subunit of photosystem I (PS I) binds two [4Fe-4S] clusters, F(A) and F(B), functioning as electron carriers between F(X) and soluble ferredoxin. To resolve the issue whether F(A) or F(B) is proximal to F(X), we used single-turnover flashes to promote step-by-step electron transfer between electron carriers in control (both F(A) and F(B) present) and HgCl2-treated (F(B)-less) PS I complexes from Synechococcus sp. PCC 6301 and analyzed the kinetics of P700+ reduction by monitoring the absorbance changes at 832 nm in the presence of a fast electron donor (phenazine methosulfate (PMS)). In control PS I complexes exogenously added ferredoxin, or flavodoxin could be photoreduced on each flash, thus allowing P700+ to be reduced from PMS. In F(B)-less complexes, both in the presence and in the absence of ferredoxin or flavodoxin, P700+ was reduced from PMS only on the first flash and was reduced from F(X)- on the following flashes, indicating lack of electron transfer to ferredoxin or flavodoxin. In the F(B)-less complexes, a normal level of P700 photooxidation was detected accompanied by a high yield of charge recombination between P700+ and F(A)- in the presence of a slow donor, 2,6-dichlorophenol-indophenol. This recombination remained the only pathway of F(A)- reoxidation in the presence of added ferredoxin, consistent with the lack of forward electron transfer. F(A)- could be reoxidized by methyl viologen in F(B)-less PS I complexes, although at a concentration two orders of magnitude higher than is required in wild-type PS I complexes, thus implying the presence of a diffusion barrier. The inhibition of electron transfer to ferredoxin and flavodoxin was completely reversed after reconstituting the F(B) cluster. Using rate versus distance estimates for electron transfer rates from F(X) to ferredoxin for two possible orientations of PsaC, we conclude that the kinetic data are best compatible with PsaC being oriented with F(A) as the cluster proximal to F(X) and F(B) as the distal cluster that donates electrons to ferredoxin.     

An outer membrane protein A (ompA) homologue from the photosynthetic purple sulfur bacterium Allochromatium vinosum

A 991 bp DNA fragment, consisting of a 225 amino acid reading frame homologous to outer membrane protein coding ompA gene, was cloned from a purple sulfur bacterium Allochromatium vinosum. The homology analysis revealed up to 51% similarity to other bacterial species. The absence of branching within diazotrophs or other taxonomically related groups shows the structural importance of the protein regardless of the metabolism and evolution of the species.     

The structure of the purple membrane from Halobacterium hallobium: analysis of the X-ray diffraction pattern.

An X-ray diffraction analysis of oriented specimens of the purple membrane from Halobacterium halobium shows that the protein and lipid components are packed in a P3 hexagonal lattice, with one protein molecule per asymmetric unit. The structure is made up of a single layer of the protein molecules, oriented vectorially in the same direction across the membrane.The presence of strong diffraction peaks equatorially centred at 10 Å, and axially at 5 Å and 1.5 Å, show that the protein molecules, which make up most of the mass of the membrane, are composed to a considerable extent of α-helices, 25 to 35 Å long, arranged roughly perpendicular to the plane of the membrane to form superhelical groupings of the “coiled-coil” type.The surface of the membrane is flat, with no bumps or dimples large enough to affect the X-ray pattern when the electron density of the suspending medium is altered. The phospholipids may be less exactly positioned in the lattice than the protein, since the presence of uranyl acetate, which is expected to co-ordinate with the acidic phosphate groups, produces intensity changes only at low resolution.     

Heparan sulfate proteoglycan from human tubular basement membrane. Comparison with this component from the glomerular basement membrane

Heparan sulfate proteoglycan (HSPG) was extracted from human tubular basement membrane (TBM) with guanidine and purified by ion-exchange chromatography and gel filtration. The glycoconjugate was sensitive to heparitinase and resistant to chondroitinase ABC, had an apparent molecular mass of 200-400 kDa and consisted of 70% protein and 30% glycosaminoglycan. The amino acid composition was characterized by its high content of glycine, proline, alanine and glutamic acid. Hydrolysis with trifluoromethanesulfonic acid yielded core proteins of 160 and 110 kDa. The heparan sulfate (HS) chains obtained after alkaline NaBH4 treatment had a molecular mass of about 18 kDa. Results of heparitinase digestion and HNO2 treatment suggest a clustering of sulfate groups in the distal portion of the HS side chains. These chemical data are comparable to those obtained previously on glomerular basement membrane (GBM) HSPG (Van den Heuvel et al. (1989) Biochem. J. 264, 457-465). Peptide patterns obtained after trypsin, clostripain or V8 protease digestion of TBM and GBM HSPG preparations showed a large similarity. Polyclonal antisera and a panel of monoclonal antibodies raised against both HSPG preparations and directed against the core protein showed complete cross-reactivity in ELISA and on Western blots. They stained all basement membranes in an intense linear fashion in indirect immunofluorescence studies on human kidneys. Based on these biochemical and immunological data we conclude that HSPGs from human GBM and TBM are identical, or at least very closely related, proteins.