Electric birefringence study of the purple membrane of Halobacterium halobium

An electric birefringence study was carried out on aqueous suspensions of the purple membrane of Halobacterium halobium. In addition to the characterization of both native and modified membrane samples, the dependence of electric birefringence on pH and ionic strength was also investigated. The results indicate that purple membrane shows electric birefringence at a field strength as low as 200 V/cm. The permanent dipole moment and polarizability ranged from 20,500 debyes and 1.01 × 10^?14 cm³ for a purple membrane concentration of 0.40 mg/mL to 41,000 debyes and 2.05 × 10^?14 cm³ for a concentration of 0.80 mg/mL. It was also found that removal of the retinyl group of bacteriorhodopsin substantially decreases but does not eliminate the electric birefringence of the membrane. The solubilization of the membrane by Triton X-100, however, completely abolishes the electric birefringence. These experiments indicate that there is an interaction between adjacent bacteriorhodopsin molecules within the purple membrane via the retinyl chromophore moiety that builds up the permanent dipole moment. They also suggest that there are two types of response when purple membrane suspensions are placed in an electric field. One is an alignment of the disk-shaped particles with the field. The other is a stacking of the particles following their alignment by the electric field, which is promoted by the induced dipole moment.     

Orientation of membrane fragments by electric field

Purple membrane fragments from Halobacterium halobium were oriented by a static electric field in a water suspension. It was found that an electric field of approx. 20 V/cm is sufficient to achieve practically complete orientation; the purple membranes have a permanent electric dipole moment of (6 ±1)· 10^?23 C · m, the orientation of the retinal transition moment relative to the direction of the electric dipole moment, θ, is (59 ± 1)⁰, and the purple membrane rotational diffusion constant D_rot = 0.65 s^?1. It was found that because of the electrophoretic movement of the particles a hydrodynamic velocity gradient builds up which also orients the purple membranes.     

Electric field induced conformational changes of bacteriorhodopsin in purple membrane films

Electric field-induced absorption changes of bacteriorhodopsin were studied with different samples of purple membranes which were prepared as randomly oriented and electrically oriented films of purple as well as cation-depleted blue bacteriorhodopsin. The absorption changes were proportional to the square of the field strength up to 300 kV/cm. The electric field from the intracellular side to the extracellular side of the purple bacteriorhodopsin induces a spectrum change, resulting in a spectrum similar to that of the cation-depleted blue bacteriorhodopsin. When the field was removed, the purple state was regenerated. The blue state was mainly affected by an electric field in the opposite direction, suggesting a reversible interaction with the Schiff's base bond of the retinal. Since the field-induced reaction of bacteriorhodopsin was observed in the presence of a concomitant steady ion flux, it is assumed that the generation of a local diffusion potential may play an important role in these spectral reactions. Although the fragments were fixed in the dried film, electric dichroism was observed. The dichroic contribution of the total absorbance change was about 15%. The angular displacement of the retinal transition moment was calculated to be 1.5° toward the membrane normal.     

紫膜碎片的电二色性研究

悬浮在水中的嗜盐菌紫膜碎片,在外电场作用下产主定向排列.在20℃时,568nm的电二色性研究表明:外加电场为2kV/m时取向程度可达60%以上;大于5.5kV/m时,取向作用趋于饱和状态;饱和时简约电二色性为-0.437左右,视黄醛生色团的跃迁矩方向与电偶极矩方向形成60.9°夹角;紫膜的永久偶极短为9.2×10~(-24)C、M,剩余电极化率为3.0×10~(-27)m~2;紫膜的旋转扩散常数为0.53秒~(-1).曲线拟合分析表明,感应偶极对紫膜碎片的定向的贡献应予考虑.本文对紫膜碎片的定向机理进行了讨论.     

Electric field induced conformational changes of bacteriorhodopsin in purple membrane films

Electric field induced conformational changes of bacteriorhodopsin were studied in six types of dried film (randomly and electrically oriented membranes of purple as well as cation-depleted blue bacteriorhodopsin) by measuring the frequency dependence of the optical absorbance change and the dielectric dispersion and absorption. For the purple bacteriorhodopsin the optical absorbance change induced by alternating rectangular electric fields of ±300 kV/cm altered the sign twice in the frequency range from 0.001 Hz to 100 kHz (around 0.03 Hz and 100 kHz), indicating that the electric field induced conformational change in these samples consists of, at least, three steps. Similarly, it was found for the blue bacteriorhodopsin that at least two steps are involved. In accord with optical measurements, the dielectric behaviour due to alternating sinusoidal electric fields of±6kV/cm in the frequency range from 10 Hz to 10 MHz showed two broad dispersion/absorption regions, one below 1 kHz and the other around 10–100 kHz. This suggests that the conformational change of bacteriorhodopsin is also reflected by its dielectrical properties and that it is partially induced at 6 kV/cm. Including previous results obtained by analysis of the action of DC fields on purple membrane films, a model for a field-induced cyclic reaction for purple as well as blue bacteriorhodopsin is proposed. In addition it was found that there are electrical interactions among purple membrane fragments in dried films.     

Surface Charge Movements of Purple Membrane During Light-Dark Adaptation

The difference in the surface charge distribution between light-adapted and dark-adapted purple membranes was investigated with electric dichroism measurements from approximately pH 5 to pH 11. Purple membrane sheets in solution are oriented in a weak electric field by their permanent dipole moment, which is due to the charge distribution of the membrane surfaces and/or within the membrane. The degree of orientation of purple membrane sheets was obtained from the measurement of “electrical anisotropy” of retinal chromophore in the membranes. At about pH 7, there was no difference in the “electric anisotropy” between light- and dark-adapted purple membranes. At about pH 9, the electric anisotropy of dark-adapted purple membrane was larger than that of light-adapted purple membrane. But at around pH 6 the difference was opposite. Linear dichroism experiments did not show any change of retinal tilt angle with respect to the membrane normal between the two forms from approximately pH 5 to pH 10. This result indicates that the changes in the “electric anisotropy” are not due to the change of retinal tilt angle, but due to the change in the permanent dipole moment of the membrane. To estimate the change in surface charges from the permanent dipole moment, we investigated the difference of the permanent dipole moment between the native purple membrane and papain-treated purple membrane in which negative charges in the cytoplasmic-terminal part are removed. This estimation suggests that this light-dark difference at around pH 9 can be accounted for by a change of ~0.5 electric charge per bacteriorhodopsin (bR) molecule at either of the two surfaces of the membrane. We also found from pH electrode measurements that at about pH 8 or 9 light adaptation was accompanied by an uptake of ~0.1 protons per bR. A possible movement of protons during light-dark adaptation is discussed. The direction of the permanent dipole moment does not change with papain treatment. The permanent dipole moment in papain-treated purple membrane is estimated to be 27 ±2 debye/bR.     

Electric field effects in bacteriorhodopsin.

Exposure of aqueous suspensions of fragments of the purple membrane of Halobacterium halobium to electric field pulses leads to transient linear dichroism phenomena. The effects are interpreted in terms of field-induced alignments of the bacteriorhodopsin chromophore. Two observed relaxation times (tau) are attributed to rotation of the whole membrane fragments (tau s approximately 100 ms), and to a much faster reorientation of the chromophore within membrane (tau f approximately 260 microns).     

Conformational flexibility of membrane proteins in electric fields. I. Ultraviolet absorbance and light scattering of bacteriorhodopsin in purple membranes

Bacteriorhodopsin of halobacterial purple membranes exhibits conformational flexibility in high electric field pulses (1-30 x 10(5) V m(-1), 1-100 micros). High-field electric dichroism data of purple membrane suspensions indicate two kinetically different structural transitions within the protein; involving a rapid (approximately 1 micros) concerted change in the orientation of both retinal and tyrosine and/or tryptophan side chains concomitant with alterations in the local protein environment of these chromophores. as well as slower changes (approximately 100 micros) of the microenvironment of aromatic amino acid residues concomitant with pK changes in at least two types of proton-binding sites. Light scattering data are consistent with the maintenance of the random distribution of the membrane discs within the short duration of the applied electric fields. The kinetics of the electro-optic signals and the steep dependence of the relaxation amplitudes on the electric field strength suggest a saturable induced-dipole mechanism and a rather large reaction dipole moment of 1.1 x 10(-25) C m ( = 3.3 x 10(4) debye) per cooperative unit at E = 1.3 x 10(5) V m(-1), which is indicative of appreciable cooperativity in the probably unidirectional transversal displacement of ionic groups on the surfaces of and within the bacteriorhodopsin proteins of the membrane lattice. The electro-optic data of bacteriorhodopsin are suggestive of a possibly general, induced-dipole mechanism for electric field-dependent structural changes in membrane transport proteins such as the gating proteins in excitable membranes or the ATP synthetases.     

The interaction of water molecules with purple membrane suspension using 2H double-quantum filter, 1H and 2H diffusion nuclear magnetic resonance

Bacteriorhodopsin is a membrane protein of the purple membrane (PM) of Halobacterium salinarum, which is isolated as sheets of highly organized two-dimensional hexagonal microcrystals and for which water molecules play a crucial role that affects its function as a proton pump. In this paper we used single- and double-quantum (2)H NMR as well as (1)H and (2)H diffusion NMR to characterize the interaction of water molecules with the PM in D(2)O suspensions. We found that, under the influence of a strong magnetic field on a concentrated PM sample (0.61 mM), the PM sheets affect the entire water population and a residual quadrupolar splitting (upsilon(q) approximately 5.5 Hz, 298 K, at 11.7 T) is observed for the D(2)O molecules. We found that the residual quadrupolar coupling, the creation time in which a maximal DQF signal was obtained (tau(max)), and the relative intensity of the (2)H DQF spectrum of the water molecules in the PM samples (referred to herein as NMR order parameters) are very sensitive to temperature, dilution, and chemical modifications of the PM. In concentrated PM samples in D(2)O, these NMR parameters seem to reflect the relative organization of the PM. Interestingly, we have observed that some of these parameters are sensitive to the efficiency of the trimer packing, as concluded from the apo-membrane behavior. The data for dionized blue membrane, partially delipidated sample, and detergent-treated PM show that these D(2)O NMR order parameters, which are magnetic field dependent, are sensitive to the structural integrity of the PM. In addition, we revealed that heating the PM sample inside or outside the NMR magnet has, after cooling, a different effect on the NMR characteristics of the water molecules in the concentrated PM suspensions. The difference in the D(2)O NMR order parameters for the PM samples, which were heated and cooled in the presence and in the absence of a strong magnetic field, corroborates the conclusions that the above D(2)O order parameters are indirect reflections of both microscopic and macroscopic order of the PM samples. In addition, (1)H NMR diffusion measurements showed that at least three distinct water populations could be identified, based on their diffusion coefficients. These water populations seem to correlate with different water populations previously reported for the PM system.     

Electric dichroism in the purple membrane of Halobacterium halobium.

Aqueous suspensions of fragments of the purple membrane of Halobacterium halobium are exposed to short electric field pulses. The relaxation kinetics of the induced dichroism are studied as a function of environmental factors such as temperature, medium viscosity, and treatment of the membranes with glutaraldehyde and dimethylsulfoxide. The data indicate that the alignment of the retinyl chromophore is due to orientation of the whole membrane fragments with their planes parallel to the electric field, as well as to an intramembrane orientation of bacteriorhodopsin molecules (or of a part of such molecules). Wavelength effects on the dichroic ratio show that weak, out of (membrane) plane components contribute to the chromophore spectrum on the red side (lambda greater than 560 nm) of the main (alpha) absorption band as well as the range of the beta band (lambda less than 480 nm). The former effect is attributed to exciton interactions, while the latter is assigned to the contribution of a transition to the lowest 1Ag+ state ("cis" band). It is also concluded that the transition moment along the short (kappa) axis, in the plane of the polyene molecule, has a substantial component perpendicular to the membrane plane.     

Electrooptical studies on proton-binding and -release of bacteriorhodopsin

Electric field induced pH changes of purple membrane suspensions were investigated in the pH range from 4.1 to 7.6 by measuring the absorbance change of pH indicators. In connection with the photocycle and proton pump ability, three different states of bacteriorhodopsin were used: (1) the native purple bacteriorhodopsin (magnesium and calcium ions are bound, the M intermediate exists in the photocycle and protons are pumped), (2) the cation-depleted blue bacteriorhodopsin (no M intermediate), and (3) the regenerated purple bacteriorhodopsin which is produced either by raising the pH or by adding magnesium ions (the M intermediate exists). In the native purple bacteriorhodopsin there are, at least, two types of proton binding sites: one releases protons and the other takes up protons in the presence of the electric field. On the other hand, blue bacteriorhodopsin and the regenerated purple bacteriorhodopsin (pH increase) show neither proton release nor proton uptake. When magnesium ions are added to the suspensions; the field-induced pH change is observed again. Thus, the stability of proton binding depends strongly on the state of bacteriorhodopsin and differences in proton binding are likely to be related to differences in proton pump activity. Furthermore, it is suggested that the appearance of the M intermediate and proton pumping are not necessarily related.     

Purple-to-blue transition of bacteriorhodopsin in a neutral lipid environment.

The red shift in the absorption maximum of native purple membrane suspensions caused by deionization is missing in lipid-depleted purple membrane, and the pK of the acid-induced transition is down-shifted to pH approximately 1.4 and has become independent of cation concentration (Szundi, I., and W. Stoeckenius. 1987. Proc. Natl. Acad. Sci. USA. 84:3681-3684). However, the proton pumping function cannot be demonstrated in these membranes. When native acidic lipids of purple membrane are exchanged for egg phosphatidylcholine or digalactosyldiglyceride, bacteriorhodopsin is functionally active in the modified membrane. It shows spectral shifts upon light-dark adaptation, a photocycle with M-intermediate and complex decay kinetics; when reconstituted into vesicles with the same neutral lipids, it pumps protons. Unlike native purple membrane, lipid-substituted modified membranes do not show a shift of the absorption maximum to longer wavelength upon deionization. A partial shift can be induced by titration with HCl; it has a pK near 1.5 and no significant salt dependence. Titration with HNO3 and H2SO4, which causes a complete transition in the lipid-depleted membranes, i.e., it changes their colors from purple to blue, does not cause the complete transition in the lipid-substituted preparations. These results show that the purple color of bacteriorhodopsin is independent of cations and their role in the purple-to-blue transition of native membranes is indirect. The purple and blue colors of bacteriorhodopsin are interpreted as two conformational states of the protein, rather than different protonation states of a counterion to the protonated Schiff base.     

Fabrication and photoelectric response of poly(allylamine hydrochloride)/PM thin films by layer-by-layer deposition technique

Thin films of poly(allylamine hydrochloride) (PAH) and bacteriorhodopsin (bR) embedded in purple membrane (PM) have been prepared by layer-by-layer (LBL) self-assembly technique. The results obtained by UV-Vis spectroscopy and atomic force microscopy (AFM) analysis showed that the biological activity of bR was preserved and PM fragments could be well oriented onto the ITO substrate. A photo-electrochemical cell with the structure of ITO/(PAH/PM)(n)/electrolyte (0.5M KCl)/Pt was fabricated and studied. The photocurrent peaks of (PAH/PM)(6) corresponding to light-on and light-off were about 200 and 100 nA/cm(2), respectively, with the former enhanced 30% higher than that of the reference films made of (PDAC/PM)(6).     

紫膜LB膜的结构特性研究

研究了紫膜LB膜中的紫膜碎片的结构特性。扫描电子显微镜观察表明,紫膜LB膜中单个紫膜碎片的直径大约为0.3微米。表面轮廓测量仪(简称台阶仪)观察到紫膜LB膜中的紫膜碎片的厚度为40—50。在不同的表面压和不同紫膜含量时测量了紫膜碎片在紫膜LB膜中的形态学分布,当表面压为30mN/m或紫膜与大豆磷脂的重量比大于20:1时,紫膜碎片容易重叠或凝聚。     

Electrooptical analysis of blue and of cation-regenerated bacteriorhodopsin

Blue bacteriorhodopsin was prepared by electrodialysis, cation-exchange chromatography and acidification. The electrooptical properties of these preparations compared to those of the native purple bacteriorhodopsin suggest that the blue bacteriorhodopsin has a smaller induced dipole moment than the native purple bacteriorhodopsin and that bound cations in the native bacteriorhodopsin stabilize the protein conformation in the membrane.Purple bacteriorhodopsin was regenerated by addition of potassium, magnesium or ferric ions to blue bacteriorhodopsin. Both spectrscopically and electrooptically the potassium- and ferric-regenerated samples are different from the native purple state. Although the magnesium-regenerated sample is spectroscopically similar to the native purple bacteriorhodopsin, the electrooptical properties are rather similar to those of the cation-depleted blue sample, suggesting that it is very difficult to re-stabilize protein structures once cations are depleted.     

Structural and spectroscopic characteristics of bacteriorhodopsin in air-water interface films

A suspension of purple membrane fragments in a solution of soya phosphatidyl-choline in hexane is spread at an air-water interface. Surface pressure and surface potential measurements indicate that the membrane fragments and lipids organize at the interface as an insoluble film. Electron microscopy of shadow-cast replicas of the film reveal that in the bacteriorhodopsin to soya PC weight ratio range of 2:1 to 10:1, these films consist of nonoverlapping membrane fragments which occupy approximately 35% of the surface area and are separated by a lipid monolayer. Furthermore, the membrane fragments are oriented with their intracellular surface towards the aqueous subphase. Nearly all the bacteriorhodopsin molecules at the interface are spectroscopically intact and exhibit visible spectral characteristics identical to those in aqueous suspensions of purple membrane and in intact bacteria. In addition, bacteriorhodopsin in air-dried interface films show spectral changes upon dark-adaptation and upon flash illumination similar to those observed in aqueous suspensions of purple membrane, but with slower kinetics. The kinetics of the spectral changes in interface films can be made nearly the same as in aqueous suspension by immersing the films in water.     

紫膜的紫外荧光及分子内的能量转移

以可见光为作用光照射天然紫膜,紫膜蛋白被280nm紫外光激发所发射的荧光强度比对照略有降低.比较天然紫膜、漂白紫膜与菌蛋白三者的紫外荧光强度,前两者无显著变化,但菌蛋白的荧光强度比天然紫膜的荧光强度大2-3倍,表明生色团对蛋白质荧光可能有猝灭作用.用280nm波长光照射紫膜的暗适应形式,可使其转变成光适应形式.若有羟胺存在,以紫外光照射也可使紫膜漂白.光漂白的作用光谱,其紫外部分与紫膜蛋白部分的吸收光谱重合得很好.上述实验证明紫膜蛋白部分吸收的能量可以转移到生色团上,即紫膜存在分子内的能量转移     

Anisotropic electric properties of purple membrane and their change during the photoreaction cycle

Purple membrane suspension shows two different orientations in electric fields of different frequencies. The orientation at low frequencies (less than or equal to approximately 10 Hz), with the membrane surface perpendicular to the electric field, is due to permanent dipole moment of the membrane and the orientation at high frequencies (greater than or equal to approximately 100 Hz), with the surface parallel to the electric field, is due to induced dipole moment. By quantitative analysis of these orientations, we determined the permanent dipole moment and the polarizability. Both values varied according to the membrane size: the permanent dipole moment ranged from 500 kD to 10 MD and was proportional to the square of the diameter of the membrane. The polarizability ranged from 1 X 10(-13) to 1 X 10(-11)cm3 and was proportional to the third to fourth power of the diameter. Because the permanent dipole moment was proportional to the area of the membrane, we could determine permanent dipole moment per bacteriorhodopsin. By determining the actual membrane size under electron microscopy, we got 98 D/bacteriorhodopsin. We also concluded that the direction of the permanent dipole moment was from the cytoplasmic to the extracellular side. These values, however, were strongly dependent on the ionic strength in the medium, suggesting a screening effect due to counter ions near the membrane surface. We evaluated the screening effect and showed about a four-charge difference between the two sides of the purple membrane. Under illumination, we found that the permanent dipole moment decreased from 98 to 63 D/bacteriorhodopsin. From the best-oriented sample, we also concluded that the angle of retinal against the axis normal to the membrane surface was greater than 68.6 degrees.     

Characterization of the binding of valinomycin to bacteriorhodopsin

Circular dichroism spectroscopy has been used to investigate the binding of valinomycin to bacteriorhodopsin in purple membrane suspensions. Addition of valinomycin to purple membrane suspensions obtained from Halobacterium halobium causes the circular dichroism spectrum to shift from an aggregate spectrum to one resembling a monomer spectrum, indicating a loss of chromophore-chromophore interactions. By observing the spectral change upon titration of valinomycin, an apparent dissociation constant of 30–40 M for valinomycin binding was determined. Kinetics of dark adaptation for valinomycin-treated purple membrane are comparable to those for monomeric bacteriorhodopsin. Centrifugation studies demonstrate that valinomycin-treated purple membrane sediments the same as untreated purple membrane suspensions. These results are consistent with a model in which valinomycin binds specifically to bacteriorhodopsin without disrupting the purple membrane fragments.Abbreviations BR bacteriorhodopsin - CD circular dichroism - Tricine N-[tris-(hydroxymethyl) methyl] glycine     

The relationship between the chromophore moiety and the cation binding sites in bacteriorhodopsin

Bleaching of the purple membrane strongly reduces the number of divalent cation binding sites as well as their affinities. Conversely, deionization of the bleached membrane drastically inhibits the chromophore regeneration. Proteolysis experiments using bromelain show that the bleached membrane has an additional cleavage site probably located at the fifth loop, whereas in the blue membrane, the C-terminal tail is no longer susceptible to proteolysis. It is suggested that there exists a close relationship between the retinal environment and one or more of the cation binding sites.