Light-induced conductivity changes in purple membrane suspensions

Small light-induced changes in the conductivity of light-adapted purple membrane suspended in strong electrolyte solutions were detected. The method used involved modulated light and a phase sensitive detector and it allowed us to detect accurately changes as small as 0.0001% in the conductivity of the suspension. The light-induced conductivity changes turned out to be composed of at least two different events: a small fast increase in conductivity (t ∼ 2 ms) followed by a slower and larger decrease in this parameter (Τ=70 ms-80 ms). The effects of pH and temperature on these changes were studied. Both events reached maximal values around neutral pH and approached zero at both high and low pH's. Heating the suspension decreased the photoconductivity change and Arrhenius plots of the data showed breaks around 31‡ C. It is suggested that the conductivity changes reflect changes in the surface charge of the membrane and can be used to follow the kinetics of the conformational changes occuring in the system.     

The surface potential on the purple membrane measured using a modified bacteriorhodopsin chromophore as the spectroscopic probe

The surface potential of the purple membrane was measured by a novel method by using an artificial bacteriorhodopsin whose chromophore was 13-CF₃ retinal instead of retinal. When attached to the apoprotein by a Schiff base, the intrinsic pK of the 13-CF₃ chromophore is around 7.3. The apparent p_K of this pigment depends on the surface potential and thus on the electrolyte concentration. This allowed us to determine the surface charge density using the Gouy-Chapman equation. The surface charge density was found to be −1.65 ± 0.15 × 10⁻³ electronic charges per Ų or about 2 negative charges/bacteriorhodopsin. This large value for the surface potential probably explains both part of the strong apparent association of divalent cations with the membrane and the effect of low salt concentrations on light-induced proton release from the purple membrane.     

Structure of the purple membrane from Halobacterium halobium

European Biophysics Journal -     

Resonance raman spectroscopy of chemically modified and isotopically labelled purple membranes. I. A critical examination of the carbon-nitrogen vibrational modes

Resonance Raman spectra of bacteriorhodopsin are compared to the spectra of this protein modified in the following ways: (1) selective deuteration at the C-15 carbon atom of retinal, (2) full deuteration of the retinal, (3) the addition of a conjugated double bond in the β-ionone ring (3-dehydroretinal), (4) full deuteration of the protein and lipid components, (5) ¹⁵N enrichment of the entire membrane and (6) deuteration of the entire membrane (including the retinal). A detailed comparison of the ¹⁵N-enriched membrane and naturally occurring purple membrane from 800 cm^?1 to 1700 cm^?1 reveals that ¹⁵N enrichment affects the frequency of only two vibrational modes. These occur at 1642 cm^?1 and 1620 cm^?1 in naturally occurring purple membrane and at 1628 cm^?1 and 1615 cm^?1 in the ¹⁵N-enriched samples. Therefore, this pair of bands reflects the states of protonation of the Schiff base. However, our data also indicate that neither of these modes are simple, localized $\text{C}\text{=}\textcolor{red}{}\text{?}\text{H}$ or C=N stretching vibrations. In the case of the 1642 cm^?1 band motions of the retinal chain beyond C-15 are not significantly involved. On the other hand, in the 1620 cm^?1 band atomic motions in the isoprenoid chain beyond C-15 are involved.     

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.     

Fast electric response signals in the bacteriorhodopsin photocycle

The time behavior of flash-induced charge movements during the first steps in the bacteriorhodopsin photocycle was measured on a suspension of purple membranes oriented by an electric field. The experiments were done in the temperature range 80–278 K. During the formation of the intermediate K, two negative (with respect to the direction of the proton pump) components of the response signal are well resolved with time constants τ₁ < 3 μs and $\text{τ}{}_2\text{? 150 μ}\text{s}$ at 200 K. The distances of the charge displacements responsible for the electric signals are estimated. On the basis of the results the two components are assigned to two steps in the trans-cis isomerization of the retinal. A third negative component appears at higher temperatures which is related by time constant measurements to the K → L transition.     

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.     

Energy transfer in the purple membrane of Halobacterium halobium.

The absorption spectrum of the primary photoproduct (the bathoproduct, or K) of the purple membrane protein (PM) at-196 degrees C has a maximum at 628 nm and an extinction coefficient of 87,000. Knowing the absorption spectrum allowed us to calculate the quantum efficiencies for PM to K and K to PM conversion at -196 degrees C. Direct measurements of these quantum yeilds at -196 degrees C gave 0.33 +/- 0.05 and 0.67 +/- 0.04, respectively. Determination of relative quantum efficiencies for PM to K and K to PM conversion by analysis of the absorption spectra of several photostationary-state mixtures of PM and K at -196 degrees C, however, gave wavelength-dependent quantum efficiencies that appear to be greater than 1. These anomolous results can be readily explained in terms of energy transfer from PM to K within the trimer clusters of pigment molecules which exist in the purple membrane. A model for such a transfer predicts an efficiency of energy transfer from PM to K of about 43%.     

Improved isolation procedures for the purple membrane of Halobacterium halobium.

Techniques for purifying teh purple membrane of Halobacterium halobium are given. This purple membrane contains a chromoprotein with a retinal prosthetic group similar to rhodopsin, the chromprotein found in the visual systems of higher invertebrates and vertebrates. The described purple membrane isolation procedures yield a highly purified preparation as determined by transmitting electron microscopy and gel electrophoresis. Critical analysis of the absorption spectra of the purple membrane was also employed to establish criteria of purity for the preparation. The visible absorption spectra of the purified purple membrane preparation in buffer was found to have a maximum at 559 nm which shifted to 567 nm on light exposure. No indication of any spectral perturbation arising from bacterioruberin-containing membrane, the major contaminant in purple membrane preparations, was found. Furthermore, the ratio of protein aromatic amino acid absorbance at 280 nm to chromophore absorbance at 567 nm was found to be 1.5 in light-exposed preparations compared to the previously reported ratio of 2.3.-3 The decrease in the value of this ratio is also indicative of an increase in the purity of the purple membrane preparation.     

Electro-optical measurements on aqueous suspension of purple membrane from Halobacterium halobium

The permanent dipole moment, polarizability, and the retinal angle of Halobacterium halobium purple membranes were determined at different pH values. All of the parameters have a maximum between pH 5 and 6. There is a reversal in the direction of the permanent dipole moment near pH 5. The value of permanent dipole moment was determined to be 60 D/protein at pH 6.6, and the value obtained for polarizability was 3 X 10(-28) Fm2/membrane fragment. The retinal angle of all-trans retinal was 0.8 degrees smaller than that of the 13-cis conformation.     

Studies of an acid-induced species of purple membrane from Halobacterium halobium.

A new spectral species of the purple membrane of Halobacterium halobium has been observed below pH 3.2. The formation of this new species is temperature-dependent and is favoured by increasing temperature up to the physiological range of the organism. The rate of formation at pH 3.0 and 22 degrees C is 7.9 x 10-3s-1. The spectral distribution and temperature-dependence of the new species suggest that it may be phototransiet O, stabilized by low pH. Flash-photolytic experiments in the pH range 7.2-2.7 show a pH-dependence corresponding to the static events and are consistent with a single protonation of bacteriorhodopsin below pH 3.22. These results can also be interpreted in terms of the stabilization of phototransient O at low pH. The temperature-dependence of the formation of the acid-induced species may reflect a relationship with the phase transition of the membrane.     

Phase transitions of the purple membrane and the brown holo-membrane X-ray diffraction,circular dichroism spectrum and absorption spectrum studies

The phase transition of the purple membrane observed by differential scanning calorimetry (Jackson, M.B. and Sturtevant, J.M. (1978) Biochemistry 17, 911–915) has been investigated by X-ray diffraction, circular dichroism and absorption spectrum, in comparison with the phase transition in the brown holo-membrane. The two-dimensional crystal of bacteriorhodopsin transformed into two-dimensional liquid around 74–78°C in the purple membrane and around 50–60°C in the brown holo-membrane. The X-ray diffraction patterns obtained at 78°C for the purple membrane and at 60°C for the brown holo-membrane exhibit several broad peaks. Analysis of the pattern suggests that bacteriorhodopsin molecules aggregate in trimers even above the phase transition temperature. The negative circular dichroism band in the visible region is still present at 80°C in the purple membrane and at 60°C in the brown holo-membrane, but becomes negligibly small at 70°C in the brown holo-membrane. The 560 nm absorption peak due to bacteriorhodopsin changes its position and height drastically around 80°C in the brown holo-membrane as in the purple membrane. X-ray diffraction studies have been made on membranes of total lipids extracted from the purple membrane. No indication of the phase transition has been found between ?81°C and 77°C.     

Effects of light adaptation on the purple membrane structure of Halobacterium halobium.

Absorption, circular dichroism and optical rotatory dispersion of the bacteriorhodopsin containing purple membrane form Halobacterium halobium were studied in regard to the structural stability of this membrane during the photoisomerization of the retinal of the bacteriorhodopsin from the 13-cis to the all-trans configuration. The following conclusions were reached: (a) the macromolecular structure (protein-protein interaction which may result in the possible exciton interaction of the retinal pi-pi* (NV1) transition moments and protein-lipid interaction) are not significantly altered, (b) possibilities of delocalized conformation changes of the apoprotein involving secondary and/or tertiary structure can be ruled out, (c) localized secondary structure conformation changes of the apoprotein must be limited to the involvement of no more than one or two amino acid residues and localized tertiary structure conformation changes of the apoprotein must be limited to a very short segment of the protein chain containing only a few aromatic amino acid residues, and (d) the interaction between the apoprotein and retinal seems to be relatively more pronounced when the retinal is in the all-trans form than the 13-cis from and also the apoprotein seems to impose a more pronounced dissymmetric constraint on the retinal in the all-trans form than in the 13-cis form.     

Reaction of the purple membrane with a carbodimide

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 (Δh?) and decay of photointermediate M) show changes indicative of slowed proton uptake. The Δh? 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 Δh? is not apparent in detergent-solubilized membranes. Ethyl dimethylaminopropyl-carbodiimide 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 different type of site and appears to inhibit the proton pump.     

Effect of water on the structure of bacteriorhodopsin and photochemical processes in purple membranes

Visible and infrared spectra of bacteriorhodopsin films under different humidities at room and low temperatures are investigated. On dehydration of purple membranes at room temperatures an additional chromophore state with the absorption band at 506 nm is revealed. The photocycle of purple membranes in the dry state is devoid of the 550 nm intermediate and involves the long-lived intermediate at 412 nm. As water is removed, the 550 nm intermediate becomes undetectable. The analysis of the infrared spectra shows that dehydration does not affect the ordering of the main network of the interpeptide hydrogen bonds which stabilizes the -helical conformation (slightly distorted in the initial humid dark- and light-adapted state); light adaptation (cis-trans isomerization) of bacteriorhodopsin results in an increase of sorbed water in purple membranes. Dehydration of purple membranes decreases the reaction rate of cis-trans isomerization.     

Phase transitions of the purple membranes of Halobacterium halobium

Purple membranes of Halobacterium halobium were studied by differential scanning calorimetry. No transition was detected at temperatures below 70 degrees C. A small endothermic transition was seen at about 80 degrees C and a larger one at 100 degrees C. The larger transition is the irreversible denaturation of bacteriorhodopsin. The smaller transition is accompanied by a change in the visible absorption spectrum and is believed to be reversible, involving a cooperative change in crystalline structure of the membrane.     

Contrasting molecular dynamics in red and purple membrane fractions of the Halobacterium halobium.

2H-nuclear magnetic resonance (NMR) has been used to study the dynamics of amino acid residues in bacteriorhodopsin with results that depend on the method of sample preparation. We show here that in [2H]-leucine-labeled samples the intensity of the isotropic signal varies according to the degree of residual contamination of the sample with red membrane. We conclude that few of the surface leucine residues of bacteriorhodopsin are moving isotropically on the 2H-NMR time scale.     

Influence of stray capacitance and sample resistance on the kinetics of fast photovoltages from oriented purple membranes

Flash-induced photovoltages were measured with metal electrodes in two experimental systems of purple membranes oriented by an electric field. One system consisted of a suspension of purple membranes cooled to 80 K. The photovoltage evoked by a xenon flash lamp displayed a single phase with a fast rise and a slow RC-decay. The signal shape is consistent with a fast charge separation occurring before the decay of the K-intermediate. The other system consisted of purple membranes embedded and stabilized in polyacrylamide gel. At room temperature, the photovoltage, evoked by a 10 ns laser flash, displayed a negative phase in the submicrosecond range and a slower positive one. The shape of the signals were altered in a complex manner by the stray capacitance and the ionic strength. The rise-time of the negative phase was approx. 14 and approx. 40 ns at ionic strengths of 10 and 1 mM, respectively. The initial peak amplitudes of the photovoltage from both experimental systems depended on the external capacitance in an inverse manner, indicating that both experimental systems were not impedance-matched. The evaluation of kinetic data of molecular reactions from measurements of the photovoltage is discussed.