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.     

A spin label study of purple membranes from Halobacterium halobium.

Mammary tumors induced in Sprague-Dawley Rats by the carcinogen 7,12-dimethylbenz(a)anthracene contain a DNA polymerase similar to that found in RNA tumor viruses. It has a molecular weight of 105,000 daltons and is active on the synthetic templates poly(rA):oligo(dT) and poly(rC):-oligo(dG) but is inactive on poly(dA):oligo(dT). This polymerase may be purified more than 300 fold with a 25% yield by ammonium sulfate precipitation, phosphocellulose chromatography and hydroxyapatite chromatography. A similar polymerase is also found in lactating normal rat mammary tissues.     

Photoelectric signals from dried oriented purple membranes of Halobacterium halobium

In dried oriented samples of purple membrane isolated from Halobacterium halobium, the photoelectric activity decreases and the light adaptation vanishes when the water content of the sample is lowered. In the photocycle the first steps of the proton movement were accelerated with decreasing humidity, while the last steps of the photocycle could not be observed. From the analysis of the photoelectric signal we conclude that at low humidities the protons move forward in the L decay and return to their original place during M decay.     

Determination of the retinal/protein molar ratios for the purple membranes of Halobacterium halobium and Halobacterium cutirubrum

Previously reported values of the retinal/protein molar ratios for the purple membranes from halobacteria are confusing. For $\text{H. halobium}$ values of approximately 1.00 and 0.45 and for $\text{H. cutirubrum}$ values of 0.45 and 0.43 have been published. A redetermination of these ratios in our hands has yielded identical values of 0.99 ± 0.01 for the membranes of both species. This is in agreement with expectations that the ratio be the same for the two species since the similarity of their membrane structure has been established and that every apoprotein molecule be complexed with a retinal since the uniqueness and equivalence of the membrane protein, bacteriorhodopsin, has been demonstrated.     

Biogenesis of the purple membrane of Halobacterium halobium

A protein closely resembling the purple membrane protein pre-exists in the cell membrane of H. halobium prior to the appearance of functional bacteriorhodopsin. It is associated with a differentiated membranous structure which has been isolated on a sucrose gradient and appears to be a precursor of the purple membrane. The identity of the precursor protein as a form of the purple membrane protein was established in different ways: (1) The cell proteins were labelled in vivo with ¹⁴C-proline during dark aerobic growth, the label was chased, and the cells transferred to the illuminated near-anaerobic conditions under which purple membrane is optimally synthesised (induction conditions). Cell lysates were fractionated on sucrose gradients at different times after induction. Label first found in the precursor fraction appeared within 24 h in the purple membrane fraction. (2) SDS-urea-acrylamide gel electrophoresis of the purple membrane protein and the precursor showed only one protein band whose migration coincided with that of the purple membrane band. (3) The amino-acid analysis of the purified precursor was very similar to that of the purple membrane.The absorption spectrum of the precursor showed little of the characteristic absorption of bacteriorhodopsin at 570 nm. A major band appears at 412 nm, the exact nature of which is not known. The difference spectrum (reduced versus oxidised) of a purified fraction showed only traces of cytochrome. Thin-layer chromatography of an acetone-soluble lipid extract indicated the presence of retinal and -carotene. Cells grown in the presence of nicotine did not develop purple membrane after induction: the species absorbing at 412 nm was much less abundant than in non-inhibited cells, but a new fraction was present with a sharp peak at 345 nm consisting mainly of lycopene.Abbreviations CTAB cetyltrimethyl ammonium bromide - SDS sodium dodecyl sulfate - CAP chloramphenicol - TLC thin layer chromatography - CD circular dichroism     

Structure of the purple membrane from Halobacterium halobium

European Biophysics Journal -     

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%.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Passive potassium ion permeability of Halobacterium halobium cell envelope membranes.

Cell envelope vesicles, prepared from Halobacterium halobium, were loaded with 3 M KCl, suspended in 3 M NaCl, and the loss of K+ was followed at various temperatures. The Arrhenius plot of the K+-efflux rates shows a break at 30 degrees C, with higher energy of activation above the break. This temperature dependence is consistent with earlier studies of chain motions in liposomes prepared from isolated lipids. The efflux of K+ is more rapid with increasing pH between pH 5 and 7. Since these vesicles do not respire under the experimental conditions it was expected that the K+-efflux data would be related to the passive permeability of the membranes to K+. The apparent K+ permeability at 30 degrees C is 1--2 - 10(-10) cm - s-1. This value corresponds to a 5-h half-life for retained K+ in the envelope vesicles and to a probably much longer half-life in whole cells. The previously observed ability of Halobacterium to retain K+ in the absence of metabolism can thus be explained solely by the permeability characteristics of the membranes.     

Spectroscopic discrimination of the three rhodopsinlike pigments in Halobacterium halobium membranes.

Membranes of Halobacterium halobium contain two photochemically reactive retinal pigments in addition to the proton pump bacteriorhodopsin. One, halorhodopsin, is also an electrogenic ion pump with a fast (on a scale of milliseconds) photoreaction cycle. The other, s-rhodopsin, is active in the same spectral region, but has a much slower photoreaction cycle (on a scale of seconds). S-rhodopsin is not an electrogenic ion pump and its properties suggest it functions as the receptor pigment for phototaxis. All three pigments have very similar absorption spectra. The recent isolation of mutants deficient in both bacteriorhodopsin and halorhodopsin and in retinal synthesis has allowed us to resolve the absorption spectra of s-rhodopsin and halorhodopsin. At neutral pH s-rhodopsin has an absorption maximum at 587 +/- 2 nm and halorhodopsin at 578 +/- 2 nm. At pH 10.8, lambda max for s-rhodopsin is shifted to 552 nm and extinction decreases slightly (15%) while halorhodopsin loses all extinction above 500 nm. Both effects are fully reversible and allow determination of the amounts of s-rhodopsin and halorhodopsin in membrane preparations containing both pigments. Both pigments were present in earlier studies of H. halobium membranes, and in view of these findings, several observations must be reinterpreted.     

The quantum efficiency of proton pumping by the purple membrane of Halobacterium halobium.

The quantum yield of H+ release in purple membrane (PM) sheets, and H+ uptake in phospholipid (egg phosphatidylcholine, PC) vesicles containing PM, was measured in single turnover light flashes using a pH-sensitive dye, p-nitrophenol, with rhodopsin as an actinometer. We have also calculated the ratio of H+ released per M412 formed (an unprotonated Shiff-base intermediate formed during the photocycle). In PM sheets, the quantum yield of H+ release depends on the medium. The quantum yield of M412 is independent of salt concentration. The ratio H+/M412 is approximately 1.8 M KC; and approximately 0.64 in 10 mM KCl. Direct measurements of the quantum yield of H+ give approximately 0.7 when the PM is suspended in 0.5 M KC; and 0.25 in 10 mM KCl. Using a quantum yield for M412 formation of 0.3 (Becher and Ebrey, 1977 Biophys J. 17:185.), these measurements also give a H+/M412 approximately 2 at high salt. In PM/PC vesicles, the H+/M412 is approximately 2 at all salt concentrations. The M412 decay is biphasic and the dye absorption change is monophasic. The dissipation of the proton gradient is very slow, taking on the order of seconds. Addition of nigericin (H+/K+ antiporter) drastically reduces the pH changes observed in PM/PC vesicles. This and the observation that the proton relaxation time is much longer than the photochemical cycling time suggest that the protons are pumped across the membrane and there is no contribution as a result of reversible binding and release of protons on just one side of the membrane.     

Chemosensory responses of Halobacterium halobium.

Responses of Halobacterium halobium cells to chemical stimuli have been shown by a capillary technique. Cells were attacted by D-glucose and several amino acids and repelled by phenol. Certain chemicals, such as acetate, benzoate, indole, and NiSO4, that are known to act as repellents of Escherichia coli cells served as attractants for Halobacterium. In the presence of ethionine, sensitivity to attractants was reduced. Arsenate prevented the attraction by glucose without lowering the cellular adenosine 5'-triphosphate level. The ability for chemo-accumulation toward glucose and histidine was interfered with by the formation of photosensory systems. Light-induced motor responses and chemosensory behavior toward glucose and histidine became detectable in the late stationary growth phase only. The behavior toward acetate and indole was not connected to photobehavior in that way: both substances acted as attractants already in the late log phase. Inhibition of bacteriorhodopsin synthesis by L-nicotine allowed chemo-accumulation toward glucose and histidine already in the late logarithmic phase.