Structure of the purple membrane from Halobacterium halobium

European Biophysics Journal -     

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     

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.     

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.     

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.     

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.     

Electrophoretic mobility of membrane fragments on a sucrose gradient. Application to isolated purple membrane fragments from Halobacterium halobium.

A simple technique for electrophoresis of particles is presented. The technique is based on running charged particles in a vertical tube along a sucrose gradient (20–50%). Purple membrane fragments from Halobacterium halobium were used to demonstrate the method. The migration of the fragments was linear with time in the region of 20 to 40% sucrose. Electrophoresis of purple membrane fragments under illumination, darkness, or darkness interrupted by short periods of illumination showed that at pH 4.5 the dark-adapted form of bacteriorhodopsin is less negative than its light-adapted form. At pH 6.5 and 8.5 no difference between these forms could be detected.     

Picosecond kinetics of the fluorescence from the chromophore of the purple membrane protein of Halobacterium halobium.

The fluorescence emission kinetics at 740 nm of the retinylidence chromophore of the purple membrane protein of Halobacterium halobium have been studied. Using picosecond laser pulses and an optical Kerr gate, the fluorescence risetime is found to be less than 8 ps and its lifetime is 40 +/- 5 ps at 90 degrees K and is estimated to be less than 3 ps at room temperature.     

Photoreceptor protein from the purple membrane of Halobacterium halobium. Molecular weight and retinal binding site.

The apparent molecular weight of the purple membrane protein of Halobacterium halobium was found to be 20 000 by sodium dodecyl sulfate gel electrophoresis and by gel filtration in sodium dodecyl sulfate. However, the molecular weight value determined by gel filtration in 6 M guanidine was 28 000. To resolve this discrepancy, methods insensitive to or independent of the conformation of the protein were used to estimate the molecular weight. Analytical ultracentrifugation of the sodium dodecyl sulfate-protein complex, peptide mapping, and amino acid analysis all gave values of 25 000 +/- 1000, a figure in agreement with a recent x-ray study. Borohydride reduction was used to attach the retinal cofactor covalently to a lysine residue. After digestion with thermolysin, peptide maps were prepared of the protein labeled at lysine residues with [14C] succinic anhydride both before and after reduction. Comparison of the maps showed one radioactive peptide with changed mobility. This peptide was isolated and shown to have the sequence Val-Ser-Asp-Pro-Asp-Lys-Lys with only one of the two lysine residues alkylated. Solid-phase sequencing showed the succinyl group to be at position 6 and hence the retinal group to be at position 7. It was possible that a small amount of retinal was also bound to Lys-6. There was no apparent homology with the corresponding peptide of vertebrate rhodopsin. No evidence of chain heterogeneity was found by radiochemical peptide mapping and sequence analysis of peptides containing lysine residues indicating that all protein chains of purple membrane are very similar or identical.     

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.     

Comparison of purple membrane from Halobacterium cutirubrum and Halobacterium halabium.

Direct comparison of purple membrane preparations from Halobacterium cutirubrum and Halobacterium halobium was carried out. Both preparations were found to be essentially identical with respect to their molecular weight, retinal content, lipid composition, fingerprinting of peptides from peptide digestion, electron micrographs and X-ray diffraction patterns, and behaviour as a light-activated proton pump. Thus, there would appear to be no species differences in the purple membranes from these two bacteria.     

Investigations into the effect of acid on the spectral and kinetic properties of purple membrane from Halobacterium halobium.

The formation and reversal of the acid species of purple membrane generated below pH 4.00 (22 degrees C) is studied together with the photochemical cycle over the pH range 6.40--3.20. The buffering capacity of the membrane reaches a peak at pH 4.30, indicating the possibility of a conformational change taking place. The generation of the new spectral species can take place in the dark and is unaffected by the addition of reducing agents. Kinetic parameters measured indicate that the group being titrated below pH 4.00 could be the same as that protonated in the formation of intermediate O. The temporal placement of intermediate O after M in the photochemical cycle is shown to be incompatible with the data presented here. Reneutralization of acidified purple membrane shows that the spectral changes in acid are reversible but the phototransient properties are altered.     

Effects of volatile anesthetics on bacteriorhodopsin in purple membrane, Halobacterium halobium cells and reconstituted vesicles.

In this study, we have investigated effects of volatile anesthetics on absorption spectra, proton pumping activity and decay of photointermediate M of bacteriorhodopsin (bR) in differently aggregated states. Anesthetics used in this study are ether-type general anesthetics; enflurane and sevoflurane. The observed effects on bR depend not only on variety or concentration of anesthetics but also strongly on the aggregation state of bR molecules in the membrane. In purple membrane (PM), bR having maximum light absorption at 567 nm (bR567) is formed in the presence of sevoflurane or a small amount of enflurane, while a species absorbing maximally at 480 nm (bR480) is formed upon the addition of large amounts of enflurane. X-ray diffraction studies show that the former species maintains crystallinity of PM, but the latter does not. In reconstituted vesicles where bR molecules exist as monomer, even sevoflurane forms bR480. Flash photolysis experiments show that bR567 contains a shorter-lived M intermediate absorbing maximally at 412 nm in the photoreaction cycle than bR does and that bR480 contains at least two long-lived M intermediates which seem to absorb maximally near and at lower than 380 nm. The measurements of light-induced pH changes of the whole cells and of the reconstituted vesicles in the presence of the anesthetics indicate that bR567 has a enhanced proton pumping efficiency, while bR480 has a quite low or no activity. No significant difference was observed in the anesthetic action between two inversely pumping vesicles. These observations suggest that on the formation of bR480, anesthetics enter into the membrane and affect the protein-lipid interaction.     

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.     

Comparative studies on the fine structure of purple membrane fromHalobacterium cutirubrum andHalobacterium halobium

Summary Direct comparison of the absorption and circular dichroic spectra of dark- and light-adapted purple membrane fromHalobacterium cutirubrum andHalobacterium halobium indicated no apparent species differences. In addition, sequential bleaching and regeneration of the purple membrane with concomitant monitoring of the absorption and circular dichroic spectra showed no species differences as well. Furthermore, perturbation of the structure of the purple membrane from either species with a detergent, Triton X-100, yielded similar spectral changes. It was concluded: (i) no apparent differences exist in the molecular organization and protein fine structure of the two purple membranes, (ii) if exciton interaction among the retinal chromophores is a reasonable possibility in the case of the purple membrane fromHalobacterium halobium, it must be similarly so for the membrane fromHalobacterium cutirubrum, (iii) the effects of light adaptation on the membrane structure of both species are essentially the same, and (iv) the underlying molecular mechanisms for the bleaching and regenerative processes must be similar, if not identical, for the purple membranes of the two species.