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     

Regulation of bacteriorhodopsin synthesis by growth rate in continuous cultures of Halobacterium halobium

The independent effects of oxygen tension and growth rate on bacteriorhodopsin synthesis in Halobacterium halobium have been studied in chemostat cultures. Bacteriorhodopsin synthesis occurs only at low growth rates and is stimulated by low oxygen tension. Fast growth rates override the stimulatory effects of oxygen tension, with the result that bacteriorhodopsin can scarcely be detected. Illumination of cultures maintained at low growth rate and low oxygen tension significantly increases the steady state cell yield. This finding suggests that under these conditions the purple membrane proton pump is coupled to energy transduction.     

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.     

Acyl chain conformations in phospholipid bilayers: a comparative study of docosahexaenoic acid and saturated fatty acids

A variety of experimental methods indicate unique biophysical properties of membranes containing the highly polyunsaturated ω-3 fatty acid, docosahexaenoic acid (DHA). In the following we review the atomically detailed picture of DHA acyl chains structure and dynamics that has emerged from computational studies of this system in our lab. A comprehensive approach, beginning with ab-initio quantum chemical studies of model compounds representing segments of DHA and ending with large scale classical molecular dynamics simulations of DHA-containing bilayers, is described with particular attention paid to contrasting the properties of DHA with those of saturated fatty acids. Connection with experiment is made primarily through comparison with Nuclear Magnetic Resonance (NMR) studies, particularly those that probe details of the chain structure and dynamics. Our computational results suggest that low torsional energy barriers, comparable to kT at physiological conditions, for the rotatable bonds in the DHA chain are the key to the differences observed between polyunsaturated and saturated acyl chains.     

Interaction of bovine rhodopsin with calcium ions

The formation of metarhodopsin II in various bovine rhodopsin preparations (rod outer segment (ROS) suspensions and rhodopsin-detergent solutions) was measured by means of flash spectrophotometry. The half-lifetime and formation of metarhodopsin II in ROS did not depend on the calcium concentration in the range of less than 10^–9 M (using EGTA or EDTA) to 15×10^–3 M calcium at pH values of 5.0, 7.1, and 9.0 (Table 1).The regeneration of rhodopsin from opsin by adding 11-cis retinal to ROS-suspensions and rhodopsin digitonin solutions was measured spectrophotometrically. It was not substantially different in either saline, one containing less than 10^–7 M calcium (by adding EGTA), the other containing 10^–3 M calcium (Table 2).Abbreviations A absorption - A absorption change - CTAB N-Cetyl-N,N,N-trimethylammoniumbromide - E₇₀₀ extinction at =700 nm - EDTA ethylenediamine-NNNN-tetraacetic acid - EGTA 2,2-ethylenedioxybis [ethyliminodi (acetic acid)] - MI metarhodopsin I - MII metarhodopsin II - Rh rhodopsin - ROS rod outer segment This work is based upon a Ph. D. dissertation (Nöll, 1974) and was presented in part at the Jahrestagung der Deutschen Gesellschaft für Biophysik, Freiburg, Germany, October 1974     

The transverse location of tryptophan residues in the purple membranes of Halobacterium halobium studied by fluorescence quenching and energy transfer

Fluorescence quenching by a series of spin-labelled fatty acids is used to map the transverse disposition of tryptophan residues in bacteriorhodopsin (the sole protein in the purple membranes of Halobacterium halobium). A new method of data analysis is employed which takes into account differences in the uptake of the quenchers into the membrane. Energy transfer from tryptophan to a set of $\text{n-(9-}\text{anthroyloxy)}$ fatty acids is used as a second technique to confirm the transverse map of tryptophan residues revealed by the quenching experiments. The relative efficiencies of quenching and energy transfer obtained experimentally are compared with those predicted on the basis of current models of bacteriorhodopsin structure. Most of the tryptophan fluorescence is located near the surface of the purple membrane. When the retinal chromophore of bacteriorhodopsin is removed, tryptophan residues deep in the membrane become fluorescent. These results indicate that the deeper residues transfer their energy to retinal in the native membrane. The retinal moiety is therefore located deep within the membrane rather than at the membrane surface.     

QM/MM study of the reaction mechanism of Cl-cis,cis-muconate with muconate lactonizing enzyme

The lactonization process of Cl-cis,cis-muconate catalyzed by anti-muconate lactonizing enzyme (anti-MLE) was studied theoretically with the aid of a combined quantum mechanics/molecular mechanics (QM/MM) approach. Two elementary processes steps involved in the lactanization process were investigated. The calculated energy barriers agree well with the experimental values. The present work provided the explicit structures of the enolate anion intermediates. The electrostatic influence analysis highlighted residues Arg51, Gln294 and TIP383 for the MLE-Cl-2 system and the residue Asn193 for the MLE-Cl-4 system as the possible mutation targets for rational design of anti-MLE in future enzyme modification.     

Proton transfers in the photochemical reaction cycle of proteorhodopsin

The spectral and photochemical properties of proteorhodopsin (PR) were determined to compare its proton transport steps to those of bacteriorhodopsin (BR). Static and time-resolved measurements on wild-type PR and several mutants were done in the visible and infrared (FTIR and FT-Raman). Assignment of the observed C=O stretch bands indicated that Asp-97 and Glu-108 serve as the proton acceptor and donor, respectively, to the retinal Schiff base, as do the residues at corresponding positions in BR, but there are numerous spectral and kinetic differences between the two proteins. There is no detectable dark-adaptation in PR, and the chromophore contains nearly entirely all-trans retinal. Because the pK(a) of Asp-97 is relatively high (7.1), the proton-transporting photocycle is produced only at alkaline pH. It contains at least seven transient states with decay times in the range from 10 micros to 200 ms, but the analysis reveals only three distinct spectral forms. The first is a red-shifted K-like state. Proton release does not occur during the very slow (several milliseconds) rise of the second, M-like, intermediate, consistent with lack of the residues facilitating extracellular proton release in BR. Proton uptake from the bulk, presumably on the cytoplasmic side, takes place prior to release (tau approximately 2 ms), and coincident with reprotonation of the retinal Schiff base. The intermediate produced by this process contains 13-cis retinal as does the N state of BR, but its absorption maximum is red-shifted relative to PR (like the O state of BR). The decay of this N-like state is coupled to reisomerization of the retinal to all-trans, and produces a state that is O-like in its C-C stretch bands, but has an absorption maximum apparently close to that of unphotolyzed PR.     

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.     

An evaluation of the charge-transfer model for the chromophores of the retinal-containing proteins,rhodopsin and bacteriorhodopsin

The chromophores of rhodopsin and bacteriorhodopsin are believed to result from an electrostatic interaction between the protonated Schiff base of retinal and amino acid side chains. It has been proposed from ESR measurements on rhodopsin (Shirane, K. (1975) Nature, 254, 722–723) and model studies using retinal and tryptophan (Ishigami, M., Maeda, Y. and Mishima, K. (1966) Biochim. Biophys. Acta 112, 372–375) that the interaction is one of charge transfer and that the amino acid involved is tryptophan. Our re-examination of this work does not support the existence of a charge-transfer complex. However, additional similarities between the model system and bateriorhodopsin were observed. It is concluded that further studies in this area may yield information about the nature of the protein chromophores.     

Thermodynamic properties of the photochemical reaction center of Heliobacterium chlorum

The thermodynamic and spectral properties of the photochemical reaction center components of Heliobacterium chlorum have been examined. The primary electron donor bacteriochlorophyll has E_m,7 = +225 mV, and the ‘primary acceptor’ E_m,10 = −510 mV. The former has an EPR signal in its oxidised form near G = 2.0025, ΔH = 0.95 mT, reminiscent of the properties of the primary donor in bacteria containing bacteriochlorophyll a. The ‘primary acceptor’ has properties similar to those of the iron-sulfur cluster acceptors of green sulfur bacteria. H. chlorum contains a c-type cytochrome (E_m,7 = +170 mV) that donates electrons to the photooxidised primary donor with . The reaction center of H. chlorum is thus very similar to that found in representative green sulfur bacteria, but the cellular architecture and photopigments of this group are quite distinct from those of H. chlorum.     

A study on the fabrication of chemically modified bacteriorhodopsin film and its photochemical properties

Chemically modified bacteriorhodopsin (BR) films embedded in gelatin matrix were fabricated. It was found that chemically modified BR/gelatin film remained the original conformation of BR and possessed homogeneity. Then, the photochemical conversion from all-trans O state to 9-cis P state in the chemically modified BR film/gelatin was investigated. For comparison purpose, the same conversion in the wild-type BR (BRwt) film was also presented here. To our know, it is the first report to show that the absorption changes of the P state in the chemically modified BR film were larger than in the BRwt film upon illumination with red light at the low actinic power density.     

The nitrate transporting photochemical reaction cycle of the pharaonis halorhodopsin

Time-resolved spectroscopy, absorption kinetic and electric signal measurement techniques were used to study the nitrate transporting photocycle of the pharaonis halorhodopsin. The spectral titration reveals two nitrate-binding constants, assigned to two independent binding sites. The high-affinity binding site (K(a) = 11 mM) contributes to the appearance of the nitrate transporting photocycle, whereas the low-affinity constant (having a K(a) of approximately 7 M) slows the last decay process in the photocycle. Although the spectra of the intermediates are not the same as those found in the chloride transporting photocycle, the sequence of the intermediates and the energy diagrams are similar. The differences in spectra and energy levels can be attributed to the difference in the size of the transported chloride or nitrate. Electric signal measurements show that a charge is transferred across the membrane during the photocycle, as expected. A new observation is an apparent release and rebinding of a small fraction of the retinal, inside the retinal pocket, during the photocycle. The release occurs during the N-to-O transition, whereas the rebinding happens in several seconds, well after the other steps of the photocycle are over.     

Stopped-flow kinetic study of the regeneration reaction of tocopheroxyl radical by reduced ubiquinone-10 in solution

Kinetic study of the reaction between tocopheroxyl (vitamin E radical) and reduced ubiquinone, n = 10) has been performed. The rates of reaction of ubiquinol with α-tocopheroxyl 1 and seven kinds of alkyl substituted tocopheroxyl radicals 2–8 in solution have been determined spectrophotometrically, using a stopped-flow technique. The result shows that the rate constants decrease as the total electron-donating capacity of the alkyl substituents on the aromatic ring of tocopheroxyls increases. For the tocopheroxyls with two alkyl substituents at ortho positions (C-5 and C-7), the second-order rate constants, k₁, obtained vary i n the order of 10², and decrease predominantly, as the size of two ortho-alkyl groups (methyl, ethyl, isopropyl and tert-buty) in tocopheroxyl increases. On the other hand, the reaction between tocopheroxyl and ubiquinone-10 (oxidized ubiquinone) has not been observed. The result indicates that ubiquinol-10 regenerates tocopherol by donating a hydrogen atom of the 1-OH and/or 4-OH group to the tocopheroxyl radical. For instance, the k₁ values obtained for α-tocopheroxyl are 3.74 · 10⁵ M^?1 · s^?1 and 2.15 · ⁵ M^?1 · s^?1 in benzene and ethanol solution at 25°C, respectively. The above reaction rates, k₁, obtained were compared with those of vitamin C with α-tocopheroxyl reported by Packer et al. (k₂ = 1.55 · 10⁶ M^?1 · s^?1) and Scarpa et al. (K₂ = 2 · 10⁵ 10⁵ M^?1 · s^?1), which is well known as a usual regeneration reaction of tocopheroxyl in biomembrane systems. The result suggests that ubiquinol-10 also regenerates the tocopheroxyl to tocopherol and prevents lipid peroxidation in various tissues and mitochondria.