Purple Membrane Of Halobacterium Halobium Research Articles

Dielectric Properties of the bR Membrane

units of the system. A large nonreversibility of the dielectric response as a result of heating and cooling has been observed in the slow processes implicating a change in the structure of the membrane stacking depending on the history of cooling and heating of the sample. Significant changes as large as 3 orders of magnitude in the mobility have been observed in this time scale. Comparison of the oriented and nonoriented bR membrane films was performed, and it was found that the oriented purple membrane has a unique liquid crystal-like ferroelectric behavior. Bacteriorhodopsin (bR) is an integral membrane protein that is found in the purple membrane of the bacterium Halobacterium salinarium. The purple membrane is a two-dimensional crystalline lattice formed by bacteriorhodopsin and lipid molecules in the cytoplasmic membrane of this bacterium. The function of bR in the purple membrane is that of a light driven proton pump. The purple membrane of Halobacterium halobium consists of 25% of the lipid bilayer and 75% of bacteriorhodopsin protein, and therefore, this purple membrane is usually called bacteriorhodopsin membrane or shortly bR membrane. The function of this system is a light-driven proton pump, and the resulting electrochemical gradient is used by the cell to drive ATP synthesis and other metabolic processes under anaerobic conditions (flash excitation). The bR molecule, with the molecular weight of 25 kD, contains seven closely packed R-helices crossing the membrane (Figure 1a) and a Retinal group playing the keynote role in the function of this protein. Every R-helix has a 50 D dipole moment and six of them compensate each other. bR molecules are packed in the membrane in very specific way. Every three macromolecules are placed close to each other in a certain position and form the triad (Figure 1b). It is known that these triads are maximally packed in a hexagonal way in the membrane. 1 The dipole moment of each macromolecule crosses the membrane in the same direction. A fragment of bR membrane is schematically presented in Figure 1b.

The biocatalytic effect of Halobacterium halobium on photoelectrochemical hydrogen production

Hydrogen gas can be produced electrochemically by leading a current through two electrodes immersed in a NaCl solution. Bacteriorhodopsin (BR) a protein found in the purple membrane of Halobacterium halobium, is known to pump protons across the membrane upon illumination. In this study, the effect of BR on photoelectrochemical hydrogen production was investigated. A batch type bio-photoelectrochemical reactor was designed and constructed. The photoelectrochemical hydrogen production experiments were performed with free H. halobium packed cells or immobilised H. halobium cells. The cells were either immobilised in polyacrylamide gel (PAG) or on cellulose acetate membrane (CAM). Experiments were also performed with purple membrane fragments of H. halobium immobilised on cellulose acetate membrane. It was found that the presence of bacteriorhodopsin (BR) in the reactor enhances the hydrogen production rate upon illumination. Immobilisation increased the amount of hydrogen produced per mole of BR. Compared to control experiments without BR, the power requirement of the photoelectrochemical reactor per amount of hydrogen produced decreased fourfold when purple membrane fragments immobilised on CAM were used. The presence of BR regulates the pH of the system, increases the hydrogen production rate and causes light-induced proton dissociation, which lowers the electrical power requirement for the electrochemical conversion.

Picosecond-millisecond dual-time-base spectroscopy of fluorescent photointermediates formed in the purple membrane of Halobacterium halobium

Dual-time-base fluorometry to measure both fluorescence lifetimes and the reaction kinetics of their origins was applied to the identification of two fluorescence (>660 nm) species T 9ps and T 62ps ( τ F=9±2 and 62±2 ps, respectively) in the purple membrane of Halobacterium halobium in the photostationary state. Photocycles were driven with a 532-nm nanosecond laser and the τ Fs were measured at delay times ( t d=0.05–90.5 ms) by a gated streak camera synchronized to a 626-nm picosecond laser. The t d dependencies of the fluorescence of T 9ps and T 62ps were identical with the behaviors of the photointermediates O and Q, respectively.

Binding of volatile anesthetics to purple membranes studied by X-ray diffraction

Volatile anesthetics, diiodomethane and trifluoroethyl iodide, acted on the purple membrane of Halobacterium halobium in two different modes depending on the concentration. At low concentration, the absorption maximum of bacteriorhodopsin shifted from 561 to 558 nm (BR558) and the M-intermediate of the photocycle decayed faster than the native one. Higher concentration induced a species absorbing maximally at 480 nm (BR480) and the long-lived M-intermediates. The X-ray study suggested that anesthetics bound specifically to the protein–lipid interfacial region within a trimer near the surface of membrane in BR558 and entered into the hydrophobic domain of the membrane in BR480.

Electron Cryomicroscopy of Bacteriorhodopsin Vesicles: Mechanism of Vesicle Formation

We obtained vesicles from purple membrane of Halobacterium halobium at different suspension compositions (pH, electrolytes, buffers), following the procedure of Kouyama et al. (1994) ( J. Mol. Biol. 236:990–994). The vesicles contained bacteriorhodopsin (bR) and halolipid, and spontaneously formed during incubation of purple membrane suspension in the presence of detergent octylthioglucoside (OTG) if the protein:OTG ratio was 2:1 by weight. The size distribution of the vesicles was precisely determined by electron cryomicroscopy and was found to be almost independent on the incubation conditions (mean radius 17.9–19 nm). The size distribution in a given sample was close to the normal one, with a standard deviation of ∼±1 nm. During dialysis for removal of the detergent, the vesicles diminished their radius by 2–2.5 nm. The results allow us to conclude that the driving force for the formation of bR vesicles is the preferential incorporation of OTG molecules in the cytoplasmic side of the membrane (with possible preferential delipidation of the extracellular side), which creates spontaneous curvature of the purple membrane. From the size distribution of the vesicles, we calculated the elasticity bending constant, K B ≈ 9 × 10 −20 J, of the vesicle wall. The results provide some insight into the possible formation mechanisms of spherical assembles in living organisms. The conditions for vesicle formation and the mechanical properties of the vesicles could also be of interest with respect to the potential technological application of the bR vesicles as light energy converters.

Experimental observation of the interference of three- and five-wave mixing processes into the signal of second harmonic generation in bacteriorhodopsin solution

The second harmonic (SH) generation with a Ti:sapphire femtosecond laser system in a randomly oriented suspension of finely fragmented purple membranes of Halobacterium halobium has been studied experimentally. The existence of two interfering contributions to the SH signal due to second- and fourth-order nonlinear processes has been established. The phase and the value of the fourth-order electronic susceptibility (hyperpolarizability) relative to the second-order ones have been determined. The analysis of the experimental data shows the existence of a coherent contribution into the SH signal.

Picosecond fluorescence spectroscopy of the purple membrane of Halobacterium halobium in alkaline suspension

The fluorescence lifetime of the light-adapted purple membrane of Halobacterium halobium in alkaline suspension was measured with a femtosecond-pulse laser-synchroscan streak camera system (< 10 ps resolution time, 620 nm excitation) at 22°C. The lifetimes of slow, fast and very fast decay components were found to be 62 ± 2, 9 ± 2 and < 1 ps, respectively. The fastest component is attributed to bacteriorhodopsin (bR 568). We examined the effect of pH and excitation power on the amplitude of the slow component and concluded that its origin is attributable to the Q intermediate, which is a photoproduct of the N intermediate formed in the later stage of the photocycle of bR 568. We found that Q is the same species as pseudo-bacteriorhodopsin (p-bR).

Doubly forbidden second-harmonic generation from isotropic suspensions: Studies on the purple membrane of Halobacterium halobium.

In this paper we report results on second-harmonic generation (SHG) from aqueous suspensions of the purple membrane of Halobacterium halobium. A detailed theory describing this SHG signal is supported by results for the angular distribution of the harmonic emission and validated by experimental measurements with circularly polarized pump radiation---a condition that normally precludes SHG. Hence we identify the precise mechanism for second-harmonic emission in this macroscopically isotropic but microscopically ordered system, resolving confusion over whether the signal should be considered coherent SHG or incoherent hyper-Rayleigh scattering. \textcopyright{} 1996 The American Physical Society.

Use of nanogold- and fluorescent-labeled antibody Fv fragments in immunocytochemistry.

Recombinant antibody fragments are emerging as a versatile tool in both basic research and medical therapy. We describe the procedures for direct labeling of engineered antibody fragments (Fv) with fluorescein or nanogold and their use in fluorescence and immunoelectron microscopy, respectively. The Fv fragments were produced in Escherichia coli, purified by one-step Strep tag affinity chromatography, chemically labeled with the marker, and employed in microscopy to localize epitopes on the membrane protein bacteriorhodopsin in purple membranes of Halobacterium halobium and the cytochrome c oxidase of Paracoccus denitrificans. In both cases, methods involving directly labeled antibody fragments show results identical to those in which antibodies or Fv fragments are detected by a secondarily labeled conjugate. The multifunctional design of the recombinant Fv fragments, however, offers more all-around applications in immunocytochemistry. The directly labeled Fv fragments, half the size of an Fab fragment, are at the molecular level the smallest antibody fragments yet described for visualization of biomolecules in microscopy.

Component analysis of the fast photoelectric signal from model bacteriorhodopsin membranes part 4. A method for isolating the B2 component and the evidence for its polarity reversal at low pH

Thin films formed from oriented purple membranes of Halobacterium halobium exhibit fast photoelectric signals upon pulsed light stimulation. These signals are generated by light-induced rapid charge separation and recombination. The fast photosignal consists of several components. We have previously developed methodology to separate these components on the basis of their pH dependence. We used the Trissl-Montal method of membrane reconstitution to generate a photosignal which contains the pH-independent B1 component and the pH-dependent B2 component, whereas we used a modified multilayered thin film method to obtain a pure B1 signal. The B2 component is prominent in the medium to high pH range (pH 7–11) and has a polarity opposite to that of B1. As the pH decreases, the B2 amplitude declines progressively. However, the B2 amplitude does not just eventually become zero but rather undergoes a polarity reversal when the pH declines below 2.7, i.e. the B2 component acquires the same polarity as the B1 component below pH 2.7. Its amplitude grows as pH decreases further. Experimentally, the polarity reversal is demonstrated by a pH titration, in which successive signals were recorded as the pH is reduced in small decrements (about 0.2–0.3 pH units). The null point of titration, where the B2 component is completely absent (pH 2.7), is determined by the superposition of the signal (from a Triss-Montal film) and the standard pure B1 signal from a multilayered thin film after normalization. The justification of this titration procedure is based on an equivalent circuit analysis previously established.

Photoinduced changes in structure and function of hexadecyl merocyanine dyes incorporated into lipid membranes

In the purple membrane of Halobacterium halobium, bacteriorhodopsin (BR) acts as a proton pump leading to a transmembrane potential difference. A biomimetic system can be formed using merocyanine derivative dye which, when incorporated into a bilayer lipid membrane, creates a potential difference across the membrane. In this system, the dye molecules in combination with the lipid molecules act as a proton transport pump due to a light-induced conformational change of the dye. In this paper, we report the photovoltaic properties and crystal structure of hexadecyl merocyanine dye, together with the change in peak intensity in the X-ray diffraction pattern of the dye and dye-lipid system after illumination. We show that, at room temperature ( σ300 K), merocyanine dyes exhibit a crystalline form with a long-chain, fibre-like structure (diameter, 2.89 nm). However, on interaction with light, the crystalline form of the dye changes but the diameter remains the same.

Fundamentals of photoelectric effects in molecular electronic thin film devices: applications to bacteriorhodopsin-based devices

This tutorial lecture focuses on the fundamental mechanistic aspects of light-induced charge movements in pigment-containing membranes. The topic is relevant to molecular electronics because many prototypes optoelectronic devices are configured as pigment-containing thin films. We use reconstituted bacteriorhodopsin membranes as an example to illustrate the underlying principle of measurements and data interpretation. Bacteriorhodopsin, a light-driven proton pump, is the only protein component in the purple membrane of Halobacterium halobium. It resembles the visual pigment rhodopsin chemically but performs the function of photosynthesis. Bacteriorhodopsin thus offers an unprecedented opportunity for us to compare the visual photoreceptor and the photosynthetic apparatus from a mechanistic point of view. Bacteriorhodopsin, well known for its exceptional chemical and mechanical stability, is also a popular advanced biomaterial for molecular device construction. The tutorial approaches the subject from two angles. First, the fundamental photoelectric properties are exploited for device construction. Second, basic design principles for photosensors and photon energy converters can be elucidated via ‘reverse engineering’. The concept of molecular intelligence and the principle of biomimetic science are discussed.

Theory of second harmonic generation in randomly oriented species

Abstract It is well known that second harmonic generation (SHG) is a process forbidden within atomic and molecular fluids. Nonetheless recent experimental observations of second harmonic evolution in suspensions of randomly oriented Halobacterium halobium purple membranes have raised new questions about the precise criteria which determine prohibition of the second harmonic. To address the problem a theoretical framework for SHG is developed that specifically deals with molecular systems, and is therefore cast in terms of molecular properties with more regard to the influence of the local structure. This contrasts with the classical approach based on bulk susceptibilities, which has not proved adequate to explain the conflicting experimental results. By properly formulating the detailed procedure for dealing with the necessary orientational averages, the present theory discloses a relationship between the coherent process of second harmonic generation and a directed component of its incoherent counterpart, hyper-Rayleigh scattering. Inter alia, the theory explains the SHG detected in purple membrane suspensions. The polarisation features of the harmonic evolution are also considered more generally, and in particular it is shown that the SHG signal will persist under conditions of circularly polarised pumping. This specific polarisation feature will allow experimental validation of the theory.

LASER PHOTOLYSIS OF THE PURPLE MEMBRANE OF Halobacterium halobium IN THE PHOTOSTATIONARY STATE: THE PHOTOBRANCHING PROCESS FROM THE O640 INTERMEDIATE

Abstract The photobranching process from the O640 intermediate (O) in the photocycle of bacteriorhodopsin was studied. The O form accumulated with continuous wave visible light (390–800 nm) irradiation of the acidic (pH 3.9–6.0) purple membrane of Halobacterium halobium at 22°C. The photocycle of O via an L‐like (or N‐like) intermediate was driven by 630 nm pulsed light. The newly found intermediate has an absorption band in the 450–560 nm region. The “green‐light”‐absorbing pigment, tentatively called G520, was converted to O with a time constant of (1.2 ± 0.2) ms. No M‐like species was found in the cycle. The quantum yield of the cycle was estimated to be 0.30 ± 0.15.

Probing toward atomic resolution in molecular topography.

Probing toward atomic resolution in molecular topography.

Nonlinear optical properties of a bacteriorhodopsin film in a Fabry–Perot cavity

Bacteriorhodopsin is a photosensitive molecule found in the purple membrane of Halobacterium halobium. To investigate the possibility of using photoinduced changes in its refractive index in the 650-800-nm region for photonic switching, we prepared solid dry bacteriorhodopsin films with low scattering and examined their optical properties in a Fabry-Perot cavity. Interference fringes, generated on irradiation of the cavity with a near-IR laser beam at 792 nm, were shifted by simultaneous exposure to a 5-mW/mm(2) beam at 543.5 nm. This result indicates that such a Fabry-Perot cavity may be used as a photonically controlled spatial light modulator.

Time-Resolved Second Harmonic Generation in the Randomly Oriented Purple Membrane

We have observed second harmonic generation (SHG) at 532 nm in randomly oriented suspensions of the purple membrane of Halobacterium halobium. The intensity of the second harmonic can be switched by photoexcitation of the bacteriorhodopsin (BR) chromophore at 570 nm. The SHG intensity decreases in <1 μs and reaches a minimum in <1 ms with the formation ofthe M state of BR. For dilute samples, we have observed photoinduccd changes in the SHG intensity of up to 90%. At low concentrations, the second harmonic intensity in light- adapted BR depends linearly on concentration, while showing saturation at higher concentrations. The linear concentration dependence shows that coherence of the second-order polarization between membrane particles is weak

Dynamical transition of bacteriorhodopsin in purple membranes revealed by neutron scattering: a relation between structure, dynamics and function

The internal motions of bacteriorhodopsin, the integral membrane protein in purple membranes of Halobacterium halobium, have been studied by neutron scattering as a function of different external conditions. The internal dynamics exhibit a transition above a critical temperature of 230 K from a harmonic to an anharmonic regime, provided the hydration of the membranes is sufficient. We found this onset of large amplitude motions correlated with the appearance of protein function under certain conditions of temperature and relative humidity.

Recording of transient gratings using the short lived bacteriorhodopsin photocycle intermediates

Bacteriorhodopsin, a light‐transducing protein in the purple membrane of Halobacterium halobium, has unique properties that make it a potentially attractive material for use in optical imaging and processing. A transient diffraction grating based on the K intermediate of the bacteriorhodopsin photocycle has been produced on the nanosecond time scale. The diffraction efficiency, which would be sufficient for pattern recognition for example, was measured and the intensity dependence assessed.

Thermal motions and function of bacteriorhodopsin in purple membranes: effects of temperature and hydration studied by neutron scattering.

The internal dynamics of bacteriorhodopsin, the light-driven proton pump in the purple membrane of Halobacterium halobium, has been studied by inelastic neutron scattering for various conditions of temperature and hydration. Light activation can take place when the membrane is vibrating harmonically. The ability of the protein to functionally relax and complete the photocycle initiated by the absorption of a photon, however, is strongly correlated with the onset of low-frequency, large-amplitude anharmonic atomic motions in the membrane. For a normally hydrated sample, this occurs at about 230 K, where a dynamical transition from a low-temperature harmonic regime is observed. In moderately dry samples, on the other hand, in which the photocycle is slowed down by several orders of magnitude, no transition is observed and protein motions remain approximately harmonic up to room temperature. These results support the hypothesis, made from previous neutron diffraction studies, that the "softness" of the membrane modulates the function of bacteriorhodopsin by allowing or not allowing large-amplitude motions in the protein.