Deposition of bacteriorhodopsin protein in a purple membrane form on nitrocellulose membranes for enhanced photoelectric response.

Bacteriorhodopsin protein (bR)-based systems are one of the simplest known biological energy converters. The robust chemical, thermal and electrochemical properties of bR have made it an attractive material for photoelectric devices. This study demonstrates the photoelectric response of a dry bR layer deposited on a nitrocellulose membrane with indium tin oxide (ITO) electrodes. Light-induced electrical current as well as potential and impedance changes of dried bR film were recorded as the function of illumination. We have also tested bR in solution and found that the electrical properties are strongly dependent on light intensity changing locally proton concentration and thus pH of the solution. Experimental data support the assumption that bR protein on a positively charged nitrocellulose membrane (PNM) can be used as highly sensitive photo- and pH detector. Here the bR layer facilitates proton translocation and acts as an ultrafast optoelectric signal transducer. It is therefore useful in applications related to bioelectronics, biosensors, bio-optics devices and current carrying junction devices.

Potential applications of bacteriorhodopsin mutants.

Bacteriorhodopsin (BR), a model system in biotechnology, is a G-protein dependent trans membrane protein which serves as a light driven proton pump in the cell membrane of Halobacterium salinarum. Due to the linkage of retinal to the protein, it seems colored and has numbers of versatile properties. As in vitro culture of the Halobacteria is very difficult, and isolation is time consuming and usually inefficient, production of genetically modified constructs of the protein is essential. There are three important characteristics based on protein catalytic cycle and molecular functions of photo-electric, photochromic and proton transporting, which makes this protein as a strategic molecule with potential applications in biotechnology. Such applications include protein films, used in artificial retinal implants, light modulators, three-dimensional optical memories, color photochromic sensors, photochromic and electrochromic papers and ink, biological camouflage and photo detectors for biodefense and non-defense purposes.

Charge transport in purple membrane monolayers: a sequential tunneling approach.

Current-voltage (I-V) characteristics in proteins are sensitive to conformational changes induced by an external stimulus (photons, chemical, etc.). This sensitivity can be used in medical and industrial applications as well as shedding new light on the microscopic structure of biological materials. Here, we show that a sequential tunneling model of carrier transfer between neighboring amino acids in a single protein is the basic mechanism responsible for the electrical properties measured over a wide range of applied potentials. We also show that such a strict correlation between the protein structure and the electrical response can lead to a new generation of nanobiosensors that mimic the sensorial activity of living species. To demonstrate the potential usefulness of protein electrical properties, we provide a microscopic interpretation of recent I-V experiments carried out in bacteriorhodopsin at a nanoscale length.

Sensitive detection of protein-lipid interaction change on bacteriorhodopsin using dodecyl ß-D-maltoside.

A light-driven proton pump bacteriorhodopsin (bR) forms a two-dimensional hexagonal lattice with about 10 archaeal lipids per monomer bR on purple membrane (PM) of Halobacterium salinarum. In this study, we found that the weakening of the bR-lipid interaction on PM by addition of alcohol can be detected as the significant increase of protein solubility in a nonionic detergent, dodecyl ß-D-maltoside (DDM). The protein solubility in DDM was also increased by bR-lipid interaction change accompanied by structural change of the apoprotein after retinal removal and was about 7 times higher in the case of completely bleached membrane than that of intact PM. Interestingly, the cyclic and milliseconds order of structural change of bR under light irradiation also led to increasing the protein solubility and had a characteristic light intensity dependence with a phase transition. These results indicate that there is a photointermediate in which bR-lipid interaction has been changed by its dynamic structural change. Because partial delipidation of PM by CHAPS gave minor influence for the change of the protein solubility compared to intact PM in both dark and light conditions, it is suggested that specific interactions of bR with some lipids which remain on PM even after delipidation treatment have a key role for the change of solubility in DDM induced by alcohol binding, ligand release, and photon absorption on bR.

Structural changes in bacteriorhodopsin in purple membranes induced by irreversible photobleaching with heterogeneous and homogeneous stability.

Kinetic studies of irreversible photobleaching of bacteriorhodopsin (bR) in purple membrane (PM) at neutral pH have previously indicated the existence of two kinds of species which differ in their structural stability. bR was shown to have kinetically slow- and fast-decayed components with the faster one increasing with changes in intra- and intermolecular structures in darkness. However, our recent work reported that photobleaching kinetics above pH 10 were characterized by a single-decay component. In order to elucidate the factors responsible for the heterogeneous or homogeneous stability of photobleaching, we conducted investigations into the structural changes in bR in PM induced by photobleaching at pH 7 and 11 by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. ATR-FTIR spectra of bR photobleached at pH 7 and 11 showed that an increase in IR peak intensity at 1656 cm(-1) occurred simultaneously with decreases at 1666 cm(-1), indicating an alpha(II)-to-alpha(I) transition in transmembrane helices during photobleaching. The most significant change in IR spectra occurred at 1626 cm(-1) for samples photobleached at pH 7, and was attributed to structures formed between adjacent molecules. The origin of the heterogeneity of photobleaching is discussed on the basis of structural characteristics found in the bleached membranes.

Inter-helical hydrogen bonds are essential elements for intra-protein signal transduction: the role of Asp115 in bacteriorhodopsin transport function.

The behavior of the D115A mutant was analyzed by time-resolved UV-Vis and Fourier transformed infrared (FTIR) spectroscopies, aiming to clarify the role of Asp115 in the intra-protein signal transductions occurring during the bacteriorhodopsin photocycle. UV-Vis data on the D115A mutant show severely desynchronized photocycle kinetics. FTIR data show a poor transmission of the retinal isomerization to the chromoprotein, evidenced by strongly attenuated helical changes (amide I), the remarkable absence of environment alterations and protonation/deprotonation events related to Asp96 and direct Schiff base (SB) protonation form the bulk. This argues for the interactions of Asp115 with Leu87 (via water molecule) and Thr90 as key elements for the effective and vectorial proton path between Asp96 and the SB, in the cytoplasmic half of bacteriorhodopsin. The results strongly suggest the presence of a regulation motif enclosed in helices C and D (Thr90-Pro91/Asp115) which drives properly the dynamics of helix C through a set of interactions. It also supports the idea that intra-helical hydrogen bonding clusters in the buried regions of transmembrane proteins can be potential elements in intra-protein signal transduction.

Multivariate curve resolution of rapid-scan FTIR difference spectra of quinone photoreduction in bacterial photosynthetic membranes.

Photosynthetic reaction centres and membranes are systems of particular interest and are often taken as models to investigate the molecular mechanisms of selected bioenergetic reactions. In this work, a multivariate curve resolution by alternating least squares procedure is detailed for resolution of time-resolved difference FTIR spectra probing the evolution of quinone reduction in photosynthetic membranes from Rhodobacter sphaeroides under photoexcitation. For this purpose, different data sets were acquired in the same time range and spectroscopic domain under slightly different experimental conditions. To enable resolution and provide meaningful results the different data sets were arranged in an augmented matrix. This strategy enabled recovery of three different species despite rank-deficiency conditions. It also results in better definition (identity and evolution) of the contributions. From the resolved spectra, the species have been attributed to: 1. the formation of ubiquinol, more precisely the disappearance of Q/appearance of QH(2); 2. conformational change of the protein in the surrounding biological medium; 3. oxidation of diaminodurene, a redox mediator. Because, moreover, results obtained from augmented data sets strategies enable quantitative and qualitative interpretation of concentration profiles, other effects, for example the consequence of repeated light excitation of the same sample, choice of illumination power, or the number of spectra accumulated could be compared and discussed.

Measurement of the optical parameters of purple membrane and plant light-harvesting complex films with optical waveguide lightmode spectroscopy.

Purple membrane (bacteriorhodopsin) and plant light-harvesting complexes (LHCII) were dried on the optical waveguide sensor with varying thicknesses in a wide range (from 20 to several hundreds of nanometers) and the optical parameters were studied with optical waveguide lightmode spectroscopy. It was found that applying the approximate 4-layer mode equations for the measured effective refractive indices resulted in unacceptable results for the optical parameters: with increasing thickness the refractive index decreased monotonously from 1.5 to 1.1. Therefore an inverse waveguide numerical method was developed and used to obtain reliable results from the experiments. The inverse method yielded an approximately constant (1.53) refractive index independently of the thickness for the purple membrane and LHCII films. Light-induced changes in the optical parameters of the purple membrane and LHCII films were also studied. For purple membrane films the most significant effect is the change in refractive index and absorption. For LHCII films prolonged illumination induced irreversible structural changes, most probably of thermo-optic origin.

High-contrast, all-optical switching in bacteriorhodopsin films.

We report experiments with nonlinear-absorption-based, high-contrast, all-optical switching in photochromic bacteriorhodopsin (BR) films. The switching action is accomplished by control of the transmission of a weak probe beam through a BR sample with the help of strong pump beam illumination at 532 nm wavelength. We found that the switching properties of BR films depend on several experimentally controllable parameters such as probe wavelength, pump beam intensity, and excitation rate. A comparative study of the switching behavior and other parameters of practical use was carried out at three probe wavelengths (543, 594, and 633 nm) and various beam powers and pump excitation rates. The results are presented for commercially available wild-type and D96N variant BR films.

Photocurrents generated by bacteriorhodopsin adsorbed on nano-black lipid membranes.

Purple membranes were adsorbed on freestanding lipid bilayers, termed nano-black lipid membranes (nano-BLMs), suspending the pores of porous alumina substrates with average pore diameters of 280 nm. Nano-BLMs were obtained by first coating the upper surface of the highly ordered porous alumina substrates with a thin gold layer followed by chemisorption of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol and subsequent addition of a droplet of 1,2-diphytanoyl-sn-glycero-3-phosphocholine and octadecylamine dissolved in n-decane onto the hydrophobic submonolayer. By means of impedance spectroscopy, the quality of the nano-BLMs was verified. The electrical parameters confirm the formation of single lipid bilayers with high membrane resistances covering the porous matrix. Adsorption of purple membranes on the nano-BLMs was followed by recording the photocurrents generated by bacteriorhodopsin upon continuous light illumination. The membrane system exhibits a very high long-term stability with the advantage that not only transient but also stationary currents are recordable. By adding the proton ionophore carbonyl cyanide-m-chlorophenylhydrazone the conductivity of the nano-BLMs increases, resulting in a higher stationary current, which proves that proton conductance occurs across the nano-BLMs.

Photoelectric response of polarization sensitive bacteriorhodopsin films.

Polarization sensitivity is introduced into oriented bacteriorhodopsin (BR) films through a photochemical bleaching process, which chemically modifies the structure of the purple membrane by breaking the intrinsic symmetry of the membrane-bound BR trimers. The resulting photovoltage generated in an indium-tin oxide (ITO)/BR/ITO detector is found to be anisotropic with respect to cross-polarized probe beams. A model, based on the polarization dependent photoselection of the BR molecules qualitatively explains the photochemical bleaching process and the observed anisotropic response. The effect reported here can be used to construct a polarization sensitive BR-based bio-photoreceiver.

Light adaptation of bacteriorhodopsin correlates with dielectric spectral kinetics in purple membrane.

The retinal protein, bacteriorhodopsin (bR), has several potential bioelectronic applications and it is considered as a model for G-protein coupled receptors. Its electrical parameters, therefore, deserve particular attention. Such parameters could be determined by virtue of studying its dielectric spectrum in the low frequency range (20 Hz-1 MHz). The kinetics of dark-light adaptation of bR is reported in terms of electrical parameters of the purple membrane (PM) containing bR. The data have exhibited sudden pronounced increase in the ac-conductivity, upon illuminating the dark-adapted bR (DA-bR), which may be considered in further implications of bR for biotechnological applications. These changes turned out to be composed of, at least, two growing exponential components: one relatively fast followed by slower one. Their lifetime ratio exhibited decreases with increasing the frequency; meanwhile, their amplitude ratio displayed very exciting behavior at significant frequencies. This may correlate the kinetics of light adaptation to relaxations in PM. Moreover, the light adaptation has been observed to cause initial fast and large decreases in dc-conductivity with subsequent slower and smaller decreases. Changing the conductivity during the time of light adaptation reflects changes in the surface charge of the PM. The lifetimes of these events, therefore, help follow the kinetics of the pathway of conformational changes that might be occurring during light adaptation. The dipole moment (permanent and induced) of PM, in addition to, its size showed one exponential growth of comparable lifetime (approximately 7 min) during the light adaptation. The variation in PM size from dark to light state should be in keeping with that diffusion may influence the three-dimensional data storage in data processing based on bR.

Proton transfer dynamics at the membrane/water interface: dependence on the fixed and mobile pH buffers, on the size and form of membrane particles, and on the interfacial potential barrier.

Crossing the membrane/water interface is an indispensable step in the transmembrane proton transfer. Elsewhere we have shown that the low dielectric permittivity of the surface water gives rise to a potential barrier for ions, so that the surface pH can deviate from that in the bulk water at steady operation of proton pumps. Here we addressed the retardation in the pulsed proton transfer across the interface as observed when light-triggered membrane proton pumps ejected or captured protons. By solving the system of diffusion equations we analyzed how the proton relaxation depends on the concentration of mobile pH buffers, on the surface buffer capacity, on the form and size of membrane particles, and on the height of the potential barrier. The fit of experimental data on proton relaxation in chromatophore vesicles from phototropic bacteria and in bacteriorhodopsin-containing membranes yielded estimates for the interfacial potential barrier for H(+)/OH(-) ions of approximately 120 meV. We analyzed published data on the acceleration of proton equilibration by anionic pH buffers and found that the height of the interfacial barrier correlated with their electric charge ranging from 90 to 120 meV for the singly charged species to >360 meV for the tetra-charged pyranine.

Characteristics and mechanisms of the two types of photoelectric differential response of bacteriorhodopsin-based photocell.

Bacteriorhodopsin (BR)-based photocells have been assigned possessing differential photoelectric response. But recently we found that the differential response described before, which occurred in milliseconds to seconds, outputting a positive pulse when light was on and a negative pulse when light was off, was not the intrinsic property of the BR molecule. It was partially caused by the measuring circuit. By measuring the photoelectric response signal of the BR film photocell to a short laser pulse, the impulse response function of the BR film photocell was obtained by data fitting with MATLAB software. A simulation system was accordingly developed. The output response signals of the BR film photocell under different stepping incident light were calculated. By simulation and analysis, it was concluded that the differential response caused by the intrinsic property of the BR molecule happened in microseconds time scale, and it produced a negative pulse when light was on and a positive pulse when light was off. It was much faster but much weaker than that described before.

Late events in the photocycle of bacteriorhodopsin mutant L93A.

In the photocycle of bacteriorhodopsin (bR) from Halobacterium salinarum mutant L93A, the O-intermediate accumulates and the cycling time is increased approximately 200 times. Nevertheless, under continuous illumination, the protein pumps protons at near wild-type rates. We excited the mutant L93A in purple membrane with single or triple laser flashes and quasicontinuous illumination, (i.e., light for a few seconds) and recorded proton release and uptake, electric signals, and absorbance changes. We found long-living, correlated, kinetic components in all three measurements, which-with exception of the absorbance changes-had not been seen in earlier investigations. At room temperature, the O-intermediate decays to bR in two transitions with rate constants of 350 and 1800 ms. Proton uptake from the cytoplasmic surface continues with similar kinetics until the bR state is reestablished. An analysis of the data from quasicontinuous illumination and multiple flash excitation led to the conclusion that acceleration of the photocycle in continuous light is due to excitation of the N-component in the fast N<-->O equilibrium, which is established at the beginning of the severe cycle slowdown. This conclusion was confirmed by an action spectrum.

Photoexcitation of the O-intermediate in bacteriorhodopsin mutant L93A.

During the extended lifetime of the O-state in bacteriorhodopsin (bR) mutant L93A, two substates have been distinguished. The first O-intermediate (OI) is in rapid equilibrium with N and apparently still has a 13-cis chromophore. OI undergoes a photoreaction with a small absorbance change, positive charge transport in the pumping direction, and proton release and uptake. None of these effects was detected after photoexcitation of the late O (OII). The most likely interpretation of the effects seen is an accelerated return of the molecule from the OI- to the bR-state. However, with a lifetime approximately 140 ms, the reaction cannot account for the observed high pumping efficiency of the mutant under continuous illumination. We suggest that OII corresponds to the O-intermediate with a twisted all-trans chromophore seen in the photocycle of wild-type bR, where the 13-cis OI-intermediate under the usual conditions does not accumulate in easily detectable amounts and, therefore, has generally been overlooked. Both the OI- and OII-decays are apparently strongly inhibited in the mutant.

Bacteriorhodopsin analog regenerated with 13-desmethyl-13-iodoretinal.

The retinal analog 13-desmethyl-13-iodoretinal (13-iodoretinal) was newly synthesized and incorporated into apomembranes to reconstitute bacteriorhodopsin analog 13-I-bR. The absorption maximum was 598 nm and 97% of the chromophore was an all-trans isomer in the dark- and light-adapted state. Upon flash illumination, 13-I-bR underwent a transient spectral change in which a shorter wavelength intermediate (lambda(max) = 426 nm) similar to the M species of the native bR developed. Also, 13-I-bR showed light-induced proton pumping with rates and extents comparable to those seen in the native bR. The ultraviolet circular dichroism (CD) spectrum originating from the aromatic groups was different from that of the native bR, indicating that the substituted bulky iodine atom strongly interacts with neighboring amino acids. A projection difference Fourier map showed the labeled iodine was in the vicinity of helix C. 13-I-bR is an advantageous specimen for kinetic investigations of light-induced structural changes associated with the proton pumping cycle by x-ray diffraction.

Mechanisms of pulse response and differential response of bacteriorhodopsin and their relations.

Bacteriorhodopsin (BR) films are oriented and deposited on indium tin oxide conductive glass by using electrophoretic sedimentation and Langmuir-Blodgett methods to construct sandwich-type photocells, respectively. The pulse response photoelectric signal of the BR photocell under pulsed laser and the differential response photoelectric signal under irradiation of interval light are measured. The origins of these two types of photoelectric responses and their correlations are analyzed. The pulse response signal initiates from the ultrafast charge separation of the retinal and the proton translocation followed by the deprotonation and reprotonation of the Schiff base and its surrounding amino acids. This is a quick response and is the preceding reaction of the differential response. The differential response signal is caused by the charging and discharging of the continuous proton current of the BR light-driven proton pump at light-on and light-off, which is a slow process. The differential response is related to not only the construction of the BR photocell but also the coupling mode of measurement. To observe the differential response signal, the BR photocell must have large enough B3 and B3' components in its pulse response as well as an alternative coupling mode to measure it.

Specific damage induced by X-ray radiation and structural changes in the primary photoreaction of bacteriorhodopsin.

Bacteriorhodopsin, the sole membrane protein of the purple membrane of Halobacterium salinarum, functions as a light-driven proton pump. A 3-D crystal of bacteriorhodopsin, which was prepared by the membrane fusion method, was used to investigate structural changes in the primary photoreaction. It was observed that when a frozen crystal was exposed to a low flux of X-ray radiation (5 x 10(14)photons mm(-2)), nearly half of the protein was converted into an orange species, exhibiting absorption peaks at 450 nm, 478 nm and 510 nm. The remainder retained the normal photochemical activity until Asp85 in the active site was decarboxlyated by a higher flux of X-ray radiation (10(16)photons mm(-2)). The procedure of diffraction measurement was improved so as to minimize the effects of the radiation damage and determine the true structural change associated with the primary photoreaction. Our structural model of the K intermediate indicates that the Schiff base linkage and the adjacent bonds in the polyene chain of retinal are largely twisted so that the Schiff base nitrogen atom still interacts with a water molecule located near Asp85. With respect to the other part of the protein, no appreciable displacement is induced in the primary photoreaction.