Purple Membrane Fragments Research Articles

Lee's transient protonic capacitor cannot explain the surface proton current observed in bacteriorhodopsin purple membranes

Recently in this Journal, James Lee employed his transmembrane electrostatically localized proton (TELP) hypothesis and the notion of a transient protonic capacitor to explain the force holding protons at the surface of bacteriorhodopsin purple membrane fragments. Here we show that purple membrane fragments cannot maintain the requisite transient non-zero transmembrane potential, and even if they could, it would not support the surface proton current moving from the P side to the N side that was reported by Heberle et al. (Nature, 1994). Currently accepted models explain the force keeping protons at the membrane surface by invoking the unusual structure of water at the interface which serves to stabilize the proton (energy well) and/or raise the activation ∆G‡ (energy barrier) for release to the bulk phase. Any future invocations of TELP should be required to include experimental measurements carried out at the surfaces of lipid bilayer membranes and/or biological membranes.

Enhanced hydrogen production of Rhodobacter sphaeroides promoted by extracellular H+ of Halobacterium salinarum

PurposeThis study utilized the principle that the bacteriorhodopsin (BR) produced by Halobacterium salinarum could increase the hydrogen production of Rhodobacter sphaeroides. H. salinarum are co-cultured with R. sphaeroides to determine the impact of purple membrane fragments (PM) on R. sphaeroides and improve its hydrogen production capacity.MethodsIn this study, low-salinity in 14 % NaCl domesticates H salinarum. Then, 0–160 nmol of different concentration gradient groups of bacteriorhodopsin (BR) and R. sphaeroides was co-cultivated, and the hydrogen production and pH are measured; then, R. sphaeroides and immobilized BR of different concentrations are used to produce hydrogen to detect the amount of hydrogen. Two-chamber microbial hydrogen production system with proton exchange membrane-assisted proton flow was established, and the system was operated. As additional electricity added under 0.3 V, the hydrogen production rate increased with voltages in the coupled system.ResultsH salinarum can still grow well after low salt in 14% NaCl domestication. When the BR concentration is 80 nmol, the highest hydrogen production reached 217 mL per hour. Both immobilized PC (packed cells) and immobilized PM (purple membrane) of H. salinarum could promote hydrogen production of R. sphaeroides to some extent. The highest production of hydrogen was obtained by the coupled system with 40 nmol BR of immobilized PC, which increased from 127 to 232 mL, and the maximum H2 production rate was 18.2 mL−1 h−1 L culture. In the 192 h experiment time, when the potential is 0.3 V, the hydrogen production amount can reach 920 mL, which is 50.3% higher than the control group.ConclusionsThe stability of the system greatly improved after PC was immobilized, and the time for hydrogen production of R. sphaeroides significantly extended on same condition. As additional electricity added under 0.3 V, the hydrogen production rate increased with voltages in the coupled system. These results are helpful to build a hydrogen production-coupled system by nitrogenase of R. sphaeroides and proton pump of H. salinarum.Graphical abstract

Structure retention of silica gel-encapsulated bacteriorhodopsin in purple membrane and in lipid nanodiscs

The integral membrane protein, bacteriorhodopsin (BR) was encapsulated in sol-gel derived porous silica gel monoliths in native purple membrane (BR-PM) and synthetic lipid nanodisc (BR nanodisc) environments. BR nanodiscs were synthesized by solubilizing purple membrane in discoidal phospholipid bilayer stabilized by amphipathic Styrene-Maleic Acid (SMA) copolymer. UV–vis absorbance spectroscopy and dynamic-light scattering indicated the formation of BR monomers solubilized in lipid nanodiscs 10.2 ± 0.7 nm in average diameter. Fluorescence and absorbance spectroscopic techniques were utilized to probe conformational, environmental, and rotational changes associated with the tryptophan residues and the covalently-bound retinal moiety of BR upon entrapment in the silica matrix. We show that the immobilized BR in both membrane environments retained its bound retinal cofactor and the ability of the cofactor to undergo conformational changes upon light illumination necessary for BR’s activity as a proton transporter. For purple membrane fragments, the results indicated that the local pH in the pores around BR after encapsulation was important for its stability at temperatures higher than 50 °C. Under the same buffering conditions, retinal was released from silica-encapsulated BR-PM and BR nanodiscs beginning at 80 °C (without a conformational change) and 50 °C (with a conformational change), respectively, reflecting differences in protein-protein (trimeric vs. monomeric) and protein-lipid interactions.

Microscopic Determination of Second-Order Nonlinear Optical Susceptibility Tensors

We demonstrate a microscopy technique that extracts tensorial information about the second-order nonlinear optical susceptibility and hyperpolarizability of molecular materials. Our technique is based on polarization-dependent second-harmonic generation and a genetic algorithm, using which the best possible match with the measured data, and the possible susceptibility tensor components are found. In contrast to existing techniques, which access only the magnitude of the nonlinear response, our technique also provides information about the phase of the tensor components, which is associated with molecular resonances. After verifying the technique using simulated model structures with well-known symmetries, we demonstrate its capabilities using model surface samples consisting of single purple membrane (PM) fragments of bacteriorhodopsin (bR) chromoproteins. Since the supramolecular structures of PM, bR, and photoactive retinal molecules are known, complex-valued tensorial information on the molecular hyper...

Measuring Photochemical Kinetics in Submonolayer Films by Transient ATR Spectroscopy on a Multimode Planar Waveguide

Understanding the kinetics of reactions in molecular thin films can aid in the molecular engineering of organic photovoltaics and biosensors. We have coupled two analytical methods, transient absorbance spectroscopy (TAS) and attenuated total reflectance (ATR), in a relatively simple arrangement when compared with previous TAS/ATR instruments to interrogate molecular structure and photochemistry at interfaces. The multimode planar waveguide geometry provides a significant path length enhancement relative to a conventional transmission geometry, making it feasible to perform measurements on low-surface-coverage films. The performance of the instrument was assessed using a thin film composed of purple membrane (PM) fragments containing bacteriorhodopsin deposited onto PDAC, a positively charged polymer. The surface coverage of retinal chromophore in this film is ∼0.1 monolayer and its orientation distribution is anisotropic, with a mean tilt angle of 68° from surface normal. After photoinduced formation of the transient M state, the chromophore decays to the ground state in 4.4 ± 0.6 ms, equivalent to the decay of suspended PM fragments, which shows that deposition on PDAC does not alter M-state photokinetics. The surface coverage of the M state is calculated to be 2 pmol/cm(2), which is ∼1% of a close-packed monolayer. This work demonstrates that TAS/ATR can be used to probe structure and photochemical kinetics in molecular films at extremely low surface coverages.

Casein Aggregates Built Step-by-Step on Charged Polyelectrolyte Film Surfaces Are Calcium Phosphate-cemented

The possible mechanism of casein aggregation and micelle buildup was studied in a new approach by letting α-casein adsorb from low concentration (0.1 mg·ml(-1)) solutions onto the charged surfaces of polyelectrolyte films. It was found that α-casein could adsorb onto both positively and negatively charged surfaces. However, only when its negative phosphoseryl clusters remained free, i.e. when it adsorbed onto a negative surface, could calcium phosphate (CaP) nanoclusters bind to the casein molecules. Once the CaP clusters were in place, step-by-step building of multilayered casein architectures became possible. The presence of CaP was essential; neither Ca(2+) nor phosphate could alone facilitate casein aggregation. Thus, it seems that CaP is the organizing motive in the casein micelle formation. Atomic force microscopy revealed that even a single adsorbed casein layer was composed of very small (in the range of tens of nanometers) spherical forms. The stiffness of the adsorbed casein layer largely increased in the presence of CaP. On this basis, we can imagine that casein micelles emerge according to the following scheme. The amphipathic casein monomers aggregate into oligomers via hydrophobic interactions even in the absence of CaP. Full scale, CaP-carrying micelles could materialize by interlocking these casein oligomers with CaP nanoclusters. Such a mechanism would not contradict former experimental results and could offer a synthesis between the submicelle and the block copolymer models of casein micelles.

Unfolding study of native bacteriorhodopsin under acidic condition

In this study, we measured the structural properties of “blue membrane”, which is specific formation of a purple membrane (PM) upon acid titration or removal of cations, by the force curve measurement mode of an atomic force microscopy. The PM fragments were immobilized on a glass substrate and force curve measurements were carried out on the fragments under neutral (pH 7.2) and acidic (pH 2.4) condition. The results revealed that peak positions of the unfolding spectra obtained under acidic condition were shifted to the shorter extension region than those at neutral pH and that the relative position of only the first peak was changed by about 5 nm. These results suggest the possibility that the specific secondary structure is formed at the site from its C-terminus to helices F in acidified PM.

Ultrasharp and high aspect ratio carbon nanotube atomic force microscopy probes forenhanced surface potential imaging

The resolution of scanning surface potential microscopy (SSPM) is mainly limited bynon-local electrostatic interactions due to the finite probe size. Here we present highresolution surface potential imaging with ultrasharp and high aspect ratio carbon nanotube(CNT) atomic force microscopy (AFM) probes fabricated via dielectrophoresis.Enhancement of surface potential contrast by several factors is reported for integratedcircuit structures and purple membrane fragments for these CNT AFM probesas compared to conventional probes. In particular, ultrahigh lateral resolution(∼2 nm) surface potential images of self-assembled bacteriorhodopsin proteins are reported atambient conditions, with the implication of label-free protein detection by SSPMtechniques.

Hydrogen gas production by combined systems of Rhodobacter sphaeroides O.U.001 and Halobacterium salinarum in a photobioreactor

Rhodobacter sphaeroides O.U.001 is a photosynthetic non-sulfur bacterium which produces hydrogen from organic compounds under anaerobic conditions. Halobacterium salinarum is an archaeon and lives under extremely halophilic conditions (4 M NaCl). H. salinarum contains a retinal protein bacteriorhodopsin in its purple membrane which acts as a light-driven proton pump. In this study the Rhodobacter sphaeroides O.U.001 culture was combined with different amounts of packed cells of H. salinarum S9 or isolated purple membrane fragments in order to increase the photofermentative hydrogen gas production. The packed cells of H. salinarum have the ability to pump protons upon illumination due to the presence of bacteriorhodopsin. The proton gradient produced may be used for the formation of ATP or protons may be used for H 2 production by R. sphaeroides. Similar to intact cells purple membrane fragments may also form vesicles around certain ions and may act like closed systems. The hydrogen production experiments were carried out using 400 ml water-jacketed-glass column stirred photobioreactors. In combined systems 10–200 nmol of bacteriorhodopsin was used. Hydrogen gas production was enhanced by four- to sixfold in combined systems of H. salinarum packed cells with R. sphaeroides O.U.001 cell. Stirring both increased the total gas produced and enhanced the rate of hydrogen production. The light energy conversion efficiency was increased from 0.6% to 2.25% in combined systems.

Fabrication and photoresponse of novel carboxymethylcellulose (CMC) based bacteriorhodopsin (bR) sensor

A single-pixel dry-type reconstituted bacteriorhodopsin (bR) sensor (Au/CMC + bR/ITO) incorporating carboxymethylcellulose (CMC) as the artificial membrane is fabricated according to a protocol developed to investigate signal acquisition from the proposed biosensor. Droplets of reconstituted purple membrane (PM) containing bR applied onto a gel-like solution of CMC placed on gold electrodes (anode) are allowed to dry under an electric field, utilising the PM fragment’s net negative charge and electric dipole moment. The resulting bR matrix thin film with high molecular orientation is finally sandwiched together with a semi-transparent indium titanium oxide (ITO) counter electrode and proper electrical connections made to form a single pixel bR-CMC photosensor. Efficient and reproducible photoresponse observed upon illumination clearly exhibits the potential of the proposed biosensor for future photosensing works.

Studies on poly(ethylenimine)/purple membrane multilayer films fabricated by layer-by-layer self-assembly

In the present work, we have obtained organized PEI/PM (poly(ethylenimine)/purple membrane) multilayer films by layer-by-layer alternate adsorptions with positively charged polycation PEI and negatively charged purple membrane fragments. The results observed by UV–vis spectroscopy show that each transfer amount of PM fragments onto the substrate is equal and the characteristic absorption bands of bacteriorhodopsin (bR) are still preserved. AFM and SEM analysis investigated the morphology and cross-section of PM films, respectively. The photocurrent peaks of (PEI/PM) 6 multilayer films corresponding to light-on and light-off are ca. 440 and 250 nA/cm 2.

15N T2' relaxation times of bacteriorhodopsin transmembrane amide nitrogens.

15N T(2)' relaxation times of bacteriorhodopsin (BR) amide nitrogens were determined in the temperature range from 40 to -60 degrees C using a Hahn echo pulse sequence and proton decoupling during the echo and detection times. Using oriented membrane samples, with their bilayer normal parallel to the external magnetic field, the (15)N amide nitrogens belonging to the transmembrane helices could be selected for the analysis. The experiments were performed on purple membrane fragments (in which BR is organized in a 2D crystalline network) and on BR reconstituted into dimyristoylphosphatidylcholine at a 1:150 molar ratio (in which BR is in a freely diffusing monomeric state at 40 degrees C and in an aggregated state at 4 degrees C and below). The results are discussed in terms of helix dynamics, mosaic spread and resolution of the (15)N spectra for the various samples and experimental conditions.

Two Photobioelectrochemical Applications of Self-Assembled Films on Mercury

The homogeneous, defect-free surface of a hanging mercury drop electrode was used to self-assemble films apt for the investigation of two photobioelectrochemical systems. Monolayers of straight-chain C12-C18alkane-1-thiols were anchored to a hanging mercury drop electrode and a film of chlorophyll was self-assembled on the top of them. The dependence of the photocurrents generated by illumination of the chlorophyll film with red light, on the thickness of the alkane-1-thiol monolayer and the applied potential is discussed. The photocurrents of purple membrane fragments, adsorbed on a mixed hexadecane-1-thiol/ dioleoylphosphatidylcholine bilayer self-assembled on mercury, were investigated in the presence of sodium perchlorate, chloride and acetate. The effect of the anions on the kinetics of the light-driven proton transport by bacteriorhodopsin has been determined.

Fabrication and photoelectric response of poly(allylamine hydrochloride)/PM thin films by layer-by-layer deposition technique

Thin films of poly(allylamine hydrochloride) (PAH) and bacteriorhodopsin (bR) embedded in purple membrane (PM) have been prepared by layer-by-layer (LBL) self-assembly technique. The results obtained by UV–Vis spectroscopy and atomic force microscopy (AFM) analysis showed that the biological activity of bR was preserved and PM fragments could be well oriented onto the ITO substrate. A photo-electrochemical cell with the structure of ITO/(PAH/PM) n /electrolyte (0.5 M KCl)/Pt was fabricated and studied. The photocurrent peaks of (PAH/PM) 6 corresponding to light-on and light-off were about 200 and 100 nA/cm 2, respectively, with the former enhanced 30% higher than that of the reference films made of (PDAC/PM) 6.

Ion Carriers and Channels in Metal-Supported Lipid Bilayers as Probes of Transmembrane and Dipole Potentials

Mercury-supported hydrophilic spacers with a lipid bilayer on top are used as biomembrane models by incorporating the ion carrier valinomycin and the ion channel melittin in the bilayer. The same films supported by gold are used to incorporate valinomycin. The extrathermodynamic absolute potential difference across the mercury/water interphase is estimated from the difference in charge density between a bare mercury electrode and a mercury electrode coated with a self-assembled dioleoylphosphatidylcholine (DOPC) monolayer. This estimate is confirmed by the potential dependence of the stationary light-on current of purple membrane fragments adsorbed at the DOPC-coated mercury electrode. The surface dipole potential of a mercury-supported self-assembled monolayer of a hydrophilic spacer is estimated from diffuse-layer effects and from the previously determined value of the absolute potential difference across the mercury/water interphase. The zero potential difference across a DOPC bilayer freely suspended on top of the above hydrophilic spacer is estimated from the potential dependence of the conductance of the mercury-supported spacer/(lipid bilayer) film, upon incorporation of valinomycin and melittin. The latter measurements, besides confirming the previous estimates, are in agreement with those carried out on black lipid membranes, thus showing the validity of this mercury-supported biomembrane model. Finally, the absolute potential difference across the gold/water interphase is estimated from a comparison between the potential dependence of the conductance of mercury- and gold-supported spacer/(lipid bilayer) films incorporating valinomycin.

Photoelectric response of purple membrane fragments adsorbed on a lipid monolayer supported by mercury and characterization of the resulting interphase

Purple membrane (PM) fragments were adsorbed on a dioleoylphosphatidylcholine (DOPC) monolayer supported by mercury to investigate the kinetics of light-driven proton transport by bacteriorhodopsin (bR). PM fragments were also adsorbed on a mercury-supported triethyleneoxythiol (TET) monolayer. On both monolayers, the light-on current exhibits a finite, potential dependent stationary component that decreases linearly with a positive shift in the applied potential. The light-on and light-off capacitive photocurrents were interpreted on the basis of a simple equivalent circuit, which accounts for the potential dependence of the stationary light-on current. The potential of zero stationary current is about equal to +0.010 V vs. saturated calomel electrode (SCE) on DOPC-coated mercury. The absolute potential difference across the PM fragments adsorbed at this applied potential was estimated on the basis of extrathermodynamic considerations and amounts to about +260 mV; it compares favorably with the value, +250 mV, of the transmembrane potential of zero stationary current across an oocyte plasma membrane incorporating bR [Biophys. J. 74 (1998) 403.]. The effect of the proton pumping activity of photoexcited PM fragments on the electroreduction kinetics of ubiquinone-10 incorporated in the DOPC monolayer underlying the PM fragments was investigated.

Photocurrents Generated by Bacteriorhodopsin Adsorbed on Thiol/Lipid Bilayers Supported by Mercury

The kinetics of light-driven proton transport by bacteriorhodopsin (bR) were investigated over a broad pH range upon adsorbing purple membrane (PM) fragments on a mercury-supported mixed alkanethiol/phospholipid bilayer. The light-on and light-off capacitive photocurrents were measured under short-circuit conditions in the absence of photoartifacts. Using dioleoylphosphatidylcholine as the lipid monolayer, a bell-shaped curve of the peak current versus pH, with a maximum in the proximity of 6, was obtained. The analysis of the biphasic decay kinetics of the light-on and light-off currents allows an estimate of the pKa values for the steps releasing protons to, and taking up protons from, the bathing solution. In particular, the pKa values obtained from the light-off current (pK1 = 3.5, pK2 = 5.3, pK3 = 7.5, and pK4 = 9.0) suggest a mechanism similar to that proposed by Balashov et al. for dark adaptation, albeit in the opposite direction (Balashov, S. P.; Imasheva, E. S.; Govindjee, R.; Sheves, M.; Ebrey, T. G. Biophys. J. 1996, 70, 473). The time dependence of the light-on and light-off currents in the proximity of pH 6 is interpreted on the basis of both a simple equivalent circuit and a kinetic model making use of spectroscopic data available in the literature. When using dioleoylphosphatidylserine (DOPS) as the lipid monolayer, an inversion in the sign of both light-on and light-off currents, as well as a change in their shape and magnitude, was observed by increasing the pH above 9 and then, at all pH values from 9 to 1, by subsequently decreasing the pH on the same mercury-supported mixed alkanethiol/DOPS bilayer. The normal situation was restored only by adding sodium azide. This inversion in current and the notable hysteresis observed under these conditions are critically discussed.

Some bioelectrochemical applications of self-assembled films on mercury

The homogeneous, defect-free surface of a hanging mercury drop electrode (HMDE) is used to self-assemble films apt to the investigation of problems of biological interest. The electrochemical behavior in the dark of a film of chlorophyll (Chl) molecules self-assembled on mercury and the peculiar features of the photocurrents recorded upon illuminating this film at different applied potentials are discussed. The limiting flux of hydrated Tl + ions through the channel-forming polypeptide gramicidin (GR), incorporated in a dioleoylphosphatidylcholine (DOPC) monolayer self-assembled on mercury, is shown to be controlled both by diffusion and by a dehydration step, while the limiting flux of dehydrated Tl + ions stemming from Tl amalgam electrooxidation is exclusively controlled by the diffusion of thallium atoms within the amalgam. The photocurrents of purple membrane (PM) fragments adsorbed on a DOPC monolayer and on a mixed alkanethiol/DOPC bilayer self-assembled on mercury are examined to investigate the kinetics of light-driven proton transport by bacteriorhodopsin (bR). The photocurrents on the mixed bilayer are interpreted on the basis of a simple equivalent circuit and their pH dependence is analyzed to estimate the p K a values of the transitions releasing protons to, and taking up protons from the solution.

Orientationally Enhanced Photocurrent and Electrooptic Modulation Effects in Bacteriorhodopsin/Polyvinyl Alcohol Thin Films

Polymeric films of bacteriorhodopsin, formed by embedding purple membrane fragments into a poly(vinyl alcohol) matrix without field poling, exhibit modulated photocurrent and electrooptic effects when an AC modulating rectangular voltage or a sinusoidal voltage is applied across the films. When the modulating frequencies are lower than 100 Hz, both effects are enhanced by the orientation of the purple membrane fragments and exhibit two different orientational time constants. The orientational enhancement at low frequencies (∼7.1 Hz) arises from the permanent dipole moments of membranes, and the enhancement at higher frequencies (∼74.8 Hz) is attributed to the induced dipole moments of the purple membrane molecules. For quantitative analyses of these processes, a theoretical model is proposed. From the combination of model calculations and fitted experimental results, we determine that the permanent dipole moment of the membrane material is ∼33 kD and the induced dipole moment is ∼10-13 cm3. We also demonstrate that when a poling field is applied across the films, the electrooptic coefficients are increased linearly with increasing field strength.

DC photoelectric signals from bacteriorhodopsin adsorbed on lipid monolayers and thiol/lipid bilayers supported by mercury

Purple membrane (PM) fragments were adsorbed on a dioleoylphosphatidylcholine (DOPC) monolayer and on a mixed alkanethiol/DOPC bilayer supported by mercury to investigate the kinetics of light-driven proton transport by bacteriorhodopsin (bR). The light-on and light-off capacitive currents on an alkanethiol/DOPC bilayer at pH 6.4 were interpreted on the basis of a simple equivalent circuit. The pH dependence of the biphasic decay kinetics of the light-on currents was analyzed to estimate the pK a values for the transitions releasing protons to, and taking up protons from, the solution. The linear dependence of the stationary light-on current of bR on a DOPC monolayer self-assembled on mercury upon the applied potential was interpreted on the basis of an equivalent circuit.