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The lipid/protein stoichiometries of a naturally crystalline biological membrane, the purple membrane (PM) of Halobacterium salinarum, have been obtained by a combination of 31P- and 1H-NMR analyses of the lipid extract. In total, 10 lipid molecules per retinal were found to be present in the PM lipid extract: 2–3 molecules of phosphatidylglycerophosphate methyl ester (PGP-Me), 3 of glycolipid sulfate, 1 of phosphatidylglycerol, 1 of archaeal glycocardiolipin (GlyC), 2 of squalene plus minor amounts of phosphatidylglycerosulfate (PGS) and bisphosphatidylglycerol (archaeal cardiolipin) (BPG) and a negligible amount of vitamin MK8. The novel data of the present study are necessary to identify the lipids in the electron density map, and to shed light on the structural relationships of the lipid and protein components of the PM. —Corcelli, A., V. M. T. Lattanzio, G. Mascolo, P. Papadia, and F. Fanizzi. Lipid-protein stoichiometries in a crystalline biological membrane: NMR quantitative analysis of the lipid extract of the purple membrane. J. Lipid Res. 2002. 43: 132–140.

It marked half a century since the discovery of bacteriorhodopsin two years ago. On this occasion, I have revisited historically important diffraction studies of this membrane protein, based on my recollections. X-ray diffraction and electron diffraction, and electron microscopy, described the low-resolution structure of bacteriorhodopsin within the purple membrane. Neutron diffraction was effective to assign the helical regions in the primary structure with 7 rods revealed by low-resolution structure as well as to describe the retinal position. Substantial conformational changes upon light illumination were clarified by the structures of various photointermediates. Early trials of time-resolved studies were also introduced. Models for the mechanism of light-driven proton pump based on the low-resolution structural studies are also described. Significantly, they are not far from the today's understanding. I believe that the spirit of the early research scientists in this field and the essence of their studies, which constitute the foundations of the field, still actively fertilizes current membrane protein research., Competing Interests: None., (2023 THE BIOPHYSICAL SOCIETY OF JAPAN.)

Bacteriorhodopsin (bR) is a trans-membrane proton pump found in the purple membrane of Halobacterium salinarum. This protein has high photochemical and photoelectric conversion efficiency and thermal stability, allowing it to withstand high temperatures, high salinity, and nutritionally-limited environments. The ability of this protein to convert light energy into chemical energy has applications that are mainly therapeutic/diagnostic and research-oriented. There is increasing demand for bacteriorhodopsin production in different fields. The present study maximized bacteriorhodopsin production using H. salinarum. The physical parameters of illumination, agitation speed, temperature, and nitrogen source were studied using a fractional factorial design to determine the optimal levels of each. The most suitable nitrogen source was determined to be peptone from meat. The optimal temperature was 39 °C, agitation speed was 150 rpm, and light intensity was 6300 lux for bR production. Under these conditions, the maximum bR yield was 196 mg/l, which is about 4.23 fold greater than those obtained with basal medium. The proposed strategies could be used for bR production using this archaeobacterium; the results are the highest reported thus far from a batch culture of H. salinarum. [ABSTRACT FROM AUTHOR]

• Abrupt, intramolecular charge displacement inside a biological membrane. • Electric response of the diffuse double layer. • Primary light-induced electric signals of bacteriorhodopsin. • Bipolar voltage decay as a consequence of capacity change of diffuse double layer. • Possible physiological consequences are discussed. In order to elucidate the old, still unsolved problem of how the diffuse electric double layer responds to an abrupt, intramolecular charge displacement inside a biological membrane, we investigated the fastest components of the light-induced electric signals of bacteriorhodopsin and its mutants, in numerous ionic and buffer solutions. The obtained data for temperature and solute concentration dependence were interpreted as a consequence of changes in the capacity of the diffuse double layer surrounding the purple membrane. The possible physiological consequences of this so far not demonstrated phenomenon are discussed. [ABSTRACT FROM AUTHOR]

Bactenorhodopsin (BR), the only protein in the purple membrane (PM) of certain extreme halophilic microorganisms, functions as a light-driven proton pump using light energy to generate transmembrane proton gradient for ATP synthesis. BR naturally aggregates in a highly ordered two-dimensional hexagonal array of trimers in the PM of Halobacterium. The BR in the isolated PM can be employed to generate a photocurrent in a photocell. However, delipidated BR (deBR) has been reported be more efficient than BR for photocurrent generation. In the present work, detergent CHAPS was included in anaqueous three-phase system (A3PS) to remove the lipids in the out layer of the BR trimer during the preparation of deBR. A3PS that consisted of polypropyleneglycol (PPG), polyethyleneglycol (PEG), and phosphate buffer purified deBR directly from the cell lysate of Halobacterium salinarum with a recovery yield of 89.7%. CHAPS along with the contaminant bacterioruberin pigment were partitioned into the top PPG-rich phase while deBR was mainly located at the interface between PEG-rich phase and the lower phosphate phase. After further purification by using ultrafiltration to remove PEG, the purified deBR when immobilized on indium tin oxide (ITO) glass was able to generate 60% higher photocurrent density. [ABSTRACT FROM AUTHOR]

Highlights: [•] Graphene oxide was complexed with oxidized avidin to serve as a linker between affinity-immobilized purple membranes and aminated supports. [•] Addition of graphene oxide improved substrate properties as well as the structure of immobilized purple membranes. [•] An improved photocurrent was stably and repeatedly generated by the composite photoelectrical chip. [Copyright &y& Elsevier]

Bacteriorhodopsin is a heptahelical protein found in the purple membrane of Halobacterium salinarum. The performance of bacteriorhodopsin was evaluated as a sensitizer in dye-sensitized solar cells (DSSCs). Bacteriorhodopsin was efficiently immobilized on the titanium dioxide nanoparticles and then tested for its ability to convert solar radiation to electricity. The photovoltaic performance of DSSC based on the bacteriorhodopsin sensitizer has been examined. Under AM1.5 irradiation, a short-circuit current of 0.28 mA cm, open-circuit voltages of 0.51 V, fill factor of 0.62, and an overall energy conversion efficiency of 0.09 % are achieved employing platinum as a counter electrode. Carbon was used as a counter electrode instead of platinum to reduce costs. Based on carbon electrode, a short-circuit current of 0.21 mA cm and open-circuit voltages of 0.52 V were obtained. [ABSTRACT FROM AUTHOR]

Purple membrane (PM) has been extracted and purified from archaebacteria for thin film development. The purified purple membrane is isolated in 1 % polyvinyl alcohol solution for making thin film within gelatin and organically modified silicate matrices. For thin film within gelatin matrix, homogenized purple membrane suspension is mixed with 8 % gelatin and poured into a specially designed block with desired thickness of spacer having hydrophobicity followed by gelatinization of the same over home-made thermostatic control unit at 38 °C. The gelatinized matrix is then allowed to dry under controlled conditions of humidity and temperature. The films of varying thicknesses ranging between 40, 50, and 60 μ are used for photo-electrochemical measurements. The results on photo-electrochemistry of non-oriented purple membrane film provides valuable information on the generation of forward (light on) and backward (light off) photocurrent as a function of: (a) applied potential and (b) film thickness. An increase in applied negative potential increases the amplitude of photocurrent whereas decrease in film thickness facilitates the reversibility of photocurrent response. [ABSTRACT FROM AUTHOR]

Abstract: Novel methanol-blocking polymer electrolyte composite membranes were prepared by inserting purple membrane (PM) to the bulk phase of Nafion® resin. Results of scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) analyses show that PM was well-dispersed in Nafion® membrane. Compared with recast Nafion®, blending PM changed the microstructure of Nafion® membrane and significantly improved the methanol-blocking efficiency by 31.6% in maximum. With PM content of 0.3wt% in the composite, the highest membrane selectivity factor and single cell power density of 62.1mWcm−2 were achieved. This satisfactory cell performance has given a promising prospect of Nafion®/PM composite membranes in the application in DMFC. [Copyright &y& Elsevier]

We have used new kinetic fitting procedures to obtain infrared (IR) absolute spectra for intermediates of the main bacteriorhodopsin (bR) photocycle(s). The linear-algebra-based procedures of Hendler et al. (J. Phys. Chem. B, 105, 3319–3228 (2001) for obtaining clean absolute visible spectra of bR photocycle intermediates were adapted for use with IR data. This led to isolation, for the first time, of corresponding clean absolute IR spectra, including the separation of the M intermediate into its MF and MS components from parallel photocycles. This in turn permitted the computation of clean IR difference spectra between pairs of successive intermediates, allowing for the most rigorous analysis to date of changes occurring at each step of the photocycle. The statistical accuracy of the spectral calculation methods allows us to identify, with great confidence, new spectral features. One of these is a very strong differential IR band at 1650 cm−1 for the L intermediate at room temperature that is not present in analogous L spectra measured at cryogenic temperatures. This band, in one of the noisiest spectral regions, has not been identified in any previous time-resolved IR papers, although retrospectively it is apparent as one of the strongest L absorbance changes in their raw data, considered collectively. Additionally, our results are most consistent with Arg82 as the primary proton-release group (PRG), rather than a protonated water cluster or H-bonded grouping of carboxylic residues. Notably, the Arg82 deprotonation occurs exclusively in the MF pathway of the parallel cycles model of the photocycle. [ABSTRACT FROM AUTHOR]

Abstract: In this study, purple membrane (PM) was applied as a methanol-blocking agent on Nafion® membranes. A series of well-organized poly(diallyldimethylammonium chloride)/PM multilayer films were obtained on a Nafion® 212 surface (PDDA/PM/Nafion®) to form composite membranes by the electrostatic layer-by-layer (LbL) self-assembly method. The effect of the PDDA/PM/Nafion® heterogenic interface on proton conductivity and methanol permeability was studied by alternating the deposited surface. With five PDDA/PM bilayers, double-sided modification (PDDA/PM)DF-5 and single-side modification (PDDA/PM)SF-5 resulted in excellent methanol blocking with a 73.4% and 64.7% reduction in methanol permeability in comparison with unmodified Nafion® 212, respectively. Indeed, the involvement of PM suppressed the methanol crossover. With regard to the selectivity factor (PDDA/PM)DF-1 composite membranes showed approximately 2-fold improvement as compared with unmodified Nafion® membranes. The cell performance of (PDDA/PM)SF-1 composite membranes achieved a power density of 27.0mWcm−2 which is 48.4% increase compared to cells using unmodified Nafion® 212 membranes. Moreover, with (PDDA/PM)DF-1, we achieved a power density of 34.4mWcm−2. This study highlights the potential application of PM as multifunctional protein membrane in direct methanol fuel cells. [ABSTRACT FROM AUTHOR]

Abstract: A poly-l-lysine (PLL)/bacteriorhodopsin-embedded purple membrane (bR-PM) multilayer film has been successfully constructed by a layer-by-layer (LbL) assembly process to enhance the photoelectric response of bR. The assembly conditions were investigated and optimized. The PLL/bR-PM adsorption process was in situ studied by surface plasmon resonance and the growth of multilayer was further characterized by UV–vis absorption spectroscopy. The results indicate that the amount of adsorbed bR-PM vs. the assembled layer number exhibits linear relationship. The atomic force microscopy images of sequentially assembled PLL/bR-PM bilayers show that the patch structure of bR-PM in the structure is well preserved and the roughness increases with increase of the bilayer number. The peak photocurrent generated from PLL/bR-PM film increases with increase of the PLL/bR-PM bilayers until achieving a maximum value. The photocurrent of bR-PM from the film through PLL assembler is higher than those assembled by other polycations, thus rendering a new platform to effectively enhance the bR photoelectric responses. [Copyright &y& Elsevier]

The simplest proton pump known in biological systems, bacteriorhodopsin (bR), is the first ion-transporting membrane protein, the function of which can be described at the atomic level, with the aid of molecular dynamics calculations. To get additional experimental support for the proposed atomic level description of the function of bR, we studied a quasi-stable state of the protein molecule, the so-called M intermediate that plays a crucial role in the proton pumping process. The temperature dependence of the light-induced events occurring in the photocycle of wild-type bacteriorhodopsin and its mutant D96N were followed in detail. Absorbance changes, electric signals generated by charge motion inside the protein, and movement of protons in the protein solution interface either forward (proton release due to excitation of bR) or backward (uptake of protons due to the M excitation: “back-take”) were monitored. The obtained Arrhenius parameters indicate that the proton back-take is triggered by charge rearrangements in the protein similar to the proton release triggered by those during the L → M transition. The time necessary for proton back-take determines the reconstitution time of the bR ground state. The data are expected to be used in theoretical modeling of the bR function. Based on these results, a more detailed photocycle model is established to describe the proton pumping mechanism, implying a formal principle ("domino model") that is expected to hold also for other charge transfer proteins. [ABSTRACT FROM AUTHOR]

Abstract: 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 5nm. 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. [Copyright &y& Elsevier]

Abstract: This work examined the biotin modification of bacteriorhodopsin (BR) in the purple membrane (PM). The results of flash kinetic absorption measurements showed that photocycle was maintained in biotinylated BR. Biotinylated BR also maintained its photoelectric activity, as indicated by the photoelectric response of the bilayer lipid membrane (BLM). Atomic force microscopy (AFM) of stretavidiin-bound biotin revealed that biotin molecules covered both surfaces of the, but the amount of biotinylated BR on the extracellular (EC) surface was markedly higher than on the cytoplasmic (CP) surface. Further studies showed that, after reaction with fluorescamine (FL), biotin labeling occurred only on the CP surface. These results are informative for future work on bioconjugation of BR as well as work on oriented assembly and the design of BR-based photoelectric devices. [Copyright &y& Elsevier]

Abstract: Components of biological macromolecules, complexes and membranes are animated by motions occurring over a wide range of time and length scales, the synergy of which is at the basis of biological activity. Understanding biological function thus requires a detailed analysis of the underlying dynamical heterogeneity. Neutron scattering, using specific isotope labeling, and molecular dynamics simulations were combined in order to study the dynamics of specific amino acid types in bacteriorhodopsin within the purple membrane (PM) of Halobacterium salinarum. Motions of leucine, isoleucine and tyrosine residues on the pico- to nanosecond time scale were examined separately as a function of temperature from 20 to 300 K. The dynamics of the three residue types displayed different temperature dependence: isoleucine residues have larger displacements compared to the global PM above 120 K; leucine residues have displacements similar to that of PM in the entire temperature range studied; and tyrosine residues have displacements smaller than that of the average membrane in an intermediate temperature range. Experimental features were mostly well reproduced by molecular dynamics simulations performed at five temperatures, which allowed the dynamical characterisation of the amino acids under study as a function of local environment. The resulting dynamical map of bacteriorhodopsin revealed that movements of a specific residue are determined by both its environment and its residue type. [Copyright &y& Elsevier]

The function and dynamics of proteins depend on their direct environment, and much evidence has pointed to a strong coupling between water and protein motions. Recently however, neutron scattering measurements on deuterated and natural-abundance purple membrane (PM), hydrated in H2O and D2O, respectively, revealed that membrane and water motions on the ns–ps time scale are not directly coupled below 260 K (Wood et al. in Proc Natl Acad Sci USA 104:18049–18054, ). In the initial study, samples with a high level of hydration were measured. Here, we have measured the dynamics of PM and water separately, at a low-hydration level corresponding to the first layer of hydration water only. As in the case of the higher hydration samples previously studied, the dynamics of PM and water display different temperature dependencies, with a transition in the hydration water at 200 K not triggering a transition in the membrane at the same temperature. Furthermore, neutron diffraction experiments were carried out to monitor the lamellar spacing of a flash-cooled deuterated PM stack hydrated in H2O as a function of temperature. At 200 K, a sudden decrease in lamellar spacing indicated the onset of long-range translational water diffusion in the second hydration layer as has already been observed on flash-cooled natural-abundance PM stacks hydrated in D2O (Weik et al. in J Mol Biol 275:632–634, ), excluding thus a notable isotope effect. Our results reinforce the notion that membrane-protein dynamics may be less strongly coupled to hydration water motions than the dynamics of soluble proteins. [ABSTRACT FROM AUTHOR]

The dynamical coupling between proteins and their hydration water is important for the understanding of macromolecular function in a cellular context. In the case of membrane proteins, the environment is heterogeneous, composed of lipids and hydration water, and the dynamical coupling might be more complex than in the case of the extensively studied soluble proteins. Here, we examine the dynamical coupling between a biological membrane, the purple membrane (PM), and its hydration water by a combination of elastic incoherent neutron scattering, specific deuteration, and molecular dynamics simulations. Examining completely deuterated PM, hydrated in H2O, allowed the direct experimental exploration of water dynamics. The study of natural abundance PM in D2O focused on membrane dynamics. The temperature-dependence of atomic mean-square displacements shows inflections at 120 K and 260 K for the membrane and at 200 K and 260 K for the hydration water. Because transition temperatures are different for PM and hydration water, we conclude that ps-ns hydration water dynamics are not directly coupled to membrane motions on the same time scale at temperatures <260 K. Molecular-dynamics simulations of hydrated PM in the temperature range from 100 to 296 K revealed an onset of hydration-water translational diffusion at 200 K, but no transition in the PM at the same temperature. Our results suggest that, in contrast to soluble proteins, the dynamics of the membrane protein is not controlled by that of hydration water at temperatures <260 K. Lipid dynamics may have a stronger impact on membrane protein dynamics than hydration water. [ABSTRACT FROM AUTHOR]

Abstract: The organic–inorganic interface and its proper structural adjustment are of central importance for the fabrication of hybrid material systems from biomolecules and semiconductors. Such material hybrids are currently under development for several advanced applications, in particular for biomolecular sensing. An investigation of biomolecular immobilization on semiconductor surfaces by X-ray reflectometry (XRR) will be presented. Complete biomembrane patches of purple membrane (PM) from Halobacterium salinarum were immobilized on oxidized and nitrided silicon wafers. A covalent immobilization protocol based on 3-aminopropyltriethoxysilane (APTS) and glutaric dialdehyde (GD) was applied for cross-linking the biomolecules to the semiconductor surface. XRR could be shown to yield the relevant morphological parameters of biomolecular monolayers such as layer thickness, interface roughness and coverage. Synchrotron radiation was not required, but a laboratory rotating anode set-up was sufficient to study the prepared stacking of organic monolayers. According to the measurement and analysis of XRR patterns both cross-linking layers APTS and GD are required for bonding purple membrane patches to SiO2/Si, whereas GD alone suffices for cross-linking to Si3N4/Si. This distinct behavior offers a pathway for nanopatterning of biomolecules on Si surfaces by selective passivation. [Copyright &y& Elsevier]

Fluorescence and absorption spectra were used to study the temperature effect on the conformation of bacteriorhodopsin (bR) in the blue and purple membranes (termed as bRb and bRp respectively). The maximum emission wavelengths of tryptophan fluorescence in both proteins at room temperature are 340 nm, and the fluorescence quantum yield of bRb is about 1.4 fold higher than that of bRp. As temperature increases, the tryptophan fluorescence of bRb decreases, while the tryptophan fluorescence of bRp increases. The binding study of extrinsic fluorescent probe bis-ANS indicated that the probe can bind only to bRb, but not to bRp. These results suggest that significant structural difference existed between bRb and bRp. It was also found that both kinds of bR are highly thermal stable. The maximum wavelength of the protein fluorescence emission only shifted from 340 nm to 346 nm at 100 °C. More interestingly, as temperature increased, the characteristic absorption peak of bRb at 605 nm decreased and a new absorption peak at 380 nm formed. The transition occurred at a narrow temperature range (65 °C–70 °C). These facts indicated that an intermediate can be induced by high temperature. This phenomenon has not been reported before. Edited by Chun-He XU [ABSTRACT FROM AUTHOR]

Proline residues in transmembrane helices have been found to have important roles in the functioning of membrane proteins. Moreover, Pro residues occur with high frequency in transmembrane α-helices, as compared to α-helices for soluble proteins. Here, we report several properties of the bacteriorhodopsin mutants P50A (helix B), P91A (helix C) and P186A (helix F). Compared to wild type, strongly perturbed behaviour has been found for these mutants. In the resting state, increased hydroxylamine accessibility and altered Asp-85 pK a and light-dark adaptation were observed. On light activation, hydroxylamine accessibility was increased and proton transport activity, M formation kinetics and FTIR difference spectra of M and N intermediates showed clear distortions. On the basis of these alterations and the near identity of the crystalline structures of mutants with that of wild type, we conclude that the transmembrane proline residues of bacteriorhodopsin fulfil a dynamic role in both the resting and the light-activated states. Our results are consistent with the notion that mutation of Pro to Ala allows the helix to increase its flexibility towards the direction originally hindered by the steric clash between the ring Cγ and the carbonyl O of the i-4 residue, at the same time decreasing the mobility towards the opposite direction. Due to their properties, transmembrane Pro residues may serve as transmission elements of conformational changes during the transport process. We propose that these concepts can be extended to other transmembrane proteins. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Pairs of PCR primers that targeted the archae/bacteriorhodopsin gene were used to clone the archaerhodopsin ( aR) gene of Halorubrum xinjiangense strain BD-1T, and this gene was sequenced and functionally expressed in Escherichia coli. Recombinant E. coli cells harboring the plasmid carrying this gene became slightly purple or blue depending on whether they were supplemented with all- trans retinal or 3,4-dihydroretinal, respectively, during induction with IPTG. The purple and blue membranes from the recombinant E. coli showed maximal absorption at 555 and 588 nm, respectively, which are different from maximal absorption at 568 nm of the wild-type purple membrane. Purple membranes from the recombinant E. coli and from strain BD-1T were investigated in parallel. The E. coli purple membrane was fabricated into films and photoelectric responses were observed that depended on the light-on and light-off stimuli. [ABSTRACT FROM AUTHOR]

Bacteriorhodopsin, a light-driven proton pump found in the purple membrane of Halobacterium salinarum, exhibits purple at neutral pH but its color is sensitive to pH. Here, structures are reported for an acid blue form and an alkaline purple form of wild-type bacteriorhodopsin. When the P622 crystal prepared at pH 5.2 was acidified with sulfuric acid, its color turned to blue with a pK a of 3.5 and a Hill coefficient of 2. Diffraction data at pH 2–5 indicated that the purple-to-blue transition accompanies a large structural change in the proton release channel; i.e. the extracellular half of helix C moves towards helix G, narrowing the proton release channel and expelling a water molecule from a micro-cavity in the vicinity of the retinal Schiff base. In this respect, the acid-induced structural change resembles the structural change observed upon formation of the M intermediate. But, the acid blue form contains a sulfate ion in a site(s) near Arg82 that is created by re-orientations of the carboxyl groups of Glu194 and Glu204, residues comprising the proton release complex. This result suggests that proton uptake by the proton release complex evokes the anion binding, which in turn induces protonation of Asp85, a key residue regulating the absorption spectrum of the chromophore. Interestingly, a pronounced structural change in the proton release complex was also observed at high pH; i.e. re-orientation of Glu194 towards Tyr83 was found to take place at around pH 10. This alkaline transition is suggested to be accompanied by proton release from the proton release complex and responsible for rapid formation of the M intermediate at high pH. [Copyright &y& Elsevier]

Phosphorus is an abundant element in living organisms. It is traceable by its X-ray absorption spectrum which shows a strong white line at its K-edge, comparable with that observed for the LIII edges of rare earth ions. With purple membrane, the variation of the imaginary part of the anomalous dispersion of phosphorus is found to be close to 20 anomalous electron units. Anomalous diffraction experiments at wavelengths near the K-absorption edge of phosphorus confirm this result. The spatial distribution of lipids derived from anomalous diffraction agrees with earlier results from neutron diffraction. Test experiments on single crystals of the carrier protein using 5.76 Å photons gave a first low-resolution diffraction pattern. Various techniques of crystal mounting were attempted. In addition, fluorescence measurements on a solution of threonine synthase appear to hint at a change of the phosphate environment of the cofactor upon activator binding. [ABSTRACT FROM AUTHOR]

Abstract: 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 250nA/cm2. [Copyright &y& Elsevier]

Abstract: The organization of bacteriorhodopsin (bR) within reconstituted purple membranes (RPM) was examined using atomic force microscopy (AFM). Five reconstituted species were examined: RPM 3 (bR/native polar lipids/dimyristoylphosphatidylcholine (DMPC) in a 1:9:14 molar ratio), RPM 4 (bR/native polar lipids in a 1:7 molar ratio), RPM 5 (bR/native polar lipids/1,2-di-O-phytanyl-sn-glycerol in a 1:3.5:6.1 molar ratio), RPM 6 (bR/native polar lipids/1,2-di-O-phytanyl-sn-glycero-3-phosphocholine in a 1:3.5:4.9 molar ratio), and RPM 7 (bR/native polar lipids/1,2-diphytanoyl-sn-glycero-3-[phospho-l-serine] in a 1:3.5:4.6 molar ratio). RPM 3 patches adsorbed onto mica exhibit domains of crystallized bR trimers arranged in a hexagonal packing structure, similar to those found in native purple membrane (NPM). These domains are enclosed by DMPC-rich regions. RPM 4 patches were observed to have larger domains of crystallized bR, with trimer orientation 30° different from that found in NPM. The bR-rich domains are enclosed by a large, protein-free, lipid-rich region. The topography of RPM 5 was difficult to resolve as the surface had no discernable patterns or structure. The topographies of RPM 6 and 7 were similar to that found in RPM 3 in that higher domains were formed within the patch adsorbed onto mica. They may contain protein-rich regions, but clear images of protein arrangement could not be obtained using AFM. This may be a result of imaging limitations or of the lack of organization of bR within these domains. [Copyright &y& Elsevier]

Bacteriorhodopsin (bR) is an attractive intelligent material. Understanding the mechanism of its light-driven proton pumping outward the cell implicates it in many technical applications, particularly, in what is called optical computers, and the biotechnology is waiting for this promised biological molecule. An ionizing radiation source handling could be computerized in radiation fields. The computer containing such biological material will not be out of reach of the fields of ionizing radiation. So it is interesting to report on the working of such biological computer if it is subjected to ionizing radiation. The functional unit in this molecule is retinal chromophore. In the present work, it is interested to assess the functionality of bR through determining the electronic transition dipole moment of its chromophore. Significant changes in the values of the absorption transition dipole moment were noticed at different doses of β-particles in the range of 0.1–0.3 kGy. Ionizing radiation-induced changes in bR were followed by intrinsic fluorescence spectroscopy. An analysis of the fluorescence data bears on the tertiary structure of bR. The emission spectrum is, however, red shifted with an increase in intensity with the different doses; in the meanwhile, gradual decrease in the visible absorbance has occurred till almost complete loss is attained. This bleaching due to ionizing radiation may offer an alternative way of data processing in such optical devices based on bR. Nevertheless, bR has proofed to be used as a biological indicator of ionizing radiation. However, the potential of bR for use as a biosensor to detect ionizing radiation should be considered. [Copyright &y& Elsevier]

We employed magnetic ACmode atomic force microscopy (MACmode AFM) as a novel dynamic force microscopy method to image surfaces of biological membranes in their native environments. The lateral resolution achieved under optimized imaging conditions was in the nanometer range, even when the sample was only weakly attached to the support. Purple membranes (PM) from Halobacterium salinarum were used as a test standard for topographical imaging. The hexagonal arrangement of the bacteriorhodopsin trimers on the cytoplasmic side of PM was resolved with 1.5 nm lateral accuracy, a resolution similar to images obtained in contact and tapping-mode AFM. Human rhinovirus 2 (HRV2) particles were attached to mica surfaces via nonspecific interactions. The capsid structure and 2 nm sized protein loops of HRV2 were routinely obtained without any displacement of the virus. Globular and filamentous structures on living and fixed endothelial cells were observed with a resolution of 5–20 nm. These examples show that MACmode AFM is a favorable method in studying the topography of soft and weakly attached biological samples with high resolution under physiological conditions. [Copyright &y& Elsevier]

The combination of high-resolution atomic force microscopy (AFM) imaging and single-molecule forcespectroscopy was employed to unfold single bacteriorhodopsins (BR) from native purple membrane patches at various physiologically relevant temperatures. The unfolding spectra reveal detailed insight into the stability of individual structural elements of BR against mechanical unfolding. Intermittent states in the unfolding process are associated with the stepwise unfolding of a-helices, whereas other states are associated with the unfolding of polypeptide loops connecting the a-helices. It was found that the unfolding forces of the secondary structures considerably decreased upon increasing the temperature from 8 to 52°C. Associated with this effect, the probability of individual unfolding pathways of BR was significantly influenced by the temperature. At lower temperatures, transmembrane α-helices and extracellular polypeptide loops exhibited sufficient stability to individually establish potential barriers against unfolding, whereas they predominantly unfolded collectively at elevated temperatures. This suggests that increasing the temperature decreases the mechanical stability of secondary structural elements and changes molecular interactions between secondary structures, thereby forcing them to act as grouped structures. [ABSTRACT FROM AUTHOR]