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Sample records for bacteriorhodopsins

  1. Absorption characteristics of bacteriorhodopsin molecules

    Indian Academy of Sciences (India)

    H K T Kumar; K Appaji Gowda

    2000-03-01

    The bacteriorhodopsin molecule absorbs light and undergoes a series of structural transformation following a well-defined photocycle. The complex photocycle is transformed to an equivalent level diagram by considering the lifetime of the intermediate states. Assuming that only and states are appreciably populated at any instant of time, the level diagram is further simplified to two-level system. Based on the rate equations for two-level system, an analytic expression for the absorption coefficient of bacteriorhodopsin molecule is derived. It is applied to study the behaviour of absorption coefficient of bacteriorhodopsin film in the visible wavelength region of 514 nm. The dependence of absorption coefficient of bacteriorhodopsin film on the thickness of the film, total number density of active molecules and initial number density of molecules in -state is presented in the graphical form.

  2. Kinetic vs. Thermodynamic Control of Bacteriorhodopsin Pumping

    Science.gov (United States)

    Gunner, Marilyn

    2011-03-01

    Bacteriorhodopsin is a transmembrane proton pump that converts light energy to a transmembrane electrochemical gradient. Retinal, bound in the center of the protein, absorbs light and isomerizes from the all-trans to 13-cis configuration. A series of conformational changes and proton transfers then restores the structure to the all-trans ground state while pumping one proton from the high pH cell interior to the low pH exterior, saving energy in an electrochemical gradient. Poorly understood gating elements control key steps where incorrect proton transfer would return the protein to the ground state without pumping. The gate's barrier height determines how much the pump leaks. Analysis of high-resolution structures trapped in different intermediates has produced ideas for how bacteriorhodopsin ensures pumping. There are two contrasting strategies, one primarily thermodynamic and the other relying on kinetic control to ensure that protons are moved uphill. With thermodynamic control, residue protonation states always remain in quasi-equilibrium. Relatively slow conformational changes shift the energy landscape modifying site pKas. Residues then change ionization remaining in equilibrium in each metastable intermediate. The sequence of intermediates imparts the directionality to the transfers. Alternatively, the direction of transfer is determined by the accessibility of low energy pathways so is thus is under kinetic control. We will discuss which steps in the bacteriorhodopsin photocycle are under thermodynamic or under kinetic control. The role of three specific conformational changes (retinal isomerization, Arg82 reorientation and Glu194 and 204 separations) on the degree of proton transfer will be described. Supported by NFS MCB 1022208. Carried out with Yifan Song now at the University of Washington Department of Biochemistry.

  3. Bacteriorhodopsin: Tunable Optical Nonlinear Magnetic Response

    CERN Document Server

    Bovino, F A; Sibilia, C; Giardina, M; Váró, G; Gergely, C

    2011-01-01

    We report on a strong and tunable magnetic optical nonlinear response of Bacteriorhodopsin (BR) under "off resonance" femtosecond (fs) pulse excitation, by detecting the polarization map of the noncollinear second harmonic signal of an oriented BR film, as a function of the input beam power. BR is a light-driven proton pump with a unique photochemistry initiated by the all trans retinal chromophore embedded in the protein. An elegant application of this photonic molecular machine has been recently found in the new area of optogenetics, where genetic expression of BR in brain cells conferred a light responsivity to the cells enabling thus specific stimulation of neurons. The observed strong tunable magnetic nonlinear response of BR might trigger promising applications in the emerging area of pairing optogenetics and functional magnetic resonance imaging susceptible to provide an unprecedented complete functional mapping of neural circuits.

  4. Multi-exponentially Photoelectric Response of Bacteriorhodopsin

    Institute of Scientific and Technical Information of China (English)

    姚保利; 徐大纶; 侯洵; 胡坤生; 王敖金

    2001-01-01

    A thin oriented bacteriorhodopsin (bR) film is deposited on a stainless steel slide by use of the electrophoretic sedimentation method. A junction is made with electrolyte gels having a counterelectrode to construct a bRbased photoelectric detector. The photoelectric response signal to a 10ns laser pulse is measured. A theory on the photoelectric kinetics of bR is developed based on the concept of the charge displacement current and the bR photocycle rate equations. Comparison between the theoretical and experimental results proves that the bR photoelectric response to a short laser pulse is a multi-exponential process. The decay time constants and amplitudes of each exponential component are obtained by data fitting.

  5. Electron microscopic observation and rotational diffusion measurement of bacteriorhodopsin in lipid vesicles

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The morphology of bacteriorhodopsin reconstituted into dimyristoylphosphatidylcholine and egg-phosphatidylcholine vesicles was observed by freeze-fracture electron microscopy. The rotational diffusion of bacteriorhodopsin at different concentrations of melittin was measured by observing flash-induced transient dichroism in dimyristoylphosphatidylcholine vesicles. In the presence of melittin, bacteriorhodopsin molecules in dimyristoylphosphatidylcholine vesicles were aggregated into large particles or patches, and the ability of rotational diffusion of bacteriorhodop sin in vesicles was decreased. This suggests that melittin produces its effect via direct electrostatic interaction with bacteriorhodopsin. Low temperature-induced aggregation of bacteriorhodopsin was also observed in dimyristoylphosphatidylcholine vesicles. Low temperature may cause phase separation. Bacteriorhodopsin was also successfully reconstituted into egg-phosphatidylcholine vesicles, but Iow temperature-induced aggregation of bacteriorhodopsin in dimyristoylphosphati dylcholine cannot appear in egg-phosphatidylcholine vesicles. This suggests that different lipids have different effects on bacteriorhodopsin in vesicles.

  6. Reaction cycle and thermodynamics in bacteriorhodopsin

    Science.gov (United States)

    Lanyi, J. K.

    1992-01-01

    Light causes the all-trans to 13-cis isomerization of the retinal in bacteriorhodopsin; the thermal relaxation leading back to the initial state drives proton transport first via proton transfer between the retinal Schiff base and D85 and then between the Schiff base and D96. The reaction sequence and thermodynamics of this photocycle are described by measuring time-resolved absorption changes with a gated multichannel analyzer between 100 ns and 100 ms, at six temperatures between 5 degrees C and 30 degrees C. Analysis of the energetics of the chromophore reaction sequence is on the basis of a recently proposed model (Varo & Lanyi, Biochemistry 30, 5016-5022, 1991) which consists of a single cycle and many reversible reactions: BR -hv-->KLM1-->M2NO-->BR. The existence of the M1-->M2 reaction, which functions as the switch in the proton transfer, is confirmed by spectroscopic evidence. The calculated thermodynamic parameters indicate that the exchange of free energy between the protein and the protons is at the switch step. Further, a large entropy decrease at this reaction suggests a protein conformation change which will conserve delta G for driving the completion of the reaction cycle. The results provide insights to mechanism and energy coupling in this system, with possible relevance to the general question of how ion pumps function.

  7. Computer-generated holograms recorded in bacteriorhodopsin

    Science.gov (United States)

    Guessous, Fouad; Juchem, Thorsten; Hampp, Norbert A.

    2004-06-01

    Computer-generated holograms (CGH's) of phase modulation type have been designed and fabricated in the biological material Bacteriorhodopsin (BR). BR is a photochromic retinal protein which may be used in optical data storage and security applications. Using the permanent light-inducible refractive index change of BR, we demonstrate that both analog and digital optical data can be stored in this material in a write-once-read-many (WORM) mode. The calculation and the optimization of the phase function of the CGH's have been accomplished with iterative Fourier transform algorithm methods (IFTA) such as error reduction algorithms. In the fabrication procedure the optimized phase functions of the CGH's have been recorded in BR which was coated onto a glass substrate. A direct laser writing process employing the 532 nm line of a cw-Nd:YAG laser was used for recording the CGH as a modulation of the absorption coefficient as well as of the refractive index. The design and fabrication method of the CGHs with a pixel pitch of 20 μm and a total size of 10 mm x 10 mm are presented.

  8. Measurements of photoinduced refractive index changes in bacteriorhodopsin films

    Indian Academy of Sciences (India)

    Ravinder Kumar Banyal; B Raghavendra Prasad

    2007-03-01

    We report the pump-probe measurements of nonlinear refractive index changes in photochromic bacteriorhodopsin films. The photoinduced absorption is caused by pump beam at 532 nm and the accompanying refractive index changes are studied using a probe beam at 633 nm. The proposed technique is based on a convenient and accurate determination of optical path difference using digital interferometry-based local fringe shift. The results are presented for the wild-type as well as genetically modified D96N variant of the bacteriorhodopsin.

  9. Optical CDMA system using bacteriorhodopsin for optical data storage

    Science.gov (United States)

    Bae; Yang; Jin; Lee; Park

    1999-11-01

    An optical CDMA (code division multiple access) system for the optical data storage using bacteriorhodopsin (BR) is reported as an application of the BR materials. The desired signal of multiple input can be recorded and reconstructed by use of orthogonal codes. An experimental setup is proposed and demonstrated.

  10. Aspartic acid substitutions affect proton translocation by bacteriorhodopsin.

    Science.gov (United States)

    Mogi, T; Stern, L J; Marti, T; Chao, B H; Khorana, H G

    1988-01-01

    We have substituted each of the aspartic acid residues in bacteriorhodopsin to determine their possible role in proton translocation by this protein. The aspartic acid residues were replaced by asparagines; in addition, Asp-85, -96, -115, and -112 were changed to glutamic acid and Asp-212 was also replaced by alanine. The mutant bacteriorhodopsin genes were expressed in Escherichia coli and the proteins were purified. The mutant proteins all regenerated bacteriorhodopsin-like chromophores when treated with a detergent-phospholipid mixture and retinal. However, the rates of regeneration of the chromophores and their lambda max varied widely. No support was obtained for the external point charge model for the opsin shift. The Asp-85----Asn mutant showed not detectable proton pumping, the Asp-96----Asn and Asp-212----Glu mutants showed less than 10% and the Asp-115----Glu mutant showed approximately equal to 30% of the normal proton pumping. The implications of these findings for possible mechanisms of proton translocation by bacteriorhodopsin are discussed. PMID:3288985

  11. Energy transfer processes in semiconductor quantum dots: bacteriorhodopsin hybrid system

    Science.gov (United States)

    Rakovich, Aliaksandra; Sukhanova, Alyona; Bouchonville, Nicolas; Molinari, Michael; Troyon, Michel; Cohen, Jacques H. M.; Rakovich, Yury; Donegan, John F.; Nabiev, Igor

    2009-05-01

    The potential impact of nanoscience on energy transfer processes in biomolecules was investigated on the example of a complex between fluorescent semiconductor nanocrystals and photochromic membrane protein. The interactions between colloidal CdTe quantum dots (QDs) and bacteriorhodopsin (bR) protein were studied by a variety of spectroscopic techniques, including integrated and time-resolved fluorescence spectroscopies, zeta potential and size measurement, and fluorescence correlation spectroscopy. QDs' luminescence was found to be strongly modulated by bacteriorhodopsin, but in a controllable way. Decreasing emission lifetimes and blue shifts in QDs' emission at increasing protein concentrations suggest that quenching occurs via Förster resonance energy transfer. On the other hand, concave Stern-Volmer plots and sigmoidal photoluminescence quenching curves imply that the self-assembling of NCs and bR exists, and the number of nanocrystals (NCs) per bacteriorhodopsin contributing to energy transfer can be determined from the inflection points of sigmoidal curves. This number was found to be highly dependent not only on the spectral overlap between NC emission and bR absorption bands, but also on nanocrystal surface charge. These results demonstrate the potential of how inorganic nanoscale materials can be employed to improve the generic molecular functions of biomolecules. The observed interactions between CdTe nanocrystals and bacteriorhodopsin can provide the basis for the development of novel functional materials with unique photonic properties and applications in areas such as all-optical switching, photovoltaics and data storage.

  12. Do Cation-π Interactions Exist in Bacteriorhodopsin

    Institute of Scientific and Technical Information of China (English)

    HU Kun-Sheng; WANG Guang-Yu; HE Jin-An

    2001-01-01

    Metal ions are essential to the structure and physiological functions of bacteriorhodopsin. Experimental evidence suggests the existence of specific cation binding to the negatively charged groups of Asp85 and Asp212 via an electrostatic interaction. However, only using electrostatic force is not enough to explain the role of the metal cations because the carboxylate of Asp85 is well known to be protonated in the M intermediate. Considering the presence of some aromatic amino acid residues in the vicinity of the retinal pocket, the existence of cation-π interactions between the metal cation and aromatic amino acid residues is suggested. Obviously, introduction of this kind of interaction is conducive to understanding the effects of the metal cations and aromatic amino acid residues inside the protein on the structural stability and proton pumping of bacteriorhodopsin.

  13. Illumination-dependent changes in the intrinsic fluorescence of bacteriorhodopsin

    Science.gov (United States)

    Bogomolni, R. A.; Stubbs, L.; Lanyi, J. K.

    1978-01-01

    The paper describes the intrinsic UV fluorescence of bacteriorhodopsin in some detail and determines the changes during the rapid cyclic reaction following light flashes. The results suggest that several tryptophan residues are affected in the protein, among them one or more exposed to aqueous medium. The kinetics of the fluorescence changes coincide closely with events involving the retinal residue during the deprotonation and reprotonation of the Schiff base group.

  14. All-optical logic-gates based on bacteriorhodopsin film

    Institute of Scientific and Technical Information of China (English)

    Chen Gui-Ying; Zhang Chun-Ping; Guo Zong-Xia; Tian Jian-Guo; Zhang Guang-Yin; Song Qi-Wang

    2005-01-01

    Based on self-diffraction in bacteriorhodopsin (bR) film, we propose all-optical NOT, XOR, half adder and XNOR logic operations. Using the relation between diffraction light and the polarization states of recording beams, we demonstrate NOT and XNOR logic operations. Studying the relation of polarization states among the diffracting, recording and reading beams, we implement XOR logic and half adder operations with three inputs. The methods are simple and practicable.

  15. Light-Induced Charge Separation and Transfer in Bacteriorhodopsin

    Institute of Scientific and Technical Information of China (English)

    HUANG Yu-Hua; LI Qing-Guo; ZHAO You-Yuan; ZHANG Zhong-Bin; OU-YANG Xiao-Ping; GONG Qin-Gan; CHEN Ling-Bing; LI Fu-Ming; LIU Jian; DING Jian-Dong

    2000-01-01

    The photo-voltage signals in bacteriorhodopsin(bR) excited by 1064nm pulse laser are different from those by 532 or 355 nm. It shows that the positive and negative photoelectric signals are produced by the motion of the positive and negative charges, respectively, and more energy is needed for producing the positive charges than the negative. The mechanism of light-induced charge generation and charge transfer in bR was studied and analyzed by measuring the photoelectric signals with different impedance of measuring circuit and different pulse-width of 532 nm laser as pump light.

  16. The behaviours of optical novelty filter based on bacteriorhodopsin film

    Institute of Scientific and Technical Information of China (English)

    Chen Gui-Ying; Yuan Yi-Zhe; Liang Xin; Xu Tang; Zhang Chun-Ping; Song Qi-Wang

    2006-01-01

    The quality of the novelty filter image is investigated at different intensities of the incident blue and yellow beams irradiating a bacteriorhodopsin (bR) film. The relationship between the transmitted blue beams and the incident yellow beams is established. The results show that the contrast of the novelty filter image depends on the lifetime of longest lived photochemical state (M state). These results enable one to identify the direction of a moving object and to improve the quality of the novel filter image by prolonging the lifetime of M state.

  17. Mixed Potential Energy Surfaces of the Ultrafast Isomerization of Retinal in Bacteriorhodopsin

    Directory of Open Access Journals (Sweden)

    Prokhorenko Valentyn I.

    2013-03-01

    Full Text Available We observe, using electronic two-dimensional photon echo spectroscopy, that the cis and trans potential energy surfaces of the ultrafast isomerization of retinal in bacteriorhodopsin are mixed via the hydrogen out of plane (HOOP mode.

  18. Proton transfer and energy coupling in the bacteriorhodopsin photocycle

    Science.gov (United States)

    Lanyi, J. K.

    1992-01-01

    A description of the rate constants and the energetics of the elementary reaction steps of the photocycle of bacteriorhodopsin has been helpful in understanding the mechanism of proton transport in this light-driven pump. The evidence suggests a single unbranched reaction sequence, BR-hv----K in equilibrium with L in equilibrium with M1----M2 in equilibrium with N in equilibrium with O----BR, where coupling to the proton-motive force is at the energetically and mechanistically important M1----M2 step. The consequences of site-specific mutations expressed homologously in Halobacterium halobium have revealed characteristics of the Schiff base deprotonation in the L----M1 reaction, the reorientation of the Schiff base from the extracellular to the cytoplasmic side in the M1----M2 reaction, and the reprotonation of the Schiff base in the M2----N reaction.

  19. Structure and function in bacteriorhodopsin: the role of the interhelical loops in the folding and stability of bacteriorhodopsin.

    Science.gov (United States)

    Kim, J M; Booth, P J; Allen, S J; Khorana, H G

    2001-04-27

    Bacteriorhodopsin functions as a light-driven proton pump in Halobacterium salinarium. The functional protein consists of an apoprotein, bacterioopsin, with seven transmembrane alpha helices together with a covalently bound all-trans retinal chromophore. In order to study the role of the interhelical loop conformations in the structure and function of bacteriorhodopsin, we have constructed bacterioopsin genes where each loop is replaced, one at a time, by a peptide linker consisting of Gly-Gly-Ser- repeat sequences, which are believed to have flexible conformations. These mutant proteins have been expressed in Escherichia coli, purified and reconstituted with all-trans retinal in l-alpha-1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/3-(3-cholamidopropyl)dimethylammonio-1-propane sulfonate (CHAPS)/SDS and l-alpha-1,2-dihexanoylphosphatidylcholine (DHPC)/DMPC/SDS micelles. Wild-type-like chromophore formation was observed in all the mutants containing single loop replacements. In the BC and FG mutants, an additional chromophore band with an absorption band at about 480 nm was observed, which was in equilibrium with the 550 nm, wild-type band. The position of the equilibrium depended on temperature, SDS and relative DMPC concentration. The proton pumping activity of all of the mutants was comparable to that of wild-type bR except for the BC and FG mutants, which had lower activity. All of the loop mutants were more sensitive to denaturation by SDS than the wild-type protein, except the mutant where the DE loop was replaced. These results suggest that a specific conformation of all the loops of bR, except the DE loop, contributes to bR stability and is required for the correct folding and function of the protein. An increase in the relative proportion of DHPC in DHPC/DMPC micelles, which reduces the micelle rigidity and alters the micelle shape, resulted in lower folding yields of all loop mutants except the BC and DE mutants. This effect of micelle rigidity on

  20. Dynamic Behaviour of Self-Diffraction in Bacteriorhodopsin Film

    Institute of Scientific and Technical Information of China (English)

    GUO Zong-Xia; CHEN Gui-Ying; ZHANG Chun-Ping; TIAN Jian-Guo; Q. Wang Song; SHEN Bin; FU Guang-Hua

    2004-01-01

    @@ We investigate the dependences of the diffraction efficiency of the first order self-diffracted beam in bacteriorhodopsin (bR) films on the illumination time, the intensity and wavelength of the incident light. When the blue light (λ = 488 nm) and low intensity red light (λ = 632.8 nm) are incident on the bR film respectively,the diffraction efficiencies increase from zero to a stable value with the illumination time. When the green light (λ = 533 nm) and high-intensity red light illuminate the bR film respectively, the diffraction efficiencies increase from zero to the maximum and then decrease to a stable value with the illumination time. Rise and decay times are dependent on the intensity and wavelength of the incident light. The maximaldiffraction efficiency of the red light is twice as high as that of the green light. The highest diffraction efficiency of 5.4% is obtained at 633nm.The diffraction efficiency change with the time for the green light is larger than that for the blue and red light.

  1. Transient Fourier holography with bacteriorhodopsin films for breast cancer diagnostics

    Science.gov (United States)

    Rao, Devulapalli; Kothapalli, Sri-Rajasekar; Wu, Pengfei; Yelleswarapu, Chandra

    X-ray mammography is the current gold standard for breast cancer screening. Microcalcifications and other features which are helpful to the radiologist for early diagnostics are often buried in the noise generated by the surrounding dense tissue. So image processing techniques are required to enhance these important features to improve the sensitivity of detection. An innovative technique is demonstrated for recording a hologram of the mammogram. It is recorded on a thin polymer film of Bacteriorhodopsin (bR) as photo induced isomerization grating containing the interference pattern between the object beam containing the Fourier spatial frequency components of the mammogram and a reference beam. The hologram contains all the enhanced features of the mammogram. A significant innovation of the technique is that the enhanced components in the processed image can be viewed by the radiologist in time scale. A technician can record the movie and when the radiologist looks at the movie at his convenience, freezing the frame as and when desired, he would see the microcalcifications as the brightest and last long in time. He would also observe lesions with intensity decreasing as their size increases. The same bR film can be used repeatedly for recording holograms with different mammograms. The technique is versatile and a different frequency band can be chosen to be optimized by changing the reference beam intensity. The experimental arrangement can be used for mammograms in screen film or digital format.

  2. Electron microscopic observation and rotational diffusion measurement of bacteriorhodopsin in lipid vesicles

    Institute of Scientific and Technical Information of China (English)

    HU; Kunsheng

    2001-01-01

    [1]Stoeckenius, W.. Bacterial rhodopsins: Evolution of a mechanistic model for the ion pumps, Protein Science, 1999, 8: 447.[2]Ebrey. T. G, Light energy transduction in bacteriorhodopsin, in Thermodynamics of Membranes, Receptors and Channels (ed. Jackson. M.), New York: CRC Press, 1993. 353-387.[3]Lanyi. J. K.. Understanding structure and function in the light-driven proton pump bacteriorhodopsin, J. Struct. Biol., 1998,l24: 164.[4]Quay. S. C., Condie. C. C., Conformational studies of aqueous melittin: Thermodynamic parameters of the monomer-tetramer self-association reaction. Biochemistry, 1983, 22: 695.[5]Habermann. E.. Bee and wasp venoms. Science, 1972, 177: 314.[6]Tosteson. M. T., Holmes. S. J., Razin. M. et al., Melitton lysis of red cells, J. Membr. Biol., 1985, 87: 35.[7]Hu, K. S., Dufton, M. J., Morrison, I. E. G. et al., Cherry interaction of bee venom melittin with bacteriorhodopsin in lipid vesicles: Protein rotational diffusion measurement. Biochem. Biophys. Acta, 1985, 816(2): 358.[8]Shi, H., Hu, K. S., Huang, Y. et al., Effect of melittin on photocycle and photoresponse of purple membrane: sites of interaction between bacteriorhodopsin and melittin, Photochemistry and Photobiology, 1993, 58(3): 413.[9]Jiang. Q. X., Hu, K. S.. Shi. H., Interaction of both melittin and its site-specific mutants with bacteriorhodopsin of Halobacterium halobium: sites of electrostatic interaction on melittin Photochemistry and Photobiology, 1994, 60(2): 175.[10]Doebler, R., Basaran. N.. Goldston H. et al., Effect of protein aggregation into aqueous phase on the binding of membrane proteins to membranes, Biophys. J., 1999, 76: 928.[11]Rehorek, M., Heyn, M. P, Binding of all-trans-retinal to the purple membrane, Evidence for cooperativity and determination of the extinction coefficient, Biochemistry, 1979, 18: 4977.[12]Chen. P. S. Jr.. Toribara, T. Y., Warner, H., Microdetermination of phosphorous, Anal. Chem., t956, 28

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

    Science.gov (United States)

    Hong, F T

    1995-01-01

    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.

  4. Trapping the M sub 1 and M sub 2 substrates of bacteriorhodopsin for electron diffraction studies

    Energy Technology Data Exchange (ETDEWEB)

    Perkins, G.A.

    1992-05-01

    Visible and Fourier transform infrared (FTIR) absorption spectroscopies are used to observe protein conformational changes occuring during the bacteriorhodopsin photocycle. Spectroscopic measurements which define the conditions under which bacteriorhodopsin can be isolated and trapped in two distinct substates of the m intermediate of the photocycle, M{sub 1}, and M{sub 2}, are described. A protocol that can be used for high-resolution electron diffraction studies is presented that will trap glucose-embedded purple membrane in the M{sub 1}and M{sub 2} substates at greater than 90% concentration. It was discovered that glucose alone does not provide a fully hydrated environment for bacteriorhodopsin. Equilibration of glucose-embedded samples at high humidity can result in a physical state that is demonstrably closer to the native, fully hydrated state. An extension of the C-T Model of bacteriorhodopsin functionality (Fodor et al., 1988; Mathies et al., 1991) is proposed based on FTIR results and guided by published spectra from resonance Raman and FTIR work. 105 refs.

  5. Optical and electric signals from dried oriented purple membrane of bacteriorhodopsins.

    Science.gov (United States)

    Tóth-Boconádi, R; Dér, A; Keszthelyi, L

    2011-04-01

    All the intermediates of the bacteriorhodopsin photocycle are excitable with light of suitable wavelength. This property might regulate the activity in the cells when they are exposed in the nature to high light intensity. On the other hand this property is involved in many applications. In this study the ground state and M intermediate of dried oriented samples of wild-type bacteriorhodopsin and its mutant D96N were excited with 406 nm laser flashes. Substantial M populations were generated with quasi-continuous illumination. The decay of the absorption of M intermediate had three components: their lifetimes were very different for laser flash and quasi-continuous illuminations in cases of both bacteriorhodopsin species. The optical answer for the excitation of M intermediate had a lifetime of 2.2 ms. Electric signals for M excitation had large fast negative components and small positive components in the 100 μs time domain. The results are expected to have important implications for bioelectronic applications of bacteriorhodopsin.

  6. Quantum yields for the light adaptations in Anabaena sensory rhodopsin and bacteriorhodopsin

    Science.gov (United States)

    Wada, Yoichiro; Kawanabe, Akira; Furutani, Yuji; Kandori, Hideki; Ohtani, Hiroyuki

    2008-02-01

    Archael-type rhodopsin has an all- trans or a 13- cis retinal. The light-induced interconversion between these two forms has been found in Anabaena sensory rhodopsin, even though only the photoreaction from the 13- cis form to the all- trans form exists in bacteriorhodopsin. In this study, we obtained the quantum yields for the 13- cis → all- trans and all- trans → 13- cis reactions of Anabaena sensory rhodopsin (0.24 ± 0.03 and 0.38 ± 0.07, respectively) and concluded that these values were independent of the wavelength of the excitation light as well as bacteriorhodopsin. In other words, no excess energy effects can be found in these reactions.

  7. The 3rd-order nonlinearity of bacteriorhodopsin by four-wave mixing

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The 3rd-order nonlinear optical susceptibility X(3) and the response time of the light-transducing biomolecule bacteriorhodopsin were measured with the four-wave mixing technique and a picosecond frequency-doubled Nd:YAG laser(532nm).The X(3) and the response time measured are 10-9 esu and 20 ps,respectively.The possible mechanism for generating the 3rd-order nonlinear optical susceptibility X(3) and response time were discussed.

  8. Unraveling the mechanism of proton translocation in the extracellular half-channel of bacteriorhodopsin.

    Science.gov (United States)

    Ge, Xiaoxia; Gunner, M R

    2016-05-01

    Bacteriorhodopsin, a light activated protein that creates a proton gradient in halobacteria, has long served as a simple model of proton pumps. Within bacteriorhodopsin, several key sites undergo protonation changes during the photocycle, moving protons from the higher pH cytoplasm to the lower pH extracellular side. The mechanism underlying the long-range proton translocation between the central (the retinal Schiff base SB216, D85, and D212) and exit clusters (E194 and E204) remains elusive. To obtain a dynamic view of the key factors controlling proton translocation, a systematic study using molecular dynamics simulation was performed for eight bacteriorhodopsin models varying in retinal isomer and protonation states of the SB216, D85, D212, and E204. The side-chain orientation of R82 is determined primarily by the protonation states of the residues in the EC. The side-chain reorientation of R82 modulates the hydrogen-bond network and consequently possible pathways of proton transfer. Quantum mechanical intrinsic reaction coordinate calculations of proton-transfer in the methyl guanidinium-hydronium-hydroxide model system show that proton transfer via a guanidinium group requires an initial geometry permitting proton donation and acceptance by the same amine. In all the bacteriorhodopsin models, R82 can form proton wires with both the CC and the EC connected by the same amine. Alternatively, rare proton wires for proton transfer from the CC to the EC without involving R82 were found in an O' state where the proton on D85 is transferred to D212.

  9. Theoretical and experimental analysis of pulse delay in bacteriorhodopsin films by a saturable absorber theory.

    Science.gov (United States)

    Blaya, Salvador; Candela, Manuel; Acebal, Pablo; Carretero, Luis; Fimia, Antonio

    2014-05-19

    Time-delay of transmitted pulses with respect to the incident pulse in bacteriorhodopsin films has been studied without the use of a pump beam. Based on a modified saturable absorber model, analytical expressions of the transmitted pulse have been obtained. As a result, time delay, distortion and fractional delay have been analyzed for sinusoidal pulses with a low background. A good agreement between theory and experiences has been observed.

  10. Crystal structure of Escherichia coli-expressed Haloarcula marismortui bacteriorhodopsin I in the trimeric form.

    Directory of Open Access Journals (Sweden)

    Vitaly Shevchenko

    Full Text Available Bacteriorhodopsins are a large family of seven-helical transmembrane proteins that function as light-driven proton pumps. Here, we present the crystal structure of a new member of the family, Haloarcula marismortui bacteriorhodopsin I (HmBRI D94N mutant, at the resolution of 2.5 Å. While the HmBRI retinal-binding pocket and proton donor site are similar to those of other archaeal proton pumps, its proton release region is extended and contains additional water molecules. The protein's fold is reinforced by three novel inter-helical hydrogen bonds, two of which result from double substitutions relative to Halobacterium salinarum bacteriorhodopsin and other similar proteins. Despite the expression in Escherichia coli and consequent absence of native lipids, the protein assembles as a trimer in crystals. The unique extended loop between the helices D and E of HmBRI makes contacts with the adjacent protomer and appears to stabilize the interface. Many lipidic hydrophobic tail groups are discernible in the membrane region, and their positions are similar to those of archaeal isoprenoid lipids in the crystals of other proton pumps, isolated from native or native-like sources. All these features might explain the HmBRI properties and establish the protein as a novel model for the microbial rhodopsin proton pumping studies.

  11. Crystal structure of Escherichia coli-expressed Haloarcula marismortui bacteriorhodopsin I in the trimeric form.

    Science.gov (United States)

    Shevchenko, Vitaly; Gushchin, Ivan; Polovinkin, Vitaly; Round, Ekaterina; Borshchevskiy, Valentin; Utrobin, Petr; Popov, Alexander; Balandin, Taras; Büldt, Georg; Gordeliy, Valentin

    2014-01-01

    Bacteriorhodopsins are a large family of seven-helical transmembrane proteins that function as light-driven proton pumps. Here, we present the crystal structure of a new member of the family, Haloarcula marismortui bacteriorhodopsin I (HmBRI) D94N mutant, at the resolution of 2.5 Å. While the HmBRI retinal-binding pocket and proton donor site are similar to those of other archaeal proton pumps, its proton release region is extended and contains additional water molecules. The protein's fold is reinforced by three novel inter-helical hydrogen bonds, two of which result from double substitutions relative to Halobacterium salinarum bacteriorhodopsin and other similar proteins. Despite the expression in Escherichia coli and consequent absence of native lipids, the protein assembles as a trimer in crystals. The unique extended loop between the helices D and E of HmBRI makes contacts with the adjacent protomer and appears to stabilize the interface. Many lipidic hydrophobic tail groups are discernible in the membrane region, and their positions are similar to those of archaeal isoprenoid lipids in the crystals of other proton pumps, isolated from native or native-like sources. All these features might explain the HmBRI properties and establish the protein as a novel model for the microbial rhodopsin proton pumping studies.

  12. Femtosecond spectroscopic study of photochromic reactions of bacteriorhodopsin and visual rhodopsin.

    Science.gov (United States)

    Feldman, Tatiana B; Smitienko, Olga A; Shelaev, Ivan V; Gostev, Fedor E; Nekrasova, Oksana V; Dolgikh, Dmitriy A; Nadtochenko, Victor A; Kirpichnikov, Mikhail P; Ostrovsky, Mikhail A

    2016-11-01

    Photochromic ultrafast reactions of bacteriorhodopsin (H. salinarum) and bovine rhodopsin were conducted with a femtosecond two-pump probe pulse setup with the time resolution of 20-25fs. The dynamics of the forward and reverse photochemical reactions for both retinal-containing proteins was compared. It is demonstrated that when retinal-containing proteins are excited by femtosecond pulses, dynamics pattern of the vibrational coherent wave packets in the course of the reaction is different for bacteriorhodopsin and visual rhodopsin. As shown in these studies, the low-frequencies that form a wave packets experimentally observed in the dynamics of primary products formation as a result of retinal photoisomerization have different intensities and are clearer for bovine rhodopsin. Photo-reversible reactions for both retinal proteins were performed from the stage of the relatively stable photointermediates that appear within 3-5ps after the light pulse impact. It is demonstrated that the efficiency of the reverse phototransition K-form→bacteriorhodopsin is almost five-fold higher than that of the Batho-intermediate→visual rhodopsin phototransition. The results obtained indicate that in the course of evolution the intramolecular mechanism of the chromophore-protein interaction in visual rhodopsin becomes more perfect and specific. The decrease in the probability of the reverse chromophore photoisomerization (all-trans→11-cis retinal) in primary photo-induced rhodopsin products causes an increase in the efficiency of the photoreception process.

  13. Two-photon polarization data storage in bacteriorhodopsin films and its potential use in security applications

    Science.gov (United States)

    Imhof, Martin; Rhinow, Daniel; Hampp, Norbert

    2014-02-01

    Bacteriorhodopsin (BR) films allow write-once-read-many recording of polarization data by a two-photon-absorption (TPA) process. The optical changes in BR films induced by the TPA recording were measured and the Müller matrix of a BR film was determined. A potential application of BR films in security technology is shown. Polarization data can be angle-selective retrieved with high signal-to-noise ratio. The BR film does not only carry optical information but serves also as a linear polarizer. This enables that polarization features recorded in BR films may be retrieved by merely using polarized light from a mobile phone display.

  14. Controlling the pKa of the bacteriorhodopsin Schiff base by use of artificial retinal analogues.

    OpenAIRE

    1986-01-01

    Artificial bacteriorhodopsin pigments based on synthetic retinal analogues carrying an electron-withdrawing CF3 substituent group were prepared. The effects of CF3 on the spectra, photocycles, and Schiff base pKa values of the pigments were analyzed. A reduction of 5 units in the pKa of the Schiff base is observed when the CF3 substituent is located at the C-13 polyene position, in the vicinity of the protonated Schiff base nitrogen. The results lead to the unambiguous characterization of the...

  15. Pathways of proton transfer in the light-driven pump bacteriorhodopsin

    Science.gov (United States)

    Lanyi, J. K.

    1993-01-01

    The mechanism of proton transport in the light-driven pump bacteriorhodopsin is beginning to be understood. Light causes the all-trans to 13-cis isomerization of the retinal chromophore. This sets off a sequential and directed series of transient decreases in the pKa's of a) the retinal Schiff base, b) an extracellular proton release complex which includes asp-85, and c) a cytoplasmic proton uptake complex which includes asp-96. The timing of these pKa changes during the photoreaction cycle causes sequential proton transfers which result in the net movement of a proton across the protein, from the cytoplasmic to the extracellular surface.

  16. Photoelectrochemical Properties of Bacteriorhodopsin Langmuir-Blodgett Films on ITO Conductive Electrode

    Institute of Scientific and Technical Information of China (English)

    王建平; 李津如; 陶培德; 李兴长; 江龙

    1994-01-01

    Langmuir-Blodgett films containing bacteriorhodopsin were deposited on ITO conduc-tive electrodes.A sandwiched photocell with a junction structure of ITO/bR/electrolyte/ITO has beenconstructed,in which the bR LB film was directly put into contact with an aqueous electrolyte immobi-lized in an agar gel.Under visible light irradiation,the photocell produced a transient photocurrent due tothe change of light intensity,which characterized vision imitative material.A photoalarm device based ondifferential responsivity phenomena was fabricated.

  17. Two-photon polarization data storage in bacteriorhodopsin films and its potential use in security applications

    Energy Technology Data Exchange (ETDEWEB)

    Imhof, Martin; Hampp, Norbert, E-mail: hampp@staff.uni-marburg.de [Department of Chemistry, Material Sciences Center, University of Marburg, Hans-Meerwein-Str., D-35032 Marburg (Germany); Rhinow, Daniel [Max-Planck-Institute of Biophysics, Max-von-Laue-Straße 3, D-60438 Frankfurt (Germany)

    2014-02-24

    Bacteriorhodopsin (BR) films allow write-once-read-many recording of polarization data by a two-photon-absorption (TPA) process. The optical changes in BR films induced by the TPA recording were measured and the Müller matrix of a BR film was determined. A potential application of BR films in security technology is shown. Polarization data can be angle-selective retrieved with high signal-to-noise ratio. The BR film does not only carry optical information but serves also as a linear polarizer. This enables that polarization features recorded in BR films may be retrieved by merely using polarized light from a mobile phone display.

  18. Resonance Raman kinetic spectroscopy of bacteriorhodopsin on the microsecond time scale

    Energy Technology Data Exchange (ETDEWEB)

    Campion, A.; El-Sayed, M.A.; Terner, J.

    1977-12-01

    Using a rotating disk with a slit of variable width, a continuous wave argon ion laser, and an Optical Multichanel Analyzer for detection, a new technique is reported which should, in principle, be capable of recording resonance Raman spectra with time resolution of 100 ns. The resonance Raman spectra of the intermediates of the photosynthetic cycle of bacteriorhodopsin are recorded on the microsecond time scale. Both the kinetic results and the resonance enhancement profile suggest that deprotonation results in an intermediate preceding bM/sub 412/ that has an optical absorption maximum at a wavelength longer than that of bM/sub 412/.

  19. Trapping the M{sub 1} and M{sub 2} substrates of bacteriorhodopsin for electron diffraction studies

    Energy Technology Data Exchange (ETDEWEB)

    Perkins, G.A.

    1992-05-01

    Visible and Fourier transform infrared (FTIR) absorption spectroscopies are used to observe protein conformational changes occuring during the bacteriorhodopsin photocycle. Spectroscopic measurements which define the conditions under which bacteriorhodopsin can be isolated and trapped in two distinct substates of the m intermediate of the photocycle, M{sub 1}, and M{sub 2}, are described. A protocol that can be used for high-resolution electron diffraction studies is presented that will trap glucose-embedded purple membrane in the M{sub 1}and M{sub 2} substates at greater than 90% concentration. It was discovered that glucose alone does not provide a fully hydrated environment for bacteriorhodopsin. Equilibration of glucose-embedded samples at high humidity can result in a physical state that is demonstrably closer to the native, fully hydrated state. An extension of the C-T Model of bacteriorhodopsin functionality (Fodor et al., 1988; Mathies et al., 1991) is proposed based on FTIR results and guided by published spectra from resonance Raman and FTIR work. 105 refs.

  20. Enhancement of photoelectric response of bacteriorhodopsin by multilayered WO3 x H2O nanocrystals/PVA membrane.

    Science.gov (United States)

    Li, Rui; Hu, Fengping; Bao, Qiaoliang; Bao, Shujuan; Qiao, Yan; Yu, Shucong; Guo, Jun; Li, Chang Ming

    2010-02-01

    For the first time, a multilayered WO(3) x H(2)O/PVA membrane on bacteriorhodopsin (bR) is constructed to significantly enhance the photoelectric response of bR by the spillover effect of WO(3) x H(2)O nanocrystals, providing great potential in its important applications in bioelectronics and proton exchange membrane fuel cells.

  1. New Nano- and Biotechnological Applications of Bacterial and Animal Photoreceptor Pigments  Bacteriorhodopsin, Rhodopsin and Iodopsin

    Directory of Open Access Journals (Sweden)

    Ignat Ignatov

    2016-03-01

    Full Text Available This paper views predominately the structure and function of animal and bacterial photoreceptor pigments (rhodopsin, iodopsin, bacteriorhodopsin and new aspects of their nano- and biotechnological usage. On an example of bacteriorhodopsin was described the method of its isolation from purple membranes of photo-organotrophic halobacterium Halobacterium halobium by cellular autolysis by distilled water, processing of bacterial biomass by ultrasound at 22 KHz, alcohol extraction of low and high-weight molecular impurities, cellular RNA, carotenoids and lipids, the solubilization with 0,5 % (w/v SDS-Na and subsequent fractionation by methanol and gel filtration chromatography on Sephadex G-200 Column balanced with 0.09 M Tris-HCl buffer (pH = 6,76 with 0,1 % (w/v SDS-Na and 2,5 mM EDTA. Within the framework of the research the mechanism of color perception by the visual analyzer having the ability to analyze certain ranges of the optical spectrum, as colors was studied along with an analysis of the additive mixing of two colors. It was shown that at the mixing of electromagnetic waves with different wavelengths, the visual analyzer perceive them as separate or average wave length corresponding to mix color.

  2. Studying of Phototransformation of Light Signal by Photoreceptor Pigments - Rhodopsin, Iodopsin and Bacteriorhodopsin

    Directory of Open Access Journals (Sweden)

    Ignat Ignatov

    2014-09-01

    Full Text Available This review article views predominately the structure and function of animal and bacterial photoreceptor pigments (rhodopsin, iodopsin, bacteriorhodopsin and their aspects of nano- and biotechnological usage. On an example of bacteriorhodopsin is described the method of its isolation from purple membranes of photo-organotrophic halobacterium Halobacterium halobium by cellular autolysis by distilled water, processing of bacterial biomass by ultrasound at 22 KHz, alcohol extraction of low and high-weight molecular impurities, cellular RNA, carotenoids and lipids, the solubilization with 0,5 % (w/v SDS-Na and subsequent fractionation by methanol and gel filtration chromatography on Sephadex G-200 Column balanced with 0.09 M Tris-borate buffer (pH = 8,35 with 0,1 % (w/v SDS-Na and 2,5 mM EDTA. Within the framework of the research the mechanism of color perception by the visual analyzer having the ability to analyze certain ranges of the optical spectrum, as colors was studied along with an analysis of the additive mixing of two colors. It was shown that at the mixing of electromagnetic waves with different wavelengths, the visual analyzer perceive them as separate or average wave length corresponding to mix color.

  3. Excited-state dynamics of bacteriorhodopsin probed by broadband femtosecond fluorescence spectroscopy.

    Science.gov (United States)

    Schmidt, B; Sobotta, C; Heinz, B; Laimgruber, S; Braun, M; Gilch, P

    2005-01-07

    The impact of varying excitation densities (approximately 0.3 to approximately 40 photons per molecule) on the ultrafast fluorescence dynamics of bacteriorhodopsin has been studied in a wide spectral range (630-900 nm). For low excitation densities, the fluorescence dynamics can be approximated biexponentially with time constants of <0.15 and approximately 0.45 ps. The spectrum associated with the fastest time constant peaks at 650 nm, while the 0.45 ps component is most prominent at 750 nm. Superimposed on these kinetics is a shift of the fluorescence maximum with time (dynamic Stokes shift). Higher excitation densities alter the time constants and their amplitudes. These changes are assigned to multi-photon absorptions.

  4. Solid-state NMR studies of bacteriorhodopsin and the purple membrane

    CERN Document Server

    Mason, A J

    2001-01-01

    proteins. This technique may prove particularly useful when studying large proteins that are difficult to orient where the MAS lineshapes will remain relatively unaffected in comparison with current static NMR methods. Finally the MAOSS method was extended to the study of the lipid components of the purple membrane and the feasibility of determining structural constraints from phospholipid headgroups was assessed. The potential of using sup 3 sup 1 P NMR to observe qualitative protein-lipid interactions in both the purple membrane and reconstituted membranes containing bovine rhodopsin was also demonstrated. Following the demonstration of a new MAS NMR method for resolving orientational constraints in uni-axially oriented biological membranes (Glaubitz and Watts, 1998), experiments were performed to realise the potential of the new method on large, oriented membrane proteins. Using bacteriorhodopsin in the purple membrane as a paradigm for large membrane proteins, the protein was specifically labelled with de...

  5. An Approach to Heterologous Expression of Membrane Proteins. The Case of Bacteriorhodopsin.

    Science.gov (United States)

    Bratanov, Dmitry; Balandin, Taras; Round, Ekaterina; Shevchenko, Vitaly; Gushchin, Ivan; Polovinkin, Vitaly; Borshchevskiy, Valentin; Gordeliy, Valentin

    2015-01-01

    Heterologous overexpression of functional membrane proteins is a major bottleneck of structural biology. Bacteriorhodopsin from Halobium salinarum (bR) is a striking example of the difficulties in membrane protein overexpression. We suggest a general approach with a finite number of steps which allows one to localize the underlying problem of poor expression of a membrane protein using bR as an example. Our approach is based on constructing chimeric proteins comprising parts of a protein of interest and complementary parts of a homologous protein demonstrating advantageous expression. This complementary protein approach allowed us to increase bR expression by two orders of magnitude through the introduction of two silent mutations into bR coding DNA. For the first time the high quality crystals of bR expressed in E. Coli were obtained using the produced protein. The crystals obtained with in meso nanovolume crystallization diffracted to 1.67 Å.

  6. An Approach to Heterologous Expression of Membrane Proteins. The Case of Bacteriorhodopsin.

    Directory of Open Access Journals (Sweden)

    Dmitry Bratanov

    Full Text Available Heterologous overexpression of functional membrane proteins is a major bottleneck of structural biology. Bacteriorhodopsin from Halobium salinarum (bR is a striking example of the difficulties in membrane protein overexpression. We suggest a general approach with a finite number of steps which allows one to localize the underlying problem of poor expression of a membrane protein using bR as an example. Our approach is based on constructing chimeric proteins comprising parts of a protein of interest and complementary parts of a homologous protein demonstrating advantageous expression. This complementary protein approach allowed us to increase bR expression by two orders of magnitude through the introduction of two silent mutations into bR coding DNA. For the first time the high quality crystals of bR expressed in E. Coli were obtained using the produced protein. The crystals obtained with in meso nanovolume crystallization diffracted to 1.67 Å.

  7. Calibration of Membrane Viscosity of the Reconstituted Vesicles by Measurement of the Rotational Diffusion of Bacteriorhodopsin

    Institute of Scientific and Technical Information of China (English)

    王敖金; 胡坤生

    2002-01-01

    Membrane viscosity of the reconstituted vesicles was calibrated by rotational diffusion of bacteriorhodopsin (BR) in dimyristoylphosphatidylcholine (DMPC) and egg phosphatidylcholine (PC) vesicles. Rotational diffusion of BR in the vesicles was measured by flash-induced absorption anisotropy decay. BR was, for the first time, reconstituted successfully into DMPC and egg PC vesicles. From the measurement of flash-induced absorption anisotropy decay of BR, the value of rotational diffusion coefficient D was obtained from each curve fitting by a global fitting procedure and, in turn, membrane viscosity η was estimated from D. The results have shown that membrane viscosity is temperature-dependent. It was decreased as temperature increased, but a transition occurred in the region of the respective phase transition of DMPC and egg PC, respectively. The decrease of η was fast near the phase transition for DMPC and egg PC. Few effects of lipid/BR ratio and glycerol or sucrose in suspension medium on membrane viscosity were found.

  8. An all-optical time-delay relay based n a bacteriorhodopsin film

    Institute of Scientific and Technical Information of China (English)

    Chen Gui-Ying; Xu Xu-Xu; Zhang Chun-Ping; Qi Shen-Wen; Song Qi-Wang

    2008-01-01

    Using a special property of dynamic complementary-suppression-modulated transmission (DCSMT) in the bacteriorhodopsin (bR) film,we have demonstrated an all-optical time-delay relay.To extend our work,the relationship between the delay time of the all-optical time-delay relay and parameters of a bR film is numerically studied.We show how the delay time changes with the product of concentration and thickness (PCT) of a bR film.Furthermore,the shortest and longest delay times are given for the relay of 'switch off'.The saturable delay time and maximum delaytime of 'switch on' are also given.How the wavelengths (632.8,568,533 and 412 nm) and intensities of the illuminating light influence the delay time is also discussed.The simulation results are useful for optimizing the design of all-optical time-delay relays.

  9. Spatio-temporal study of non-degenerate two-wave mixing in bacteriorhodopsin films.

    Science.gov (United States)

    Blaya, Salvador; González, Alejandro; Acebal, Pablo; Carretero, Luis

    2016-10-31

    A spatio-temporal analysis of non-degenerate two-wave mixing in a saturable absorber, such as bacteriorhodopsin (bR) film, is performed. To do this, a theoretical model describing the temporal variation of the intensities is developed by taking into account the dielectric constant as a function of bR population. A good agreement between theory and experimental measurements is obtained. Thus, the dependence of the optical gain and the main dielectric constant parameters are studied at different intensities and frequencies. As a result, the best intensity-frequency zones where higher coupling is reached are proposed, and it is also demonstrated that non-uniform patterns, which evolve over time as a function of frequency difference, can be observed.

  10. Rigorous analysis of the propagation of sinusoidal pulses in bacteriorhodopsin films.

    Science.gov (United States)

    Acebal, Pablo; Blaya, Salvador; Carretero, Luis; Madrigal, R F; Fimia, A

    2012-11-05

    The propagation of sinusoidal pulses in bacteriorhodopsin films has been theoretically analyzed using a complete study of the photoinduced processes that take into account all the physical parameters, the coupling of rate equations with the energy transfer equation and the temperature change during the experiment. The theoretical approach was compared to experimental data and a good concordance was observed. This theoretical treatment, can be widely applied, i.e when arbitrary pump and/or signal is used or in the case of the pump and signal beams have different wavelengths. Due to we have performed a rigorous analysis, from this treatment the corresponding two level approximation has also been analyzed for these systems.

  11. Steady-State Characterization of Bacteriorhodopsin-D85N Photocycle

    Science.gov (United States)

    Timucin, Dogan A.; Downie, John D.; Norvig, Peter (Technical Monitor)

    1999-01-01

    An operational characterization of the photocycle of the genetic mutant D85N of bacteriorhodopsin, BR-D85N, is presented. Steady-state bleach spectra and pump-probe absorbance data are obtained with thick hydrated films containing BR-D85N embedded in a gelatin host. Simple two- and three-state models are used to analyze the photocycle dynamics and extract relevant information such as pure-state absorption spectra, photochemical-transition quantum efficiencies, and thermal lifetimes of dominant states appearing in the photocycle, the knowledge of which should aid in the analysis of optical recording and retrieval of data in films incorporating this photochromic material. The remarkable characteristics of this material and their implications from the viewpoint of optical data storage and processing are discussed.

  12. Development of bacteriorhodopsin analogues and studies of charge separated excited states in the photoprocesses of linear polyenes.

    Science.gov (United States)

    Singh, Anil K; Hota, Prasanta K

    2007-01-01

    Development of bacteriorhodopsin (bR) analogues employing chromophore substitution technique for the purpose of characterizing the binding site of bR and generating bR analogues with novel opto-electronic properties for applications as photoactive element in nanotechnical devices are described. Additionally, the photophysical and photochemical properties of variously substituted diarylpolyenes as models of photobiologically relevant linear polyenes are discussed. The role of charge separated dipolar excited states in the photoprocesses of linear polyenes is highlighted.

  13. Modulation of folding and assembly of the membrane protein bacteriorhodopsin by intermolecular forces within the lipid bilayer.

    Science.gov (United States)

    Curran, A R; Templer, R H; Booth, P J

    1999-07-20

    Three different lipid systems have been developed to investigate the effect of physicochemical forces within the lipid bilayer on the folding of the integral membrane protein bacteriorhodopsin. Each system consists of lipid vesicles containing two lipid species, one with phosphatidylcholine and the other with phosphatidylethanolamine headgroups, but the same hydrocarbon chains: either L-alpha-1, 2-dioleoyl, L-alpha-1,2-dipalmitoleoyl, or L-alpha-1,2-dimyristoyl. Increasing the mole fraction of the phosphatidylethanolamine lipid increases the desire of each monolayer leaflet in the bilayer to curve toward water. This increases the torque tension of such monolayers, when they are constrained to remain flat in the vesicle bilayer. Consequently, the lateral pressure in the hydrocarbon chain region increases, and we have used excimer fluorescence from pyrene-labeled phosphatidylcholine lipids to probe these pressure changes. We show that bacteriorhodopsin regenerates to about 95% yield in vesicles of 100% phosphatidylcholine. The regeneration yield decreases as the mole fraction of the corresponding phosphatidylethanolamine component is increased. The decrease in yield correlates with the increase in lateral pressure which the lipid chains exert on the refolding protein. We suggest that the increase in lipid chain pressure either hinders insertion of the denatured state of bacterioopsin into the bilayer or slows a folding step within the bilayer, to the extent that an intermediate involved in bacteriorhodopsin regeneration is effectively trapped.

  14. Studying proton pumping mechanism of bacteriorhodopsin and cytochrome c oxidase with multi-conformation continuum electrostatics

    Science.gov (United States)

    Song, Yifan

    The proton gradient across the biological membrane is important for the biological systems. Bacteriorhodopsin and cytochrome c oxidase convert different energy sources into this gradient. The focus of this thesis is to understand the mechanism of these proteins using computational methods. In bacteriorhodopsin, residue ionization states were calculated in 9 crystal structures trapped in bR, early M and late M states by Multi-Conformation Continuum Electrostatics (MCC). The three groups in the central cluster are ionized in bR structures while a proton has transferred from the SB+ to Asp 85 - in the late M structures matching prior experimental results. The proton release cluster binds one proton in bR structure which is lost to water by pH 8 in late M. Modest changes in intra-protein interactions cause the charge shifts within the clusters. Motions of Arg 82 couple the proton shift in the central cluster to proton release. Changes in the total charge of the two clusters are coupled by direct long-range interactions. Cytochrome c oxidase is a transmembrane proton pump that builds an electrochemical gradient using chemical energy from the reduction of O2. Ionization states of all residues were calculated with MCCE in seven anaerobic oxidase redox states ranging from fully oxidized to fully reduced in Rb. sphaeroides cytochrome c oxidase. At pH 7, only a hydroxide coordinated to CuB shifts its pKa from below 7 to above 7, and so picks up a proton when Heme a3 and CuB are reduced. Glu I-286, Tyr I-288, His I-334 and a second hydroxide on Heme a3 all have pKas above 7. The propionic acids near the BNC are deprotonated with pKas well below 7. This suggests electroneutrality in the BNC is not maintained during the anaerobic reduction. The electrochemical midpoint potential (E m) of Heme a is calculated to shift down when the BNC is reduced, which agrees with prior experiments. If the BNC reduction is electroneutral, then the Heme a Em is independent of the BNC redox state.

  15. Effective atomic numbers and electron densities of bacteriorhodopsin and its comprising amino acids in the energy range 1 keV-100 GeV

    Science.gov (United States)

    Ahmadi, Morteza; Lunscher, Nolan; Yeow, John T. W.

    2013-04-01

    Recently, there has been an interest in fabrication of X-ray sensors based on bacteriorhodopsin, a proton pump protein in cell membrane of Halobacterium salinarium. Therefore, a better understanding of interaction of X-ray photons with bacteriorhodopsin is required. We use WinXCom program to calculate the mass attenuation coefficient of bacteriorhodopsin and its comprising amino acids for photon energies from 1 keV to 100 GeV. These amino acids include alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, Asx1, Asx2, Glx1 and Glx2. We then use that data to calculate effective atomic number and electron densities for the same range of energy. We also emphasize on two ranges of energies (10-200 keV and 1-20 MeV) in which X-ray imaging and radiotherapy machines work.

  16. Effective atomic numbers and electron densities of bacteriorhodopsin and its comprising amino acids in the energy range 1 keV–100 GeV

    Energy Technology Data Exchange (ETDEWEB)

    Ahmadi, Morteza; Lunscher, Nolan [Waterloo Institute for Nanotechnology and Department of Systems Design Engineering, University of Waterloo, 200 University Ave., W., Waterloo, Ontario, Canada N2L 3G1 (Canada); Yeow, John T.W., E-mail: jyeow@uwaterloo.ca [Waterloo Institute for Nanotechnology and Department of Systems Design Engineering, University of Waterloo, 200 University Ave., W., Waterloo, Ontario, Canada N2L 3G1 (Canada)

    2013-04-01

    Recently, there has been an interest in fabrication of X-ray sensors based on bacteriorhodopsin, a proton pump protein in cell membrane of Halobacterium salinarium. Therefore, a better understanding of interaction of X-ray photons with bacteriorhodopsin is required. We use WinXCom program to calculate the mass attenuation coefficient of bacteriorhodopsin and its comprising amino acids for photon energies from 1 keV to 100 GeV. These amino acids include alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, Asx1, Asx2, Glx1 and Glx2. We then use that data to calculate effective atomic number and electron densities for the same range of energy. We also emphasize on two ranges of energies (10–200 keV and 1–20 MeV) in which X-ray imaging and radiotherapy machines work.

  17. All-Optical Arithmetic and Combinatorial Logic Circuits with High-Q Bacteriorhodopsin Coated Microcavities

    CERN Document Server

    Roy, Sukhdev; Topolancik, Juraj; Vollmer, Frank

    2010-01-01

    We present designs of all-optical computing circuits, namely, half-full adder/subtractor, de-multiplexer, multiplexer, and an arithmetic unit, based on bacteriorhodopsin (BR) protein coated microcavity switch in a tree architecture. The basic all-optical switch consists of an input infrared (IR) laser beam at 1310 nm in a single mode fiber (SMF-28) switched by a control pulsed laser beam at 532 nm, which triggers the change in the resonance condition on a silica bead coated with BR between two tapered fibers. We show that fast switching of 50 us can be achieved by injecting a blue laser beam at 410 nm that helps in truncating the BR photocycle at the M intermediate state. Realization of all-optical switch with BR coated microcavity switch has been done experimentally. Based on this basic switch configuration, designs of all-optical higher computing circuits have been presented. The design requires 2n-1 switches to realize n bit computation. The proposed designs require less number of switches than terahertz o...

  18. Deposition of Bacteriorhodopsin Protein in a Purple Membrane Form on Nitrocellulose Membranes for Enhanced Photoelectric Response

    Directory of Open Access Journals (Sweden)

    Chang-Hoon Nam

    2012-12-01

    Full Text Available 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.

  19. Nanoparticle surface-enhanced Raman scattering of bacteriorhodopsin stabilized by amphipol A8-35.

    Science.gov (United States)

    Polovinkin, V; Balandin, T; Volkov, O; Round, E; Borshchevskiy, V; Utrobin, P; von Stetten, D; Royant, A; Willbold, D; Arzumanyan, G; Chupin, V; Popot, J-L; Gordeliy, V

    2014-10-01

    Surface-enhanced Raman spectroscopy (SERS) has developed dramatically since its discovery in the 1970s, because of its power as an analytical tool for selective sensing of molecules adsorbed onto noble metal nanoparticles (NPs) and nanostructures, including at the single-molecule (SM) level. Despite the high importance of membrane proteins (MPs), SERS application to MPs has not really been studied, due to the great handling difficulties resulting from the amphiphilic nature of MPs. The ability of amphipols (APols) to trap MPs and keep them soluble, stable, and functional opens up onto highly interesting applications for SERS studies, possibly at the SM level. This seems to be feasible since single APol-trapped MPs can fit into gaps between noble metal NPs, or in other gap-containing SERS substrates, whereby the enhancement of Raman scattering signal may be sufficient for SM sensitivity. The goal of the present study is to give a proof of concept of SERS with APol-stabilized MPs, using bacteriorhodopsin (BR) as a model. BR trapped by APol A8-35 remains functional even after partial drying at a low humidity. A dried mixture of silver Lee-Meisel colloid NPs and BR/A8-35 complexes give rise to SERS with an average enhancement factor in excess of 10(2). SERS spectra resemble non-SERS spectra of a dried sample of BR/APol complexes.

  20. Enhanced Photocurrent Generation from Bacteriorhodopsin Photocells Using Grating-Structured Transparent Conductive Oxide Electrodes.

    Science.gov (United States)

    Kaji, Takahiro; Kasai, Katsuyuki; Haruyama, Yoshihiro; Yamada, Toshiki; Inoue, Shin-Ichiro; Tominari, Yukihiro; Ueda, Rieko; Terui, Toshifumi; Tanaka, Shukichi; Otomo, Akira

    2016-04-01

    We fabricated a grating-structured electrode made of indium-doped zinc oxide (IZO) with a high refractive index (approximately 2) for a bacteriorhodopsin (bR) photocell. We investigated the photocurrent characteristics of the bR photocell and demonstrated that the photocurrent values from the bR/IZO electrode with the grating structure with a grating period of 340 nm were more than 3.5-4 times larger than those without the grating structure. The photocurrent enhancement was attributed to the resonance effect due to light coupling to the grating structure as well as the scattering effect based on the experimental results and analysis using the photonic band structure determined using finite-difference time-domain (FDTD) simulations. The refractive index of the bR film in electrolyte solution (1.40) used in the FDTD simulations was estimated by analyzing the extinction peak wavelength of 20-nm gold colloids in the bR film. Our results indicate that the grating- or photonic-crystal-structured transparent conductive oxide (TCO) electrodes can increase the light use efficiency of various bR devices such as artificial photosynthetic devices, solar cells, and light-sensing devices.

  1. Resonant transfer of one- and two-photon excitations in quantum dot-bacteriorhodopsin complexes

    Science.gov (United States)

    Krivenkov, V. A.; Samokhvalov, P. S.; Bilan, R. S.; Chistyakov, A. A.; Nabiev, I. R.

    2017-01-01

    Light-sensitive protein bacteriorhodopsin (BR), which is capable of electrical response upon exposure to light, is a promising material for photovoltaics and optoelectronics. However, the rather narrow absorption spectrum of BR does not allow achieving efficient conversion of the light energy in the blue and infrared spectral regions. This paper summarizes the results of studies showing the possibility of extending the spectral region of the BR function by means of the Förster resonance energy transfer (FRET) from CdSe/ZnS quantum dots (QDs), which have a broad spectrum of one-photon absorption and a large twophoton absorption cross section (TPACS), to BR upon one- and two-photon excitation. In particular, it is shown that, on the basis of QDs and BR-containing purple membranes, it is possible to create electrostatically associated bio-nano hybrid systems in which FRET is implemented. In addition, the large TPACS of QDs, which is two orders of magnitude larger than those of BR and organic dyes, opens up a means for selective two-photon excitation of synthesized bio-nano hybrid complexes. On the basis of the results of this work, the spectral region in which BR converts the light energy into electrical energy can be extended from the UV to near-IR region, creating new opportunities for the use of this material in photovoltaics and optoelectronics.

  2. Schiff base switch II precedes the retinal thermal isomerization in the photocycle of bacteriorhodopsin.

    Directory of Open Access Journals (Sweden)

    Ting Wang

    Full Text Available In bacteriorhodopsin, the order of molecular events that control the cytoplasmic or extracellular accessibility of the Schiff bases (SB are not well understood. We use molecular dynamics simulations to study a process involved in the second accessibility switch of SB that occurs after its reprotonation in the N intermediate of the photocycle. We find that once protonated, the SB C15 = NZ bond switches from a cytoplasmic facing (13-cis, 15-anti configuration to an extracellular facing (13-cis, 15-syn configuration on the pico to nanosecond timescale. Significantly, rotation about the retinal's C13 = C14 double bond is not observed. The dynamics of the isomeric state transitions of the protonated SB are strongly influenced by the surrounding charges and dielectric effects of other buried ions, particularly D96 and D212. Our simulations indicate that the thermal isomerization of retinal from 13-cis back to all-trans likely occurs independently from and after the SB C15 = NZ rotation in the N-to-O transition.

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

    Science.gov (United States)

    Kim, Young Jun; Neuzil, Pavel; Nam, Chang-Hoon; Engelhard, Martin

    2012-12-27

    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.

  4. Photosensory behaviour of a bacteriorhodopsin-deficient mutant, ET-15, of Halobacterium halobium

    Energy Technology Data Exchange (ETDEWEB)

    Hildebrand, E.; Schimz, A. (Kernforschungsanlage Juelich G.m.b.H. (Germany, F.R.). Inst. fuer Neurobiologie)

    1983-05-01

    Halobacterium halobium, strain ET-15, which does not contain detectable amounts of bacteriorhodopsin (BR) shows behavioral responses to UV and yellow-green light. Attractant stimuli, i.e. light-increases in the yellow-green range or light-decreases in the UV, suppress the spontaneous reversals of the swimming direction for a certain time. Repellent stimuli, i.e. light-decreases in the yellow-green range or light-increases in the UV, elicit an additional reversal response after a few seconds. Action spectra of both sensory photosystems, PS 370 and PS 565, were measured with attractant as well as with repellent stimuli. As in BR-containing cells, maximal sensitivity was always found at 370 nm for the UV-system and at 565 nm for the long-wavelength system. Fluence-response curves at 370 and 565 nm obtained with strain ET-15 and with a BR-containing strain show that the sensitivity of both photosystems is not reduced in the absence of BR. It is concluded that BR is required neither for PS 565 nor for PS 370. Instead retinal-containing pigments different from BR have to be assumed to mediate photosensory behavior.

  5. Förster Resonance Energy Transfer between Core/Shell Quantum Dots and Bacteriorhodopsin

    Directory of Open Access Journals (Sweden)

    Mark H. Griep

    2012-01-01

    Full Text Available An energy transfer relationship between core-shell CdSe/ZnS quantum dots (QDs and the optical protein bacteriorhodopsin (bR is shown, demonstrating a distance-dependent energy transfer with 88.2% and 51.1% of the QD energy being transferred to the bR monomer at separation distances of 3.5 nm and 8.5 nm, respectively. Fluorescence lifetime measurements isolate nonradiative energy transfer, other than optical absorptive mechanisms, with the effective QD excited state lifetime reducing from 18.0 ns to 13.3 ns with bR integration, demonstrating the Förster resonance energy transfer contributes to 26.1% of the transferred QD energy at the 3.5 nm separation distance. The established direct energy transfer mechanism holds the potential to enhance the bR spectral range and sensitivity of energies that the protein can utilize, increasing its subsequent photocurrent generation, a significant potential expansion of the applicability of bR in solar cell, biosensing, biocomputing, optoelectronic, and imaging technologies.

  6. Structural analysis of bacteriorhodopsin solubilized by lipid-like phosphocholine biosurfactants with varying micelle concentrations.

    Science.gov (United States)

    Wang, Xiaoqiang; Huang, Haihong; Sun, Chenghao; Huang, Fang

    2015-01-01

    Surfactants that can provide a more natural substitute for lipid bilayers are important in the purification and in vitro study of membrane proteins. Here we investigate the structural response of a model membrane protein, bacteriorhodopsin (BR), to phosphocholine biosurfactants. Phosphocholine biosurfactants are a type of biomimetic amphiphile that are similar to phospholipids, in which membrane proteins are commonly embedded. Multiple spectroscopic and zeta potential measurements are employed to characterize the conformational change, secondary and tertiary structure, oligomeric status, surface charge distribution and the structural stability of BR solubilized with phosphocholine biosurfactants of varying tail length. The process of phosphocholine micelle formation is found to facilitate the solubilization of BR, and for long-chain phosphocholines, concentrations much higher than their critical micelle concentrations achieve good solubilization. Phosphocholine biosurfactants are shown to be mild compared with the ionic surfactant SDS or CTAB, and tend to preserve membrane protein structure during solubilization, especially at low micelle concentrations, by virtue of their phospholipid-like zwitterionic head groups. The increase of alkyl chain length is shown to obviously enhance the capability of phosphocholine biosurfactants to stabilize BR. The underlying mechanism for the favorable actions of phosphocholine biosurfactant is also discussed.

  7. Charge-controlled assembling of bacteriorhodopsin and semiconductor quantum dots for fluorescence resonance energy transfer-based nanophotonic applications

    Science.gov (United States)

    Bouchonville, Nicolas; Molinari, Michael; Sukhanova, Alyona; Artemyev, Mikhail; Oleinikov, Vladimir A.; Troyon, Michel; Nabiev, Igor

    2011-01-01

    The fluorescence resonance energy transfer (FRET) between quantum dots (QDs) and photochromic protein bacteriorhodopsin within its natural purple membrane (PM) is explored to monitor their assembling. It is shown that the efficiency of FRET may be controlled by variation of the surface charge and thickness of QD organic coating. Atomic force microscopy imaging revealed correlation between the surface charge of QDs and degree of their ordering on the surface of PM. The most FRET-efficient QD-PM complexes have the highest level of QDs ordering, and their assembling design may be further optimized to engineer hybrid materials with advanced biophotonic and photovoltaic properties.

  8. Influence of the charge at D85 on the initial steps in the photocycle of bacteriorhodopsin.

    Science.gov (United States)

    Sobotta, Constanze; Braun, Markus; Tittor, Jörg; Oesterhelt, D; Zinth, Wolfgang

    2009-07-08

    Studies have shown that trans-cis isomerization of retinal is the primary photoreaction in the photocycle of the light-driven proton pump bacteriorhodopsin (BR) from Halobacterium salinarum, as well as in the photocycle of the chloride pump halorhodopsin (HR). The transmembrane proteins HR and BR show extensive structural similarities, but differ in the electrostatic surroundings of the retinal chromophore near the protonated Schiff base. Point mutation of BR of the negatively charged aspartate D85 to a threonine T (D85T) in combination with variation of the pH value and anion concentration is used to study the ultrafast photoisomerization of BR and HR for well-defined electrostatic surroundings of the retinal chromophore. Variations of the pH value and salt concentration allow a switch in the isomerization dynamics of the BR mutant D85T between BR-like and HR-like behaviors. At low salt concentrations or a high pH value (pH 8), the mutant D85T shows a biexponential initial reaction similar to that of HR. The combination of high salt concentration and a low pH value (pH 6) leads to a subpopulation of 25% of the mutant D85T whose stationary and dynamic absorption properties are similar to those of native BR. In this sample, the combination of low pH and high salt concentration reestablishes the electrostatic surroundings originally present in native BR, but only a minor fraction of the D85T molecules have the charge located exactly at the position required for the BR-like fast isomerization reaction. The results suggest that the electrostatics in the native BR protein is optimized by evolution. The accurate location of the fixed charge at the aspartate D85 near the Schiff base in BR is essential for the high efficiency of the primary reaction.

  9. Water and carboxyl group environments in the dehydration blueshift of bacteriorhodopsin.

    Science.gov (United States)

    Renthal, R; Gracia, N; Regalado, R

    2000-11-01

    The proton channels of the bacteriorhodopsin (BR) proton pump contain bound water molecules. The channels connect the purple membrane surfaces with the protonated retinal Schiff base at the membrane center. Films of purple membrane equilibrated at low relative humidity display a shift of the 570 nm retinal absorbance maximum to 528 nm, with most of the change occurring below 15% relative humidity. Purple membrane films were dehydrated to defined humidities between about 50 and 4.5% and examined by Fourier transform infrared difference spectroscopy. In spectra of dehydrated-minus-hydrated purple membrane, troughs are observed at 3645 and 3550 cm-1, and peaks are observed at 3665 and 3500 cm-1. We attribute these changes to water dissociation from the proton uptake channel and the resulting changes in hydrogen bonding of water that remains bound. Also, in the carboxylic acid spectral region, a trough was observed at 1742 cm-1 and a peak at 1737 cm-1. The magnitude of the trough to peak difference between 1737 and 1742 cm-1 correlates linearly with the extent of the 528 nm pigment. This suggests that a carboxylic acid group or groups is undergoing a change in environment as a result of dehydration, and that this change is linked to the appearance of the 528 nm pigment. Dehydration difference spectra with BR mutants D96N and D115N show that the 1737-1742 cm-1 change is due to Asp 96 and Asp 115. A possible mechanism is suggested that links dissociation of water in the proton uptake channel to the environmental change at the Schiff base site.

  10. Volume and enthalpy changes of proton transfers in the bacteriorhodopsin photocycle studied by millisecond time-resolved photopressure measurements.

    Science.gov (United States)

    Liu, Yan; Edens, Gregory J; Grzymski, Joseph; Mauzerall, David

    2008-07-22

    The volume and enthalpy changes associated with proton translocation steps during the bacteriorhodopsin (BR) photocycle were determined by time-resolved photopressure measurements. The data at 25 degrees C show a prompt increase in volume followed by two further increases and one decrease to the original state to complete the cycle. These volume changes are decomposed into enthalpy and inherent volume changes. The positive enthalpy changes support the argument for inherent entropy-driven late steps in the BR photocycle [Ort, D. R., and Parson, W. M. (1979) Enthalpy changes during the photochemical cycle of bacteriorhodopsin. Biophys. J. 25, 355-364]. The volume change data can be interpreted by the electrostriction effect as charges are canceled and formed during the proton transfers. A simple glutamic acid-glutamate ion model or a diglutamate-arginine-protonated water charge-delocalized model for the proton-release complex (PRC) fit the data. A conformational change with a large positive volume change is required in the slower rise (M --> N of the optical cycle) step and is reversed in the decay (N --> O --> BR) steps. The large variation in the published values for both the volume and enthalpy changes is greatly ameliorated if the values are presented per absorbed photon instead of per mole of BR. Thus, it is the highly differing assumptions about the quantum or reaction yields that cause the variations in the published results.

  11. A residue substitution near the beta-ionone ring of the retinal affects the M substates of bacteriorhodopsin

    Science.gov (United States)

    Varo, G.; Zimanyi, L.; Chang, M.; Ni, B.; Needleman, R.; Lanyi, J. K.

    1992-01-01

    The switch in the bacteriorhodopsin photocycle, which reorients access of the retinal Schiff base from the extracellular to the cytoplasmic side, was suggested to be an M1----M2 reaction (Varo and Lanyi. 1991. Biochemistry. 30:5008-5015, 5016-5022). Thus, in this light-driven proton pump it is the interconversion of proposed M substates that gives direction to the transport. We find that in monomeric, although not purple membrane-lattice immobilized, D115N bacteriorhodopsin, the absorption maximum of M changes during the photocycle: in the time domain between its rise and decay it shifts 15 nm to the blue relative to the spectrum at earlier times. This large shift strongly supports the existence of two M substates. Since D115 is located near the beta-ionone ring of the retinal, the result raises questions about the possible involvement of the retinal chain or protein residues as far away as 10 A from the Schiff base in the mechanism of the switching reaction.

  12. Experimental evidence for secondary protein-chromophore interactions at the Schiff base linkage in bacteriorhodopsin: molecular mechanism for proton pumping

    Energy Technology Data Exchange (ETDEWEB)

    Lewis, A.; Marcus, M.A.; Ehrenberg, B.; Crespi, H.

    1978-10-01

    Resonance Raman spectroscopy of the retinylidene chromophore in various isotopically labeled membrane environments together with spectra of isotopically labeled model compounds demonstrates that a secondary protein interaction is present at the protonated Schiff base linkage in bacteriorhodopsin. The data indicate that although the interaction is present in all protonated bacteriorhodopsin species it is absent in unprotonated intermediates. Furthermore, kinetic resonance Raman spectroscopy has been used to monitor the dynamics of Schiff base deprotonation as a function of pH. All results are consistent with lysine as the interacting group. A structure for the interaction is proposed in which the interacting protein group in an unprotonated configuration is complexed through the Schiff base proton to the Schiff base nitrogen. These data suggest a molecular mechanism for proton pumping and ion gate molecular regulation. In this mechanism, light causes electron redistribution in the retinylidene chromophore, which results in the deprotonation of an amino acid side chain with pK > 10.2 +- 0.3 (e.g., arginine). This induces subsequent retinal and protein conformational transitions which eventually lower the pK of the Schiff base complex from > 12 before light absorption to 10.2 +- 0.3 in microseconds after photon absorption. Finally, in this low pK state the complex can reprotonate the proton-deficient high pK group generated by light, and the complex is then reprotonated from the opposite side of the membrane.

  13. Studying the Mechanism of Phototransformation of Light Signal by Various Mammal and Bacterial Photoreceptor Pigments  Rhodopsin, Iodopsin and Bacteriorhodopsin

    Directory of Open Access Journals (Sweden)

    Ignat Ignatov

    2015-06-01

    Full Text Available This review article outlines the structure and function of mammal and bacterial photoreceptor pigments (rhodopsin, iodopsin, bacteriorhodopsin and their aspects of bio-nanotechnological usage. On an example of bacteriorhodopsin is described the method of its isolation from purple membranes of photo-organotrophic halobacterium Halobacterium halobium ET 1001 by cellular autolysis by distilled water, processing of bacterial biomass by ultrasound at 22 KHz, alcohol extraction of low and high-weight molecular impurities, cellular RNA, carotenoids and lipids, the solubilization with 0,5 % (w/v SDS-Na and subsequent fractionation by methanol and gel filtration chromatography on Sephadex G-200 Column balanced with 0,09 M Tris-buffer (pH = 8,35 with 0,1 % (w/v SDS-Na and 2,5 mM EDTA. Within the framework of the research the mechanism of color perception by the visual retina analyzer having the ability to analyze certain ranges of the optical spectrum as colors, was studied along with an analysis of the additive mixing of two or more colors. It was shown that at the mixing of electromagnetic waves with different wavelengths, the visual analyzer perceives them as the separate or average wave length corresponding to the mixing color.

  14. Voltage dependence of proton pumping by bacteriorhodopsin mutants with altered lifetime of the M intermediate.

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    Sven Geibel

    Full Text Available The light-driven proton pump bacteriorhodopsin (BR from Halobacterium salinarum is tightly regulated by the [H(+] gradient and transmembrane potential. BR exhibits optoelectric properties, since spectral changes during the photocycle are kinetically controlled by voltage, which predestines BR for optical storage or processing devices. BR mutants with prolonged lifetime of the blue-shifted M intermediate would be advantageous, but the optoelectric properties of such mutants are still elusive. Using expression in Xenopus oocytes and two-electrode voltage-clamping, we analyzed photocurrents of BR mutants with kinetically destabilized (F171C, F219L or stabilized (D96N, D96G M intermediate in response to green light (to probe H(+ pumping and blue laser flashes (to probe accumulation/decay of M. These mutants have divergent M lifetimes. As for BR-WT, this strictly correlates with the voltage dependence of H(+ pumping. BR-F171C and BR-F219L showed photocurrents similar to BR-WT. Yet, BR-F171C showed a weaker voltage dependence of proton pumping. For both mutants, blue laser flashes applied during and after green-light illumination showed reduced M accumulation and shorter M lifetime. In contrast, BR-D96G and BR-D96N exhibited small photocurrents, with nonlinear current-voltage curves, which increased strongly in the presence of azide. Blue laser flashes showed heavy M accumulation and prolonged M lifetime, which accounts for the strongly reduced H(+ pumping rate. Hyperpolarizing potentials augmented these effects. The combination of M-stabilizing and -destabilizing mutations in BR-D96G/F171C/F219L (BR-tri shows that disruption of the primary proton donor Asp-96 is fatal for BR as a proton pump. Mechanistically, M destabilizing mutations cannot compensate for the disruption of Asp-96. Accordingly, BR-tri and BR-D96G photocurrents were similar. However, BR-tri showed negative blue laser flash-induced currents even without actinic green light, indicating

  15. Crystallographic structure of the K intermediate of bacteriorhodopsin: conservation of free energy after photoisomerization of the retinal.

    Science.gov (United States)

    Schobert, Brigitte; Cupp-Vickery, Jill; Hornak, Viktor; Smith, Steven; Lanyi, Janos

    2002-08-23

    The K state, an early intermediate of the bacteriorhodopsin photocycle, contains the excess free energy used for light-driven proton transport. The energy gain must reside in or near the photoisomerized retinal, but in what form has long been an open question. We produced the K intermediate in bacteriorhodopsin crystals in a photostationary state at 100K, with 40% yield, and determined its X-ray diffraction structure to 1.43 A resolution. In independent refinements of data from four crystals, the changes are confined mainly to the photoisomerized retinal. The retinal is 13-cis,15-anti, as known from vibrational spectroscopy. The C13=C14 bond is rotated nearly fully to cis from the initial trans configuration, but the C14-C15 and C15=NZ bonds are partially counter-rotated. This strained geometry keeps the direction of the Schiff base N-H bond vector roughly in the extracellular direction, but the angle of its hydrogen bond with water 402, that connects it to the anionic Asp85 and Asp212, is not optimal. Weakening of this hydrogen bond may account for many of the reported features of the infrared spectrum of K, and for its photoelectric signal, as well as the deprotonation of the Schiff base later in the cycle. Importantly, although 13-cis, the retinal does not assume the expected bent shape of this configuration. Comparison of the calculated energy of the increased angle of C12-C13=C14, that allows this distortion, with the earlier reported calorimetric measurement of the enthalpy gain of the K state indicates that a significant part of the excess energy is conserved in the bond strain at C13.

  16. The influence of a transmembrane pH gradient on protonation probabilities of bacteriorhodopsin: the structural basis of the back-pressure effect.

    Science.gov (United States)

    Calimet, Nicolas; Ullmann, G Matthias

    2004-06-01

    Bacteriorhodopsin pumps protons across a membrane using the energy of light. The proton pumping is inhibited when the transmembrane proton gradient that the protein generates becomes larger than four pH units. This phenomenon is known as the back-pressure effect. Here, we investigate the structural basis of this effect by predicting the influence of a transmembrane pH gradient on the titration behavior of bacteriorhodopsin. For this purpose we introduce a method that accounts for a pH gradient in protonation probability calculations. The method considers that in a transmembrane protein, which is exposed to two different aqueous phases, each titratable residue is accessible for protons from one side of the membrane depending on its hydrogen-bond pattern. This method is applied to several ground-state structures of bacteriorhodopsin, which residues already present complicated titration behaviors in the absence of a proton gradient. Our calculations show that a pH gradient across the membrane influences in a non-trivial manner the protonation probabilities of six titratable residues which are known to participate in the proton transfer: D85, D96, D115, E194, E204, and the Schiff base. The residues connected to one side of the membrane are influenced by the pH on the other side because of their long-range electrostatic interactions within the protein. In particular, D115 senses the pH at the cytoplasmic side of the membrane and transmits this information to D85 and the Schiff base. We propose that the strong electrostatic interactions found between D85, D115, and the Schiff base as well as the interplay of their respective protonation states under the influence of a transmembrane pH gradient are responsible for the back-pressure effect on bacteriorhodopsin.

  17. The two consecutive M substates in the photocycle of bacteriorhodopsin are affected specifically by the D85N and D96N residue replacements

    Science.gov (United States)

    Zimanyi, L.; Cao, Y.; Chang, M.; Ni, B.; Needleman, R.; Lanyi, J. K.

    1992-01-01

    The photocycle of the proton pump bacteriorhodopsin contains two consecutive intermediates in which the retinal Schiff base is unprotonated; the reaction between these states, termed M1 and M2, was suggested to be the switch in the proton transport which reorients the Schiff base from D85 on the extracellular side to D96 on the cytoplasmic side (Varo and Lanyi, Biochemistry 30, 5016-5022, 1991). At pH 10 the absorption maxima of both M1 and M2 could be determined in the recombinant D96N protein. We find that M1 absorbs at 411 nm as do M1 and M2 in wild-type bacteriorhodopsin, but M2 absorbs at 404 nm. Thus, in M2 but not M1 the unprotonated Schiff base is affected by the D96N residue replacement. The connectivity of the Schiff base to D96 in the detected M2 state, but not in M1, is thereby established. On the other hand, the photostationary state which develops during illumination of D85N bacteriorhodopsin contains an M state corresponding to M1 with an absorption maximum shifted to 400 nm, suggesting that this species in turn is affected by D85. These results are consistent with the suggestion that M1 and M2 are pre-switch and post-switch states, respectively.

  18. Protein changes associated with reprotonation of the Schiff base in the photocycle of Asp96-->Asn bacteriorhodopsin. The MN intermediate with unprotonated Schiff base but N-like protein structure

    Science.gov (United States)

    Sasaki, J.; Shichida, Y.; Lanyi, J. K.; Maeda, A.

    1992-01-01

    The difference Fourier transform infrared spectrum for the N intermediate in the photoreaction of the light-adapted form of bacteriorhodopsin can be recorded at pH 10 at 274 K (Pfefferle, J.-M., Maeda, A., Sasaki, J., and Yoshizawa, T. (1991) Biochemistry 30, 6548-6556). Under these conditions, Asp96-->Asn bacteriorhodopsin gives a photoproduct which shows changes in protein structure similar to those observed in N of wild-type bacteriorhodopsin. However, decreased intensity of the chromophore bands and the single absorbance maximum at about 400 nm indicate that the Schiff base is unprotonated, as in the M intermediate. This photoproduct was named MN. At pH 7, where the supply of proton is not as restricted as at pH 10, Asp96-->Asn bacteriorhodopsin yields N with a protonated Schiff base. The Asn96 residue, which cannot deprotonate as Asp96 in wild-type bacteriorhodopsin, is perturbed upon formation of both MN at pH 10 and N at pH 7. We suggest that the reprotonation of the Schiff base is preceded by a large change in the protein structure including perturbation of the residue at position 96.

  19. The back photoreaction of the M intermediate in the photocycle of bacteriorhodopsin: mechanism and evidence for two M species

    Science.gov (United States)

    Druckmann, S.; Friedman, N.; Lanyi, J. K.; Needleman, R.; Ottolenghi, M.; Sheves, M.

    1992-01-01

    The back photoreaction of the M intermediate in the photocycle of bacteriorhodopsin is investigated both for the native pigment and its D96N mutant. The experimental setup is based on creating the M intermediate by a first pulse, followed by a (blue) laser pulse which drives the back photoreaction of M. Experiments are carried out varying the delay between the two pulses, as well as the temperature over the -25 degrees C-20 degrees C range. It is found that the kinetic patterns of the M back photoreaction change with time after the generation of this intermediate. The data provide independent evidence for the suggestion of a photocycle mechanism based on two distinct M intermediates. They are thus in keeping with the consecutive model of Varo and Lanyi (Biochemistry 30, 5016-5022; 1991), although they cannot exclude other models such as those based on branched or parallel cycles. More generally, we offer a "photochemical" approach to discriminating between intermediate stages in the photocycle which does not depend on spectroscopic and/or kinetic data. While markedly affecting the rate of the M --> N transition in the photocycle, the rate of the thermal step in back photoreaction of M, at both room and low temperatures, is not significantly affected by the D96N mutation. It is proposed that while Asp 96 is the Schiff-base protonating moiety in the M --> N transition, another residue (most probably Asp 85) reprotonates the Schiff base following light absorption by M.

  20. Titration kinetics of Asp-85 in bacteriorhodopsin: exclusion of the retinal pocket as the color-controlling cation binding site.

    Science.gov (United States)

    Fu, X; Bressler, S; Ottolenghi, M; Eliash, T; Friedman, N; Sheves, M

    1997-10-20

    The spectrum (the purple blue transition) and function of the light-driven proton pump bacteriorhodopsin are determined by the state of protonation of the Asp-85 residue located in the vicinity of the retinal chromophore. The titration of Asp-85 is controlled by the binding/unbinding of one or two divalent metal cations (Ca2+ or Mg2+). The location of such metal binding site(s) is approached by studying the kinetics of the cation-induced titration of Asp-85 using metal ions and large molecular cations, such as quaternary ammonium ions, R4N+ (R = Et, Pr, a divalent 'bolaform ion' [Et3N+-(CH2)4-N+Et3] and the 1:3 molecular complex formed between Fe2+ and 1,10-phenanthroline (OP). The basic multi-component kinetic features of the titration, extending from 10(-2) to 10(4) s, are unaffected by the charge and size of the cation. This indicates that cation binding to bR triggers the blue --> purple titration in a fast step, which is not rate-determining. In view of the size of the cations involved, these observations indicate that the cation binding site is in an exposed location on, or close to, the membrane surface. This excludes previous models, which placed the color-controlling Ca2+ ion in the retinal binding pocket.

  1. Role of trimer-trimer interaction of bacteriorhodopsin studied by optical spectroscopy and high-speed atomic force microscopy.

    Science.gov (United States)

    Yamashita, Hayato; Inoue, Keiichi; Shibata, Mikihiro; Uchihashi, Takayuki; Sasaki, Jun; Kandori, Hideki; Ando, Toshio

    2013-10-01

    Bacteriorhodopsin (bR) trimers form a two-dimensional hexagonal lattice in the purple membrane of Halobacterium salinarum. However, the physiological significance of forming the lattice has long been elusive. Here, we study this issue by comparing properties of assembled and non-assembled bR trimers using directed mutagenesis, high-speed atomic force microscopy (HS-AFM), optical spectroscopy, and a proton pumping assay. First, we show that the bonds formed between W12 and F135 amino acid residues are responsible for trimer-trimer association that leads to lattice assembly; the lattice is completely disrupted in both W12I and F135I mutants. HS-AFM imaging reveals that both crystallized D96N and non-crystallized D96N/W12I mutants undergo a large conformational change (i.e., outward E-F loop displacement) upon light-activation. However, lattice disruption significantly reduces the rate of conformational change under continuous light illumination. Nevertheless, the quantum yield of M-state formation, measured by low-temperature UV-visible spectroscopy, and proton pumping efficiency are unaffected by lattice disruption. From these results, we conclude that trimer-trimer association plays essential roles in providing bound retinal with an appropriate environment to maintain its full photo-reactivity and in maintaining the natural photo-reaction pathway.

  2. Recent Advances in the Field of Bionanotechnology: An Insight into Optoelectric Bacteriorhodopsin, Quantum Dots, and Noble Metal Nanoclusters

    Directory of Open Access Journals (Sweden)

    Christopher Knoblauch

    2014-10-01

    Full Text Available Molecular sensors and molecular electronics are a major component of a recent research area known as bionanotechnology, which merges biology with nanotechnology. This new class of biosensors and bioelectronics has been a subject of intense research over the past decade and has found application in a wide variety of fields. The unique characteristics of these biomolecular transduction systems has been utilized in applications ranging from solar cells and single-electron transistors (SETs to fluorescent sensors capable of sensitive and selective detection of a wide variety of targets, both organic and inorganic. This review will discuss three major systems in the area of molecular sensors and electronics and their application in unique technological innovations. Firstly, the synthesis of optoelectric bacteriorhodopsin (bR and its application in the field of molecular sensors and electronics will be discussed. Next, this article will discuss recent advances in the synthesis and application of semiconductor quantum dots (QDs. Finally, this article will conclude with a review of the new and exciting field of noble metal nanoclusters and their application in the creation of a new class of fluorescent sensors.

  3. Influence of the size and protonation state of acidic residue 85 on the absorption spectrum and photoreaction of the bacteriorhodopsin chromophore

    Science.gov (United States)

    Lanyi, J. K.; Tittor, J.; Varo, G.; Krippahl, G.; Oesterhelt, D.

    1992-01-01

    The consequences of replacing Asp-85 with glutamate in bacteriorhodopsin, as expressed in Halobacterium sp. GRB, were investigated. Similarly to the in vitro mutated and in Escherichia coli expressed protein, the chromophore was found to exist as a mixture of blue (absorption maximum 615 nm) and red (532 nm) forms, depending on the pH. However, we found two widely separated pKa values (about 5.4 and 10.4 without added salt), arguing for two blue and two red forms in separate equilibria. Both blue and red forms of the protein are in the two-dimensional crystalline state. A single pKa, such as in the E. coli expressed protein, was observed only after solubilization with detergent. The photocycle of the blue forms was determined at pH 4.0 with 610 nm photoexcitation, and that of the red forms at pH 10.5 and with 520 nm photoexcitation, in the time-range of 100 ns to 1 s. The blue forms produced no M, but a K- and an L-like intermediate, whose spectra and kinetics resembled those of blue wild-type bacteriorhodopsin below pH 3. The red forms produced a K-like intermediate, as well as M and N. Only the red forms transported protons. Specific perturbation of the neighborhood of the Schiff base by the replacement of Asp-85 with glutamate was suggested by (1) the shift and splitting of the pKa for what is presumably the protonation of residue 85, (2) a 36 nm blue-shift in the absorption of the all-trans red chromophore and a 25 nm red-shift of the 13-cis N chromophore, as compared to wild-type bacteriorhodopsin and its N intermediate, and (3) significant acceleration of the deprotonation of the Schiff base at pH 7, but not of its reprotonation and the following steps in the photocycle.

  4. Aspartic acid-96 is the internal proton donor in the reprotonation of the Schiff base of bacteriorhodopsin.

    Science.gov (United States)

    Otto, H; Marti, T; Holz, M; Mogi, T; Lindau, M; Khorana, H G; Heyn, M P

    1989-01-01

    Above pH 8 the decay of the photocycle intermediate M of bacteriorhodopsin splits into two components: the usual millisecond pH-independent component and an additional slower component with a rate constant proportional to the molar concentration of H+, [H+]. In parallel, the charge translocation signal associated with the reprotonation of the Schiff base develops a similar slow component. These observations are explained by a two-step reprotonation mechanism. An internal donor first reprotonates the Schiff base in the decay of M to N and is then reprotonated from the cytoplasm in the N----O transition. The decay rate of N is proportional to [H+]. By postulating a back reaction from N to M, the M decay splits up into two components, with the slower one having the same pH dependence as the decay of N. Photocycle, photovoltage, and pH-indicator experiments with mutants in which aspartic acid-96 is replaced by asparagine or alanine, which we call D96N and D96A, suggest that Asp-96 is the internal proton donor involved in the re-uptake pathway. In both mutants the stoichiometry of proton pumping is the same as in wild type. However, the M decay is monophasic, with the logarithm of the decay time [log (tau)] linearly dependent on pH, suggesting that the internal donor is absent and that the Schiff base is directly reprotonated from the cytoplasm. Like H+, azide increases the M decay rate in D96N. The rate constant is proportional to the azide concentration and can become greater than 100 times greater than in wild type. Thus, azide functions as a mobile proton donor directly reprotonating the Schiff base in a bimolecular reaction. Both the proton and azide effects, which are absent in wild type, indicate that the internal donor is removed and that the reprotonation pathway is different from wild type in these mutants. PMID:2556706

  5. Structures of aspartic acid-96 in the L and N intermediates of bacteriorhodopsin: analysis by Fourier transform infrared spectroscopy

    Science.gov (United States)

    Maeda, A.; Sasaki, J.; Shichida, Y.; Yoshizawa, T.; Chang, M.; Ni, B.; Needleman, R.; Lanyi, J. K.

    1992-01-01

    The light-induced difference Fourier transform infrared spectrum between the L or N intermediate minus light-adapted bacteriorhodopsin (BR) was measured in order to examine the protonated states and the changes in the interactions of carboxylic acids of Asp-96 and Asp-115 in these intermediates. Vibrational bands due to the protonated and unprotonated carboxylic acid were identified by isotope shift and band depletion upon substitution of Asp-96 or -115 by asparagine. While the signal due to the deprotonation of Asp-96 was clearly observed in the N intermediate, this residue remained protonated in L. Asp-115 was partially deprotonated in L. The C = O stretching vibration of protonated Asp-96 of L showed almost no shift upon 2H2O substitution, in contrast to the corresponding band of Asp-96 or Asp-115 of BR, which shifted by 9-12 cm-1 under the same conditions. In the model system of acetic acid in organic solvents, such an absence of the shift of the C = O stretching vibration of the protonated carboxylic acid upon 2H2O substitution was seen only when the O-H of acetic acid is hydrogen-bonded. The non-hydrogen-bonded monomer showed the 2H2O-dependent shift. Thus, the O-H bond of Asp-96 enters into hydrogen bonding upon conversion of BR to L. Its increased hydrogen bonding in L is consistent with the observed downshift of the O-H stretching vibration of the carboxylic acid of Asp-96.

  6. pH-Sensitive photoinduced energy transfer from bacteriorhodopsin to single-walled carbon nanotubes in SWNT-bR hybrids.

    Science.gov (United States)

    El Hadj, Karim; Bertoncini, Patricia; Chauvet, Olivier

    2013-10-22

    Energy transfer mechanisms in noncovalently bound bacteriorhodopsin/single-walled carbon nanotube (SWNT) hybrids are investigated using optical absorption and photoluminescence excitation measurements. The morphology of the hybrids was investigated by atomic force microscopy. In this study, proteins are immobilized onto the sidewall of the carbon nanotubes using a sodium cholate suspension-dialysis method that maintains the intrinsic optical and fluorescence properties of both molecules. The hybrids are stable in aqueous solutions for pH ranging from 4.2 to 9 and exhibit photoluminescence properties that are pH-dependent. The study reveals that energy transfer from bacteriorhodopsin to carbon nanotubes takes place. So, at pH higher than 5 and up to 9, the SWNTs absorb the photons emitted by the aromatic residues of the protein, inducing a strong increase in intensity of the E11 emissions of SWNTs through their E33 and E44 excitations. From pH = 4.2 to pH = 5, the protein fluorescence is strongly quenched whatever the emission wavelengths, while additional fluorescence features appear at excitation wavelengths ranging from 660 to 680 nm and at 330 nm. The presence of these features is attributed to a resonance energy transfer mechanism that has an efficiency of 0.94 ± 0.02. More, by increasing the pH of the dispersion, the fluorescence characteristics become those observed at higher pH values and vice versa.

  7. The Improved Method for Isolation of Photochrome Trans-membrane Protein Bacteriorhodopsin from Purple Membranes of Halobacterium Halobacterium Halobium ET 1001

    Directory of Open Access Journals (Sweden)

    Oleg Mosin

    2015-12-01

    Full Text Available It was developed the improved method for isolation of photochrome trans-membraine protein bacteriorhodopsin (output – 5 mg from 100 g of wet biomass capable to transform light energy to electrochemical energy of generated protons H+ and АТP. The protein was isolated from purple membranes of photo-organotrophic halobacterium Halobacterium halobium ET 1001 by cellular autolysis by distilled water, processing of bacterial biomass by ultrasound at 22 KHz, alcohol extraction of low and high-weight molecular impurities, cellular RNA, carotenoids and lipids, solubilization with 0.5% (w/v SDS-Na, fractionation by MeOH and column gel permeation chromatography (GPC of the final protein on Sephadex G-200 with 0.1% (w/v SDS-Na and 2.5 mM ETDA. The homogeneity of the isolated bacteriorhodopsin was proved by combination of preparative and analytical methods, including elecrtophoresis in 12.5% (w/v PAAG with 0.1% (w/v SDS-Na and regeneration of apomembranes with 13-trans-retinal.

  8. An Observation of Diamond-Shaped Particle Structure in a Soya Phosphatidylcohline and Bacteriorhodopsin Composite Langmuir Blodgett Film Fabricated by Multilayer Molecular Thin Film Method

    Science.gov (United States)

    Tsujiuchi, Y.; Makino, Y.

    A composite film of soya phosphatidylcohline (soya PC) and bacteriorhodopsin (BR) was fabricated by the multilayer molecular thin film method using fatty acid and lipid on a quartz substrate. Direct Force Microscopy (DFM), UV absorption spectra and IR absorption spectra of the film were characterized on the detail of surface structure of the film. The DFM data revealed that many rhombus (diamond-shaped) particles were observed in the film. The spectroscopic data exhibited the yield of M-intermediate of BR in the film. On our modelling of molecular configuration indicate that the coexistence of the strong inter-molecular interaction and the strong inter-molecular interaction between BR trimmers attributed to form the particles.

  9. Replacement of aspartic acid-96 by asparagine in bacteriorhodopsin slows both the decay of the M intermediate and the associated proton movement.

    Science.gov (United States)

    Holz, M; Drachev, L A; Mogi, T; Otto, H; Kaulen, A D; Heyn, M P; Skulachev, V P; Khorana, H G

    1989-01-01

    The photocycle, electrical charge translocation, and release and uptake of protons from the aqueous phase and release and uptake of protons from the aqueous phase were investigated for bacteriorhodopsin mutants with aspartic acid-96 replaced by asparagine or glutamic acid. At neutral pH the main effect of the Asp-96----Asn mutation is to slow by 2 orders of magnitude the decay of the M intermediate and the concomitant charge displacement associated with the reprotonation of the Schiff base from the cytoplasmic side of the membrane. The proton uptake measured with the indicator dye pyranine is likewise slowed without affecting the stoichiometry of proton pumping. The corresponding results for the Asp-96----Glu mutant, on the other hand, are very close to those for the wild-type protein. These results provide a kinetic explanation for the fact that at pH 7 and saturating light intensities the steady-state proton pumping is almost abolished in the Asp-96----Asn mutant but is close to normal in the Asp-96----Glu mutant. Thus, the pump is simply turning over much more slowly in the Asp-96----Asn mutant. The time constants of the decay of M and the associated charge translocation increase strongly with increasing pH for the Asp-96----Asn mutant but are virtually pH-independent for the Asp-96----Glu mutant and wild-type bacteriorhodopsin. At pH 5 the M decay of the Asp-96----Asn mutant is as fast as for wild type. These results suggest that Asp-96 serves as an internal proton donor in the proton-uptake pathway from the cytoplasm to the Schiff base. PMID:2648392

  10. Vibrational spectroscopy of bacteriorhodopsin mutants: light-driven proton transport involves protonation changes of aspartic acid residues 85, 96, and 212

    Energy Technology Data Exchange (ETDEWEB)

    Braiman, M.S.; Mogi, T.; Marti, T.; Stern, L.J.; Khorana, H.G.; Rothschild, K.J.

    1988-11-15

    Fourier transform infrared (FTIR) difference spectra have been obtained for the bR----K, bR----L, and bR----M photoreactions in bacteriorhodopsin mutants in which Asp residues 85, 96, 115, and 212 have been replaced by Asn and by Glu. Difference peaks that had previously been attributed to Asp COOH groups on the basis of isotopic labeling were absent or shifted in these mutants. In general, each COOH peak was affected strongly by mutation at only one of the four residues. Thus, it was possible to assign each peak tentatively to a particular Asp. From these assignments, a model for the proton-pumping mechanism of bR is derived, which features proton transfers among Asp-85, -96, and -212, the chromophore Schiff base, and other ionizable groups within the protein. The model can explain the observed COOH peaks in the FTIR difference spectra of bR photointermediates and could also account for other recent results on site-directed mutants of bR.

  11. Identification of Specific Effect of Chloride on the Spectral Properties and Structural Stability of Multiple Extracellular Glutamic Acid Mutants of Bacteriorhodopsin

    Science.gov (United States)

    Lazarova, Tzvetana; Mlynarczyk, Krzysztof; Querol, Enric; Tenchov, Boris; Filipek, Slawomir; Padrós, Esteve

    2016-01-01

    In the present work we combine spectroscopic, DSC and computational approaches to examine the multiple extracellular Glu mutants E204Q/E194Q, E204Q/E194Q/E9Q and E204Q/E194Q/E9Q/E74Q of bacteriorhodopsin by varying solvent ionic strength and composition. Absorption spectroscopy data reveal that the absorption maxima of multiple EC Glu mutants can be tuned by the chloride concentration in the solution. Visible Circular dichroism spectra imply that the specific binding of Cl- can modulate weakened exciton chromophore coupling and reestablish wild type-like bilobe spectral features of the mutants. The DSC data display reappearance of the reversible thermal transition, higher Tm of denaturation and an increase in the enthalpy of unfolding of the mutants in 1 M KCl solutions. Molecular dynamics simulations indicate high affinity binding of Cl- to Arg82 and to Gln204 and Gln194 residues in the mutants. Analysis of the experimental data suggests that simultaneous elimination of the negatively charged side chain of Glu194 and Glu204 is the major cause for mutants’ alterations. Specific Cl- binding efficiently coordinates distorted hydrogen bonding interactions of the EC region and reconstitutes the conformation and structure stability of mutated bR in WT-like fashion. PMID:27657718

  12. Incorporation of the dopamine D2L receptor and bacteriorhodopsin within bicontinuous cubic lipid phases. 2. Relevance to in meso crystallization of integral membrane proteins in novel lipid systems

    Energy Technology Data Exchange (ETDEWEB)

    Conn, Charlotte E.; Darmanin, Connie; Sagnella, Sharon M.; Mulet, Xavier; Greaves, Tamar L.; Varghese, Joseph N.; Drummond, Calum J.

    2014-09-24

    The dopamine D2 long (D2L) receptor and bacteriorhodopsin (bR), which are integral membraneproteins, have been incorporated within bicontinuous cubic mesophases formed by the lipids anandamide and H-farnesoyl monoethanolamide, which have been specifically investigated by us for use as in mesocrystallization media. We show that the incorporated membraneprotein affects the structure of the cubic phases with the particular effect observed dependent on the geometry of the underlying cubic phase. The results are complementary to those obtained in Part 1 of this series, where we demonstrated that the structural effects observed depend on the structure of the membraneprotein. Importantly protein concentrations commonly used for crystallization can destroy the cubic phase matrix, particularly where there is a large discrepancy between the hydrophilic and the hydrophobic spans of the membraneprotein, and the hydrophilic and hydrophobic domain sizes of the cubic phase.

  13. 化学增强细菌视紫红质膜对高斯光束的自整形%Self-shaping of Gaussian Beam by Chemically Enhanced Bacteriorhodopsin Film

    Institute of Scientific and Technical Information of China (English)

    王勇; 富光华; 等

    2002-01-01

    A CW Gaussian-beam illuminates a thin film of chemically enhanced bacteriorhodopsin (bR),and then strong self-defocusing of the Gaussian beam passing the bR film will be shown when the ilumination intensity is greater than 220 W/cm2 for the sample used.Owing to this effect,the self-shaping of Gasussian beam can be realized.The analyses show the incidentbeam intensity and the thickness and concentration of the bR film influence the transverse profile of the transmitted beam.%连续激光束照射化学增强的细菌视紫红质膜.当照射强度大于某一定值,光束产生强的自散焦效应.通过这个效应可以实现高斯光束的自整形.本文讨论了入射光以及bR膜的各项参数对透过光束强度的横向轮廓的影响.

  14. Regio-selective detection of dynamic structure of transmembrane alpha-helices as revealed from (13)C NMR spectra of [3-13C]Ala-labeled bacteriorhodopsin in the presence of Mn2+ ion.

    Science.gov (United States)

    Tuzi, S; Hasegawa, J; Kawaminami, R; Naito, A; Saitô, H

    2001-07-01

    13C Nuclear magnetic resonance (NMR) spectra of [3-(13)C]Ala-labeled bacteriorhodopsin (bR) were edited to give rise to regio-selective signals from hydrophobic transmembrane alpha-helices by using NMR relaxation reagent, Mn(2+) ion. As a result of selective suppression of (13)C NMR signals from the surfaces in the presence of Mn(2+) ions, several (13)C NMR signals of Ala residues in the transmembrane alpha-helices were identified on the basis of site-directed mutagenesis without overlaps from (13)C NMR signals of residues located near the bilayer surfaces. The upper bound of the interatomic distances between (13)C nucleus in bR and Mn(2+) ions bound to the hydrophilic surface to cause suppressed peaks by the presence of Mn(2+) ion was estimated as 8.7 A to result in the signal broadening to 100 Hz and consistent with the data based on experimental finding. The Ala C(beta) (13)C NMR peaks corresponding to Ala-51, Ala-53, Ala-81, Ala-84, and Ala-215 located around the extracellular half of the proton channel and Ala-184 located at the kink in the helix F were successfully identified on the basis of (13)C NMR spectra of bR in the presence of Mn(2+) ion and site-directed replacement of Ala by Gly or Val. Utilizing these peaks as probes to observe local structure in the transmembrane alpha-helices, dynamic conformation of the extracellular half of bR at ambient temperature was examined, and the local structures of Ala-215 and 184 were compared with those elucidated at low temperature. Conformational changes in the transmembrane alpha-helices induced in D85N and E204Q and its long-range transmission from the proton release site to the site around the Schiff base in E204Q were also examined.

  15. Introduction of Deuterated Aromatic Amino Acids  [2,3,4,5,6-2H5]phenylalanine, [3,5-2H2]tyrosine and [2,4,5,6,7-2H5]tryptophan into a Molecule of Photochrome Trans-membrane Protein Bacteriorhodopsin

    Directory of Open Access Journals (Sweden)

    Oleg Mosin

    2015-09-01

    Full Text Available It was carried out the introduction of functionally important deuterated aromatic amino acids  [2,3,4,5,6-2H5]phenylalanine, [3,5-2H2]tyrosine and [2,4,5,6,7-2H5]tryptophan into a molecule of photochrome trans-membrane protein bacteriorhodopsin, synthesized by a photo-organotrophic halobacterium Halobacterium halobium ET 1001. The deuterated protein (output 810 mg was isolated from purple membranes by cellular autolysis by distilled water, processing of bacterial biomass by ultrasound at 22 KHz, alcohol extraction of low and high-weight molecular impurities, cellular RNA, carotenoids and lipids, with the subsequent solubilization of final product with 0,5 % (w/v SDS-Na and fractionation by methanol, gel filtration chromatography on Sephadex G-200, reverse-phase HPLC and EI impact mass-spectrometry of methyl esters of N-Dns-[2H]derivatives of amino acids. Deuterium was detected in all residues of aromatic amino acids. However, the presence in the EI mass spectrum of the BR hydrolysate the peaks [M]+ of semi-deuterated analogues of aromatic amino acids  phenylalanine with [M]+ at m/z = 413418, tyrosine  with [M]+ at m/z = 428430 and tryptophan  with [M]+ at m/z = 453457 with different levels of contributions to the deuterium enrichment of molecules testifies about the conservation of the minor pathways of biosynthesis of aromatic amino acids de novo.

  16. Spectral Signatures of the Pentagonal Water Cluster in Bacteriorhodopsin

    Energy Technology Data Exchange (ETDEWEB)

    Baer, M; Mathias, G; Kuo, I W; Tobias, D J; Mundy, C J; Marx, D

    2008-07-25

    The exchange of protons between basic and acidic groups within proteins often involves transient protonation of amino acids and water molecules embedded in the protein matrix. One of the best studied proteins in this respect is Bacteriorohodopsin (BR), which works in the membrane of Halobacterium salinarium as a light-driven proton pump. The pumping process is triggered in the initial bR state by a photon absorption of an all-trans retinylidene chromophore, which is linked via a protonated Schiff base (pRSB) to the sidechain of Lys216. The subsequent photocycle comprises a series of intermediate states J, K, L, M, N and O, which are characterized by conformational and absorbance changes of the chromophore accompanying several elementary proton transfer processes. Upon completion of the photocycle one net proton has been transferred from the cyctoplasmic to the extracellular side against the proton gradient across the membrane. These proton exchange reactions can be monitored by time resolved infrared (IR) spectroscopy of the BR wild type and site specific mutants, which allow the localization of absorbance changes within the protein. Furthermore, these measurements have revealed the fundamental importance of internal water molecules in these processes as supported by recent large-scale QM/MM molecular dynamics studies of anharmonic IR spectra.

  17. Theory and procedures for finding a correct kinetic model for the bacteriorhodopsin photocycle.

    Science.gov (United States)

    Hendler, R W; Shrager, R; Bose, S

    2001-04-26

    In this paper, we present the implementation and results of new methodology based on linear algebra. The theory behind these methods is covered in detail in the Supporting Information, available electronically (Shragerand Hendler). In brief, the methods presented search through all possible forward sequential submodels in order to find candidates that can be used to construct a complete model for the BR-photocycle. The methodology is limited only to forward sequential models. If no such models are compatible with the experimental data,none will be found. The procedures apply objective tests and filters to eliminate possibilities that cannot be correct, thus cutting the total number of candidate sequences to be considered. In the current application,which uses six exponentials, the total sequences were cut from 1950 to 49. The remaining sequences were further screened using known experimental criteria. The approach led to a solution which consists of a pair of sequences, one with 5 exponentials showing BR* f L(f) M(f) N O BR and the other with three exponentials showing BR* L(s) M(s) BR. The deduced complete kinetic model for the BR photocycle is thus either a single photocycle branched at the L intermediate or a pair of two parallel photocycles. Reasons for preferring the parallel photocycles are presented. Synthetic data constructed on the basis of the parallel photocycles were indistinguishable from the experimental data in a number of analytical tests that were applied.

  18. A Model for Light-Driven Proton Pump and Transfer in Bacteriorhodopsin

    Institute of Scientific and Technical Information of China (English)

    黄玉华; 赵有源; 欧阳小平; 李富铭; 丁建东; 李庆国

    2003-01-01

    A theoretical model was assumed to describe the kinetic processes of light-driven proton pump and motion. The calculation shows that the photovoltage signals are greatly affected by experimental conditions including pulse width of excitation and impedance of measurement system. At the same excitation, only a negative photovoltage signal can be observed with small impedance and both the negative and positive signals can be observed with large impedance. With the same impedance, shorter response time of the pulsed photoelectric signals is responsible for shorter pulsed excitation. All these phenomena are in good agreement with the experimental results.

  19. The interaction between purple membrane and membrane lipid

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Bacteriorhodopsin in purple membrane was reconstituted into different lipid vesicles. The effect of three different lipids on the structure and function of bacteriorhodopsin in lipid vesicles was studied by the observation on freeze-fracture eletron microscopy, the rotational diffusion of bacteriorhodopsin in lipid vesicles, the measurement of absorption spectrum, and the absorbance change with time. For these prepared samples, the results showed that DMPC was the stable lipid environment of bacteriorhodopsin; egg-pc causeed the loss of retinal chromophore of bacteriorhodopsin and it was not reversible change, cholesterol could stabilize the bacteriorhodopsin in lipid environment,but it caused the aggregation of bacteriorhodopsin.

  20. Time Resolved Optical Studies on The Plasmonic Field Enhancement of Bacteriorhodopsin Proton Photo-current: Final Technical Report Covering Aug 31, 2015–Aug 31, 2016

    Energy Technology Data Exchange (ETDEWEB)

    El-Sayed, Mostafa A. [Georgia Inst. of Technology, Atlanta, GA (United States). School of Chemistry and Biochemistry

    2016-09-15

    Our research continues to focus on the effects of plasmonic nanoparticles on organic and inorganic light-harvesting materials. Recent work has focused on the synthesis of stabilized gold nanoparticles to enhance the efficiency of dye-sensitized solar cells (DSSCs). Two major concerns in dye sensitized solar cells (DSSCs) are efficient light absorption and charge collection. Charge collection typically suffers because transport of electrons through the mesoporous TiO2 substrate is slow. Thus, one obvious way to improve charge collection is to reduce the thickness of the TiO2. Alternatively, a form of TiO2 with fewer grain boundaries, such as nanotubes, could be used in place of sintered nanospheres. Unfortunately, both of these solutions end up reducing the amount of surface area available to adsorb dye molecules. This directly reduces the percentage of photons absorbed. This problem could be avoided if dye molecules with larger absorption were designed; although synthetic chemists seem to be pushing the limits of what is achievable. Plasmonic nanoparticles offer an alternative way to boost light absorption. It is well known that plasmonic nanoparticles can enhance the local electric field of resonant frequencies of light. If this were in the same spectral region as the dye’s absorption band it would increase the percentage of absorbed photons. One concern is that if the nanoparticles are too close to the dye molecules they can quench the excited state. To avoid this problem, we prepared gold nanoparticles with a silica shell. This limited the amount of quenching while still permitting some enhancement of absorption. Unfortunately, we ran into some serious issues. The iodide based electrolyte etched the gold nanoparticles, completely dissolving them within a few hours. The silica shell should have provided protection but there were pinholes through which iodide could diffuse. Increasing the thickness of the silica to over 10 nm prevented etching but also limited any photon absorption enhancement.

  1. PHOTONIC SWITCHES BASED ON BACTERIORHODOPSIN%细菌视紫红质光开关特性实验研究

    Institute of Scientific and Technical Information of China (English)

    田燕宁; 冯晓强; 侯洵; 陈烽

    2001-01-01

    介绍实现光子开关材料BR分子的重要性。理论上分析BR分子材料光学特性,实验观测BR分子材料在400nm和632nm光照射下的相互抑制作用,最后分析BR的光子开关特性。%The importance of BR which is used in achieving photonic switches is summarized simply.The principle and ways how to realized photonic switches based on BR are analyzed in detail.Under illuminatiing by two beams at 568nm and 412nm,the transmission of two beams is moduleted by BR in a complementary fashion.Then,this paper shows the experiments of photonic switches.The datas from experiments are analyzed finally.

  2. Theoretical Calculation and Experimental Measurement of Photoinduced Refractive Index Change of Bacteriorhodopsin Film%菌紫质薄膜光致折射率变化的理论计算和实验测量

    Institute of Scientific and Technical Information of China (English)

    董卫斌; 姚保利; 门克内木乐; 王英利; 郑媛; 雷铭; 陈国夫

    2005-01-01

    基因改性细菌视紫红质BR-D96N由于M态寿命的延长而具有显著的光致变色特性.根据Kramers-Kronig变换关系,样品吸收光谱的变化会引起样品折射率的变化.本文首先从实验上测量了BR-D96N薄膜样品在光激发前后的吸收光谱,然后根据Kramers-Kronig变换关系,理论计算了对应此光致变色光谱变化的光致折射率变化光谱.实验上为了直接测量样品的光致折射率变化,采用Michelson干涉方法测量BR-D96N薄膜在不同探测波长下的光致折射率变化量,并与理论计算曲线进行了比较.

  3. Real-time UV-visible spectroscopy analysis of purple membrane-polyacrylamide film formation taking into account Fano line shapes and scattering.

    Directory of Open Access Journals (Sweden)

    María Gomariz

    Full Text Available We theoretically and experimentally analyze the formation of thick Purple Membrane (PM polyacrylamide (PA films by means of optical spectroscopy by considering the absorption of bacteriorhodopsin and scattering. We have applied semiclassical quantum mechanical techniques for the calculation of absorption spectra by taking into account the Fano effects on the ground state of bacteriorhodopsin. A model of the formation of PM-polyacrylamide films has been proposed based on the growth of polymeric chains around purple membrane. Experimentally, the temporal evolution of the polymerization process of acrylamide has been studied as function of the pH solution, obtaining a good correspondence to the proposed model. Thus, due to the formation of intermediate bacteriorhodopsin-doped nanogel, by controlling the polymerization process, an alternative methodology for the synthesis of bacteriorhodopsin-doped nanogels can be provided.

  4. Real-time UV-visible spectroscopy analysis of purple membrane-polyacrylamide film formation taking into account Fano line shapes and scattering.

    Science.gov (United States)

    Gomariz, María; Blaya, Salvador; Acebal, Pablo; Carretero, Luis

    2014-01-01

    We theoretically and experimentally analyze the formation of thick Purple Membrane (PM) polyacrylamide (PA) films by means of optical spectroscopy by considering the absorption of bacteriorhodopsin and scattering. We have applied semiclassical quantum mechanical techniques for the calculation of absorption spectra by taking into account the Fano effects on the ground state of bacteriorhodopsin. A model of the formation of PM-polyacrylamide films has been proposed based on the growth of polymeric chains around purple membrane. Experimentally, the temporal evolution of the polymerization process of acrylamide has been studied as function of the pH solution, obtaining a good correspondence to the proposed model. Thus, due to the formation of intermediate bacteriorhodopsin-doped nanogel, by controlling the polymerization process, an alternative methodology for the synthesis of bacteriorhodopsin-doped nanogels can be provided.

  5. Calcium binding to the purple membrane : A molecular dynamics study

    NARCIS (Netherlands)

    Wassenaar, Tsjerk A.; Daura, Xavier; Padros, Esteve; Mark, Alan E.

    2009-01-01

    The purple membrane (PM) is a specialized membrane patch found in halophilic archaea, containing the photoreceptor bacteriorhodopsin (bR). It is long known that calcium ions bind to the PM, but their position and role remain elusive to date. Molecular dynamics simulations in conjunction with a highl

  6. Biocompatible and Biomimetic Self-Assembly of Functional

    Science.gov (United States)

    2007-10-03

    effective interaction potential between cells and model supported lipid bilayer systems, lipid bilayer coated beads or supported lipid bilayers...bacterial proteins at the surface of yeast cells. Bacteriorhodopsin (BR) is a transmembrane photochromic protein isolated from the purple membrane of...precursors with a biologically compatible surfactant, glycerol monooleate (GMO) via dip- coating , spin- coating , drop-casting, or aerosol deposition

  7. JPRS Report Science & Technology USSR: Life Sciences

    Science.gov (United States)

    2007-11-02

    topography of bacteriorhodopsin sites in Halobacterium halobium involves removal of the apomembranes (chro- mophore) by hydroxylamine treatment of the...rabbits under hexenal anesthesia (10-15 mg/kg) to evaluate the effects of low-intensity HNL on corneal and scleral permeability of 35S-streptomycin

  8. Dead Sea rhodopsins revisited.

    Science.gov (United States)

    Bodaker, Idan; Suzuki, Marcelino T; Oren, Aharon; Béjà, Oded

    2012-12-01

    The Dead Sea is a unique hypersaline ecosystem with near toxic magnesium levels (∼2 M), dominance of divalent cations and a slightly acidic pH. Previously, we reported a haloarchaeon related to Halobacterium salinarum to dominate in a microbial bloom that developed in 1992 in the upper water layers of the lake following massive freshwater runoff. Whether this clade also dominated an earlier bloom in 1980-1982 cannot be ascertained as no samples for cultivation-independent analysis were preserved. The presence of the light-driven proton pump bacteriorhodopsin was reported in the 1980-1982 bloom of prokaryotes that had developed in the Dead Sea. To test the hypothesis that bacteriorhodopsin proton pumping may play a major role in determining what type of haloarchaea may dominate in specific bloom conditions, we compared rhodopsin genes recovered from Dead Sea biomass collected in different periods with genes coding for retinal proteins in isolated haloarchaea. Novel bacteriorhodopsin and sensory rhodopsin genes were found in samples collected in 2007 and 2010. The fact that no rhodopsin genes were recovered from samples collected during the 1992 bloom, which was dominated by a single species, suggests that different clades were present in the 1980-1982 and 1992 blooms, and that bacteriorhodopsin proton pumping did not necessarily play a determinative role in the dominance of specific halophiles in the blooms.

  9. Evolution of rhodopsin ion pumps in haloarchaea

    Directory of Open Access Journals (Sweden)

    Ford Doolittle W

    2007-05-01

    Full Text Available Abstract Background The type 1 (microbial rhodopsins are a diverse group of photochemically reactive proteins that display a broad yet patchy distribution among the three domains of life. Recent work indicates that this pattern is likely the result of lateral gene transfer (LGT of rhodopsin genes between major lineages, and even across domain boundaries. Within the lineage in which the microbial rhodopsins were initially discovered, the haloarchaea, a similar patchy distribution is observed. In this initial study, we assess the roles of LGT and gene loss in the evolution of haloarchaeal rhodopsin ion pump genes, using phylogenetics and comparative genomics approaches. Results Mapping presence/absence of rhodopsins onto the phylogeny of the RNA polymerase B' subunit (RpoB' of the haloarchaea supports previous notions that rhodopsins are patchily distributed. The phylogeny for the bacteriorhodopsin (BR protein revealed two discrepancies in comparison to the RpoB' marker, while the halorhodopsin (HR tree showed incongruence to both markers. Comparative analyses of bacteriorhodopsin-linked regions of five haloarchaeal genomes supported relationships observed in the BR tree, and also identified two open reading frames (ORFs that were more frequently linked to the bacteriorhodopsin gene than those genes previously shown to be important to the function and expression of BR. Conclusion The evidence presented here reveals a complex evolutionary history for the haloarchaeal rhodopsins, with both LGT and gene loss contributing to the patchy distribution of rhodopsins within this group. Similarities between the BR and RpoB' phylogenies provide supportive evidence for the presence of bacteriorhodopsin in the last common ancestor of haloarchaea. Furthermore, two loci that we have designated bacterio-opsin associated chaperone (bac and bacterio-opsin associated protein (bap are inferred to have important roles in BR biogenesis based on frequent linkage and co

  10. Imaging bacteriorhodopsinlike molecules of claretmembranes from Tibet halobacteria xz515 by atomic force microscope

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Halobacteria H. sp.xz 515 was isolated from a salt lake in Tibet. Although proton release-and-uptake across claret membrane is in reverse order compared to bacteriorhodopsin in purple membrane from Halobacterium Salinarum, and its efficiency of proton pump is much lower,AFM image shows that the molecules are still arranged in a two-dimensional hexagonal lattice of trimers. Primary structure of C- to G-helix of the archaerhodopsin shows that it has only 56% homology with bacteriorhodopsin. But the interactive amino acid residues at the interface between Band D-helixes are conserved. These amino acid residues are believed to play a significant role in the stability of protein oligomers.

  11. Isolation and characterization of a novel strain of Natrinema containing a bop gene

    Institute of Scientific and Technical Information of China (English)

    XU Xue-wei; WU Min; HUANG Wei-da

    2005-01-01

    A novel member of extremely halophilic archaea, strain AJ2, was isolated from Ayakekum Lake located in Altun Mountain National Nature Reserve of Xinjiang Uygur Autonomous Region in China. The strain A J2 requires at least 10% (w/v)NaCl and grows 10% to 30% (optimum at 20%). Phylogenetic analysis based on 16S rDNA sequence comparison revealed that strain A J2 clustered to three Natrinema species with less than 97.7% sequence similarities, suggesting A J2 is a novel member of Natrinema. A bacteriorhodopsin-encoding (bop) gene was subsequently detected in the A J2 genome using the polymerase chain reaction technique. The cloning and sequencing of a 401 base pairs fragment indicated the deduced amino acid sequence of bop from A J2 is different from that reported for bacteriorhodopsins. This is the first reported detection of a bop gene in Natrinema.

  12. Membrane boenergetics of salt tolerant organisms. Progress report, June 1993--June 1995

    Energy Technology Data Exchange (ETDEWEB)

    Lanyi, J.K.

    1996-06-01

    Substantial progress was made on describing the pathway of the transported proton in bacteriorhodopsin, and the thermodynamics of the proton transfers. The underlying principle of the transport was identified as the alternating access of the retinal Schiff base toward the two membrane surfaces, regulated by electrostatic interaction between the retinylidene nitrogen and its counterion. Consistent with a shared transport mechanism for both retinal proteins, bacteriorhodopsin was converted into a balorhodopsin-like chloride pump by replacing asp-85 with threonine. This region is thereby identified as the active site that determines ion specificity. Description of the metal ion-dependent kinetics of the ATP hydrolysis provided clues to the structure of active site in the halobacterial ATPase.

  13. Investigation of structural change of purple membrane in storage by transmission electron microscope and atomic force microscope

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The structural change of purple membrane during storage has been investigated by means of transmission electron microscope and atomic force microscope. It is found that many liposomes have spontaneously evolved from the purple membrane sheets isolated three years ago. The membrane proteins on the liposomes, bacteriorhodopsin, are still presented as trimers in 2-D hexagonal structure, which is the same as that in natural cell membrane. However, the cytoplasmic surface of purple membrane faced outside on the liposomes.

  14. Investigations on the electrical current-voltage response in protein light receptors

    CERN Document Server

    Alfinito, E; Reggiani, L

    2014-01-01

    We report a theoretical/computational approach for modeling the current-voltage characteristics of sensing proteins. The modeling is applied to a couple of transmembrane proteins, bacteriorhodopsin and proteorhodopsin, sensitive to visible light and promising biomaterials for the development of a new generation of photo-transducers. The agreement between theory and experiments sheds new light on the microscopic interpretation of charge transfer in proteins and biological materials in general.

  15. Quantum Mechanical/Molecular Mechanical Studies on Spectral Tuning Mechanisms of Visual Pigments and Other Photoactive Proteins†

    Science.gov (United States)

    Altun, Ahmet; Yokoyama, Shozo; Morokuma, Keiji

    2008-01-01

    The protein environments surrounding the retinal tune electronic absorption maximum from 350 to 630 nm. Hybrid quantum mechanical/molecular mechanical (QM/MM) methods can be used in calculating excitation energies of retinal in its native protein environments and in studying the molecular basis of spectral tuning. We hereby review recent QM/MM results on the phototransduction of bovine rhodopsin, bacteriorhodopsin, sensory rhodopsin II, nonretinal photoactive yellow protein and their mutants. PMID:18331400

  16. Quantum Mechanical/Molecular Mechanical Studies on Spectral Tuning Mechanisms of Visual Pigments and Other Photoactive Proteins†

    OpenAIRE

    Altun, Ahmet; Yokoyama, Shozo; Morokuma, Keiji

    2008-01-01

    The protein environments surrounding the retinal tune electronic absorption maximum from 350 to 630 nm. Hybrid quantum mechanical/molecular mechanical (QM/MM) methods can be used in calculating excitation energies of retinal in its native protein environments and in studying the molecular basis of spectral tuning. We hereby review recent QM/MM results on the phototransduction of bovine rhodopsin, bacteriorhodopsin, sensory rhodopsin II, nonretinal photoactive yellow protein and their mutants.

  17. Purple membranes from Halobacterium salinarum as building blocks for nanobiotechnology: The importance of mechanical and thermal properties for matrix and surface applications

    OpenAIRE

    Rhinow, Daniel Christopher

    2008-01-01

    Bacteriorhodopsin (BR) is a light-driven proton pump and the key protein in halobacterial photosynthesis. In its native host, the archaeon Halobacterium salinarum, BR trimers arrange into a 2-D crystalline lattice, the so-called purple membranes (PMs) which comprise BR and lipids only. Along with the PM assembly BR is astonishingly stable against thermal and chemical stress which makes it an excellent candidate for a variety of ...

  18. Azulenic Chromophores For Optical Limiting and Other Nonlinear Optical Applications

    Science.gov (United States)

    2007-11-02

    that azulene-containing polyenal analogs of retinal could be incorporated into the important retinal-protein, bacteriorhodopsin. The resulting...S. H. Liu, “Effects of Fluoro-Substituents on Excited State Properties of Conjugated Polyenes (Fluorinated Retinoids),” The Spectrum, 14, 8-12 (2002...Colmenares, A. E. Asato, and R. S. H. Liu, “Effects of Fluoro-Substituents on Excited State Properties of Conjugated Polyenes (Fluorinated Retinoids),” The

  19. H+-type and OH−-type biological protonic semiconductors and complementary devices

    OpenAIRE

    Deng, Yingxin; Josberger, Erik; Jin, Jungho; Rousdari, Anita Fadavi; Helms, Brett A.; Zhong, Chao; Anantram, M. P.; Rolandi, Marco

    2013-01-01

    Proton conduction is essential in biological systems. Oxidative phosphorylation in mitochondria, proton pumping in bacteriorhodopsin, and uncoupling membrane potentials by the antibiotic Gramicidin are examples. In these systems, H+ hop along chains of hydrogen bonds between water molecules and hydrophilic residues – proton wires. These wires also support the transport of OH− as proton holes. Discriminating between H+ and OH− transport has been elusive. Here, H+ and OH− transport is achieved ...

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

    Energy Technology Data Exchange (ETDEWEB)

    Zabut, Baker; El-Kahlout, Kamal [Department of Biochemistry, School of Science, IUG, Gaza (PS); Yuecel, Meral [Department of Biology, Middle East Technical University, 06531 Ankara (Turkey); Guenduez, Ufuk; Tuerker, Lemi [Department of Chemistry, Middle East Technical University, 06531 Ankara (Turkey); Eroglu, Inci [Department of Chemical Engineering, Middle East Technical University, 06531 Ankara (Turkey)

    2006-09-15

    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 (4M 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{sub 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 400ml water-jacketed-glass column stirred photobioreactors. In combined systems 10-200nmol 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. (author)

  1. Domain Modeling: NP_009174.1 [SAHG[Archive

    Lifescience Database Archive (English)

    Full Text Available NP_009174.1 chr6 Bacteriorhodopsin-like d1h2sa_ chr6/NP_009174.1/NP_009174.1_holo_10-249.pdb swppa 32R,35L,...36G,39P,67L,70L,71L,80M,101Y,114R,115Y,116W,121K,122G,124V,125L,143V,149W,152I,153F,156L,184K,191L,203L,...206V,207L,210V,221A,222F,229G,230L,239L,240R,243R BOG,RET 0 ...

  2. Holographic Properties of BR-D96N Film and Its Application in Hologram Aberration Correction

    Institute of Scientific and Technical Information of China (English)

    ZHENG Yuan(郑媛); YAO Bao-Li(姚保利); WANG Ying-Li(王英利); MENKE Neimule(门克内木乐); LEI Ming(雷铭); CHEN Guo-Fu(陈国夫); Norbert HAMPP

    2003-01-01

    A biophotochromic material, i.e., genetic mutant bacteriorhodopsin (BR-D96N), was experimentally studied on its holographic recording properties. The saturation absorption curve and the diffraction efficiency curve were measured respectively. As holographic storage application, reflection type polarization holograms were recorded on the BR-D96N film. The 173° configuration between the object and reference beams proves that the spatial resolution of the film is over 6000 lines/mm. By using phase conjugate wave of reference beam as reconstruction beam, the distorted object image introduced by the optical components and the defects in the recording medium can be well corrected.

  3. Adaptive Holographic Fiber-Optic Interferometer

    Science.gov (United States)

    Kozhevnikov, Nikolai M.; Lipovskaya, Margarita J.

    1990-04-01

    Interaction of phase-modulated light beams in photorefractive local inertial responce media was analysed. Interaction of this type allows to registrate phase-modulated signals adaptively under low frequency phase disturbtion. The experiments on multimode fiber-optic interferometer with demodulation element based on photorefractive bacteriorhodopsin-doped polimer film are described. As the writing of dynamic phase hologram is an inertial process the signal fluctuations with the frequencies up to 100 Hz can be canceled. The hologram efficiencies are enough to registrate high frequency phase shifts ~10-4 radn.

  4. Quantum Nanobiology and Biophysical Chemistry

    DEFF Research Database (Denmark)

    2013-01-01

    An introduction was provided in the first issue by way of an Editorial to this special two issue volume of Current Physical Chemistry – “Quantum Nanobiology and Biophysical Chemistry” [1]. The Guest Editors would like to thank all the authors and referees who have contributed to this second issue....... demonstrate extremely low detection performance of acyl-homoserine lactone in a biologically relevant system using surface enhanced Raman spectroscopy. Sugihara and Bondar evaluate the influence of methyl-groups and the protein environment on retinal geometries in rhodopsin and bacteriorhodopsin, two...

  5. Directional Self-assembly in Archaerhodopsin-Reconstituted Phospholipid Liposomes

    Institute of Scientific and Technical Information of China (English)

    吴佳; 黄力; 刘坚; 明明; 李庆国; 丁建东

    2005-01-01

    This paper reports, for the first time, that Archaerhodopsin-4 (AR4) could be reconstituted into phospholipid liposomes by self-assembly. AR4 is a new membrane protein isolated from halobacteria H.sp. xz515 in a salt lake of Tibet, China. This is a bacteriorhodopsin (bR) like protein, function as a light-driven proton pump. Experimental measurements verified that similar to bR, AR not only remains its biological activity in pmteoliposome, but also keeps a preferred orientation in self-assembly.

  6. Proton Pumps: Mechanism of Action and Applications

    Science.gov (United States)

    Lanyi, Janos K.; Pohorille, Andrew; DeVincenzi, Donald L. (Technical Monitor)

    2001-01-01

    Recent progress in understanding molecular structures and mechanisms of action of proton pumps has paved the way to their novel applications in biotechnology. Proton pumps, in particular bacteriorhodopsin and ATP synthases, are capable of continuous, renewable conversion of light to chemical, mechanical or electrical energy, which can be used in macro- or nano-scale devices. The capability of protein systems incorporated into liposomes to generate ATP, which can be further used to drive chemical reactions, and to act as molecular motors has been already demonstrated. Other possible applications of such biochemical devices include targeted drug delivery and biocatalytic re actors. All these devices might prove superior to their inorganic alternatives.

  7. Detergent-free incorporation of a seven-transmembrane receptor protein into nanosized bilayer Lipodisq particles for functional and biophysical studies.

    Science.gov (United States)

    Orwick-Rydmark, Marcella; Lovett, Janet E; Graziadei, Andrea; Lindholm, Ljubica; Hicks, Matthew R; Watts, Anthony

    2012-09-12

    SMA-Lipodisq nanoparticles, with one bacteriorhodopsin (bR) per 12 nm particle on average (protein/lipid molar ratio, 1:172), were prepared without the use of detergents. Using pulsed and continuous wave nitroxide spin label electron paramagnetic resonance, the structural and dynamic integrity of bR was retained when compared with data for bR obtained in the native membrane and in detergents and then with crystal data. This indicates the potential of Lipodisq nanoparticles as a useful membrane mimetic.

  8. Evidence that bilayer bending rigidity affects membrane protein folding.

    Science.gov (United States)

    Booth, P J; Riley, M L; Flitsch, S L; Templer, R H; Farooq, A; Curran, A R; Chadborn, N; Wright, P

    1997-01-07

    The regeneration kinetics of the integral membrane protein bacteriorhodopsin have been investigated in a lipid-based refolding system. Previous studies on bacteriorhodopsin regeneration have involved detergent-based systems, and in particular mixed dimyristoylphosphatidylcholine (DMPC)/CHAPS micelles. Here, we show that the short chain lipid dihexanoylphosphatidylcholine (DHPC) can be substituted for the detergent CHAPS and that bacteriorhodopsin can be regenerated to high yield in mixed DMPC/DHPC micelles. Bacteriorhodopsin refolding kinetics are measured in the mixed DMPC/DHPC micelles. Rapid, stopped flow mixing is employed to initiate refolding of denatured bacterioopsin in SDS micelles with mixed DMPC/DHPC micelles and time-resolved fluorescence spectroscopy to follow changes in protein fluorescence during folding. Essentially identical refolding kinetics are observed for mixed DMPC/CHAPS and mixed DMPC/DHPC micelles. Only one second-order retinal/apoprotein reaction is identified, in which retinal binds to a partially folded apoprotein intermediate, and the free energy of this retinal binding reaction is found to be the same in both types of mixed micelles. Formation of the partially folded apoprotein intermediate is a rate-limiting step in protein folding and appears to be biexponential. Both apparent rate constants are found to be dependent on the relative proportion of DMPC present in the mixed DMPC/DHPC micelles as well as on the pH of the aqueous phase. Increasing the DMPC concentration should increase the bending rigidity of the amphiphilic bilayer, and this is found to slow the rate of formation of the partially folded apoprotein intermediate. Increasing the mole fraction of DMPC from 0.3 to 0.6 slows the two apparent rate constants associated with formation of this intermediate from 0.29 and 0.031 to 0.11 and 0.013 s-1, respectively. Formation of the intermediate also slows with increasing pH, from 0.11 and 0.013 s-1 at pH 6 to 0.033 and 0.0053 s-1 at

  9. Determining the Absorbance Spectra of Photochromic Materials From Measured Spectrophotometer Data

    Science.gov (United States)

    Downie, John D.

    1998-01-01

    If a two-state photochromic material is optically bleached, the absorbance spectrum data measured by a spectrophotometer is in general comprised of components from both the ground state and the upper state. Under general conditions, it may be difficult to extract the actual upper state spectrum from the spectrum of the bleached material. A simple algorithm is presented here for the recovery of the pure absorbance spectra of the upper state of a material such as bacteriorhodopsin, given single wavelength bleaching illumination, steady-state conditions, and accurate knowledge of phototransition rates and thermal decay rates.

  10. A small basic protein from the brz-brb operon is involved in regulation of bop transcription in Halobacterium salinarum

    Directory of Open Access Journals (Sweden)

    Dyall-Smith Mike

    2011-09-01

    Full Text Available Abstract Background The halophilic archaeon Halobacterium salinarum expresses bacteriorhodopsin, a retinal-protein that allows photosynthetic growth. Transcription of the bop (bacterioopsin gene is controlled by two transcription factors, Bat and Brz that induce bop when cells are grown anaerobically and under light. Results A new gene was identified that is transcribed together with the brz gene that encodes a small basic protein designated as Brb (bacteriorhodopsin-regulating basic protein. The translation activity of the start codon of the brb gene was confirmed by BgaH reporter assays. In vivo site-directed mutagenesis of the brb gene showed that the Brb protein cooperates with Brz in the regulation of bop expression. Using a GFP reporter assay, it was demonstrated that Brb cooperates with both Brz and Bat proteins to activate bop transcription under phototrophic growth conditions. Conclusions The activation of the bop promoter was shown to be dependent not only on two major factors, Bat and Brz, but is also tuned by the small basic protein, Brb.

  11. Metabolism, Physiology and Biotechnological Applications of Halobacteria

    Directory of Open Access Journals (Sweden)

    Oleg Mosin

    2015-06-01

    Full Text Available Halophiles (lat. “salt-loving” is the taxonomic group of extreme aerobic obligate Gram-negative microorganisms that live in conditions of high salinity – in the seas, salt lakes, saline soils etc. These microorganisms are known to reddish patina on products, preserved with using large quantities of salt (NaCl. Halophiles were isolated for the first time at the beginning of the XX century from the marine flora estuary mud, but their systematic study was started only at the end of the second decade of the XX century. The internal environment of the human body is not suitable for existence of halobacteria, since none of them are known to have pathogenic forms. Halobacteria have great practical potential for using in molecular bioelectronics and bio-nanotechnology due to their unique ability to convert the energy of sunlight into electrochemical energy of protons H+ due to the presence in their cells a special photo transforming retinal containing integral protein – bacteriorhodopsin, the mechanism of action of which has been currently studied in detail. This article describes the characteristics of the metabolism and physiology of halophilic bacteria, as well as a method of biosynthesis and preparation of bacteriorhodopsin from purple membranes of cells of the extreme photoorganotrophic halobacterium Halobacterium halobium.

  12. Photocycle of Exiguobacterium sibiricum rhodopsin characterized by low-temperature trapping in the IR and time-resolved studies in the visible.

    Science.gov (United States)

    Dioumaev, Andrei K; Petrovskaya, Lada E; Wang, Jennifer M; Balashov, Sergei P; Dolgikh, Dmitriy A; Kirpichnikov, Mikhail P; Lanyi, Janos K

    2013-06-20

    The photocycle of the retinal protein from Exiguobacterium sibiricum, which differs from bacteriorhodopsin in both its primary donor and acceptor, is characterized by visible and infrared spectroscopy. At pH above pKa ~6.5, we find a bacteriorhodopsin-like photocycle, which originates from excitation of the all-trans retinal chromophore with K-, L-, M-, and N-like intermediates. At pH below pKa ~6.5, the M state, which reflects Schiff base deprotonation during proton pumping, is not accumulated. However, using the infrared band at ~1760 cm(-1) as a marker for transient protonation of the primary acceptor, we find that Schiff base deprotonation must have occurred at pH not only above but also below the pKa ~6.5. Thus, the M state is formed but not accumulated for kinetic reasons. Further, chromophore reisomerization from the 13-cis to the all-trans conformation occurs very late in the photocycle. The strongly red-shifted states that dominate the second half of the cycle are produced before the reisomerization step, and by this criterion, they are not O-like but rather N-like states. The assignment of photocycle intermediates enables reevaluation of the photocycle; its specific features are discussed in relation to the general mechanism of proton transport in retinal proteins.

  13. Specificity of anion-binding in the substrate-pocket ofbacteriorhodopsin

    Energy Technology Data Exchange (ETDEWEB)

    Facciotti, Marc T.; Cheung, Vincent S.; Lunde, Christopher S.; Rouhani, Shahab; Baliga, Nitin S.; Glaeser, Robert M.

    2003-08-30

    The structure of the D85S mutant of bacteriorhodopsin with a nitrate anion bound in the Schiff-base binding site, and the structure of the anion-free protein have been obtained in the same crystal form. Together with the previously solved structures of this anion pump, in both the anion-free state and bromide-bound state, these new structures provide insight into how this mutant of bacteriorhodopsin is able to bind a variety of different anions in the same binding pocket. The structural analysis reveals that the main structural change that accommodates different anions is the repositioning of the polar side-chain of S85. On the basis of these x-ray crystal structures, the prediction is then made that the D85S/D212N double mutant might bind similar anions and do so over a broader pH range than does the single mutant. Experimental comparison of the dissociation constants, K{sub d}, for a variety of anions confirms this prediction and demonstrates, in addition, that the binding affinity is dramatically improved by the D212N substitution.

  14. Protein-Based Three-Dimensional Memories and Associative Processors

    Science.gov (United States)

    Birge, Robert

    2008-03-01

    The field of bioelectronics has benefited from the fact that nature has often solved problems of a similar nature to those which must be solved to create molecular electronic or photonic devices that operate with efficiency and reliability. Retinal proteins show great promise in bioelectronic devices because they operate with high efficiency (˜0.65%), high cyclicity (>10^7), operate over an extended wavelength range (360 -- 630 nm) and can convert light into changes in voltage, pH, absorption or refractive index. This talk will focus on a retinal protein called bacteriorhodopsin, the proton pump of the organism Halobacterium salinarum. Two memories based on this protein will be described. The first is an optical three-dimensional memory. This memory stores information using volume elements (voxels), and provides as much as a thousand-fold improvement in effective capacity over current technology. A unique branching reaction of a variant of bacteriorhodopsin is used to turn each protein into an optically addressed latched AND gate. Although three working prototypes have been developed, a number of cost/performance and architectural issues must be resolved prior to commercialization. The major issue is that the native protein provides a very inefficient branching reaction. Genetic engineering has improved performance by nearly 500-fold, but a further order of magnitude improvement is needed. Protein-based holographic associative memories will also be discussed. The human brain stores and retrieves information via association, and human intelligence is intimately connected to the nature and enormous capacity of this associative search and retrieval process. To a first order approximation, creativity can be viewed as the association of two seemingly disparate concepts to form a totally new construct. Thus, artificial intelligence requires large scale associative memories. Current computer hardware does not provide an optimal environment for creating artificial

  15. Photoreceptors for a light biotransducer: a comparative study of the electrical responses of two (type-1)-opsins

    CERN Document Server

    Alfinito, E; Reggiani, L; Lee, K

    2013-01-01

    The increasing interest in photoactivated proteins as natural replacement of standard inorganic materials in photocells drives to the compared analysis of bacteriorhodopsin and proteorhodopsin, two widely diffused proteins belonging to the family of \\textit{type-1} opsins. These proteins share similar behaviours but exhibit relevant differences in the sequential chain of the amino acids constituting their tertiary structure. The use of an impedance network analogue to model the protein main features provides a microscopic interpretation of a set of experiments on their photoconductance properties. In particular, this model links the protein electrical responses to the tertiary structure and to the interactions among neighbouring amino acids. The same model is also used to predict the small-signal response in terms of the Nyquist plot. Interesting enough, these rhodopsins are found to behave like a wide gap semiconductor with intrinsic conductivities of the order of $10^{-7}$ S/cm.

  16. Photoenergy transduction in halobacterium halobium

    Science.gov (United States)

    Stoeckenius, W.; Bogomolni, R. A.

    1985-01-01

    The structure and function of a rhodopsin-like pigment bacteriorhodopsin, discovered and isolated from the membranes of the halophile Halobacterium halobium, were studied. Intermediatesm appearing in the cyclic photoreaction that drives the proton translocation, were spectroscopically characterized. The charge translocation in membrane monolayers and mulitlayers placed between electrodes were kinetically resolved. A model was developed for the proton translocation process, in which the isomerization of the retinal Schiff base decreases its pK to drive the proton off and simultaneaouly changes the connectivity from the cytoplasmic surface to the external surface. The stoichiometry of proton pumping in intact cells and the effect of the light generated electrochemical potential on the kinetics of the photoreaction cycle and the synthesis of ATP were investigated.

  17. MeshAndCollect: an automated multi-crystal data-collection workflow for synchrotron macromolecular crystallography beamlines

    Energy Technology Data Exchange (ETDEWEB)

    Zander, Ulrich [European Synchrotron Radiation Facility, CS 40220, 38043 Grenoble (France); Bourenkov, Gleb [European Molecular Biology Laboratory, Hamburg Outstation, Notkestrasse 85, 22607 Hamburg (Germany); Popov, Alexander N.; Sanctis, Daniele de; Svensson, Olof [European Synchrotron Radiation Facility, CS 40220, 38043 Grenoble (France); McCarthy, Andrew A. [European Molecular Biology Laboratory, Grenoble Outstation, 71 Avenue des Martyrs, CS 90181, 38042 Grenoble (France); Université Grenoble Alpes–EMBL–CNRS, 38042 Grenoble (France); Round, Ekaterina; Gordeliy, Valentin [Université Grenoble Alpes, IBS, 38044 Grenoble (France); CNRS, IBS, 38044 Grenoble (France); CEA, IBS, 38044 Grenoble (France); Institute of Complex Systems (ICS), Research Centre Juelich, 52425 Juelich (Germany); Moscow Institute of Physics and Technology, Dolgoprudniy 141700 (Russian Federation); Mueller-Dieckmann, Christoph; Leonard, Gordon A., E-mail: leonard@esrf.fr [European Synchrotron Radiation Facility, CS 40220, 38043 Grenoble (France)

    2015-10-31

    The fully automated collection and merging of partial data sets from a series of cryocooled crystals of biological macromolecules contained on the same support is presented, as are the results of test experiments carried out on various systems. Here, an automated procedure is described to identify the positions of many cryocooled crystals mounted on the same sample holder, to rapidly predict and rank their relative diffraction strengths and to collect partial X-ray diffraction data sets from as many of the crystals as desired. Subsequent hierarchical cluster analysis then allows the best combination of partial data sets, optimizing the quality of the final data set obtained. The results of applying the method developed to various systems and scenarios including the compilation of a complete data set from tiny crystals of the membrane protein bacteriorhodopsin and the collection of data sets for successful structure determination using the single-wavelength anomalous dispersion technique are also presented.

  18. MeshAndCollect: an automated multi-crystal data-collection workflow for synchrotron macromolecular crystallography beamlines.

    Science.gov (United States)

    Zander, Ulrich; Bourenkov, Gleb; Popov, Alexander N; de Sanctis, Daniele; Svensson, Olof; McCarthy, Andrew A; Round, Ekaterina; Gordeliy, Valentin; Mueller-Dieckmann, Christoph; Leonard, Gordon A

    2015-11-01

    Here, an automated procedure is described to identify the positions of many cryocooled crystals mounted on the same sample holder, to rapidly predict and rank their relative diffraction strengths and to collect partial X-ray diffraction data sets from as many of the crystals as desired. Subsequent hierarchical cluster analysis then allows the best combination of partial data sets, optimizing the quality of the final data set obtained. The results of applying the method developed to various systems and scenarios including the compilation of a complete data set from tiny crystals of the membrane protein bacteriorhodopsin and the collection of data sets for successful structure determination using the single-wavelength anomalous dispersion technique are also presented.

  19. Introduction to electron crystallography.

    Science.gov (United States)

    Kühlbrandt, Werner

    2013-01-01

    From the earliest work on regular arrays in negative stain, electron crystallography has contributed greatly to our understanding of the structure and function of biological macromolecules. The development of electron cryo-microscopy (cryo-EM) then lead to the first groundbreaking atomic models of the membrane proteins bacteriorhodopsin and light harvesting complex II within lipid bilayers. Key contributions towards cryo-EM and electron crystallography methods included specimen preparation and vitrification, liquid-helium cooling, data collection, and image processing. These methods are now applied almost routinely to both membrane and soluble proteins. Here we outline the advances and the breakthroughs that paved the way towards high-resolution structures by electron crystallography, both in terms of methods development and biological milestones.

  20. Electron crystallography and aquaporins.

    Science.gov (United States)

    Schenk, Andreas D; Hite, Richard K; Engel, Andreas; Fujiyoshi, Yoshinori; Walz, Thomas

    2010-01-01

    Electron crystallography of two-dimensional (2D) crystals can provide information on the structure of membrane proteins at near-atomic resolution. Originally developed and used to determine the structure of bacteriorhodopsin (bR), electron crystallography has recently been applied to elucidate the structure of aquaporins (AQPs), a family of membrane proteins that form pores mostly for water but also other solutes. While electron crystallography has made major contributions to our understanding of the structure and function of AQPs, structural studies on AQPs, in turn, have fostered a number of technical developments in electron crystallography. In this contribution, we summarize the insights electron crystallography has provided into the biology of AQPs, and describe technical advancements in electron crystallography that were driven by structural studies on AQP 2D crystals. In addition, we discuss some of the lessons that were learned from electron crystallographic work on AQPs.

  1. Probing how initial retinal configuration controls photochemical dynamics in retinal proteins

    Directory of Open Access Journals (Sweden)

    Sheves M.

    2013-03-01

    Full Text Available The effects of the initial retinal configuration and the active isomerization coordinate on the photochemistry of retinal proteins (RPs are assessed by comparing photochemical dynamics of two stable retinal ground state configurations (all-trans,15-anti vs. 13-cis,15-syn, within two RPs: Bacteriorhodopsin (BR and Anabaena Sensory Rhodopsin (ASR. Hyperspectral pump-probe spectroscopy shows that photochemistry starting from 13-cis retinal in both proteins is 3-10 times faster than when started in the all-trans state, suggesting that the hastening is ubiquitous to microbial RPs, regardless of their different biological functions and origin. This may also relate to the known disparity of photochemical rates between microbial RPs and visual pigments. Importance and possible underlying mechanisms are discussed as well.

  2. Lipidic cubic phase serial millisecond crystallography using synchrotron radiation

    Directory of Open Access Journals (Sweden)

    Przemyslaw Nogly

    2015-03-01

    Full Text Available Lipidic cubic phases (LCPs have emerged as successful matrixes for the crystallization of membrane proteins. Moreover, the viscous LCP also provides a highly effective delivery medium for serial femtosecond crystallography (SFX at X-ray free-electron lasers (XFELs. Here, the adaptation of this technology to perform serial millisecond crystallography (SMX at more widely available synchrotron microfocus beamlines is described. Compared with conventional microcrystallography, LCP-SMX eliminates the need for difficult handling of individual crystals and allows for data collection at room temperature. The technology is demonstrated by solving a structure of the light-driven proton-pump bacteriorhodopsin (bR at a resolution of 2.4 Å. The room-temperature structure of bR is very similar to previous cryogenic structures but shows small yet distinct differences in the retinal ligand and proton-transfer pathway.

  3. Electron transport through supported biomembranes at the nanoscale by conductive atomic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Casuso, I [Department Electronica, Universitat de Barcelona and Laboratori de Nanobioenginyeria-IBEC, Parc CientIfic de Barcelona, Barcelona (Spain); Fumagalli, L [Department Electronica, Universitat de Barcelona and Laboratori de Nanobioenginyeria-IBEC, Parc CientIfic de Barcelona, Barcelona (Spain); Samitier, J [Department Electronica, Universitat de Barcelona and Laboratori de Nanobioenginyeria-IBEC, Parc CientIfic de Barcelona, Barcelona (Spain); Padros, E [Unitat de BiofIsica, Departamento de BioquImica i de Biologia Molecular, Facultat de Medicina i Centre d' Estudis en BiofIsica, Universitat Autonoma de Barcelona, Barcelona (Spain); Reggiani, L [CNR-INFM National Nanotechnology Laboratory, Dipartimento di Ingegneria dell' Innovazione, Universita di Lecce, Lecce (Italy); Akimov, V [CNR-INFM National Nanotechnology Laboratory, Dipartimento di Ingegneria dell' Innovazione, Universita di Lecce, Lecce (Italy); Gomila, G [Department Electronica, Universitat de Barcelona and Laboratori de Nanobioenginyeria-IBEC, Parc CientIfic de Barcelona, Barcelona (Spain)

    2007-11-21

    We present a reliable methodology to perform electron transport measurements at the nanoscale on supported biomembranes by conductive atomic force microscopy (C-AFM). It allows measurement of both (a) non-destructive conductive maps and (b) force controlled current-voltage characteristics in wide voltage bias range in a reproducible way. Tests experiments were performed on purple membrane monolayers, a two-dimensional (2D) crystal lattice of the transmembrane protein bacteriorhodopsin. Non-destructive conductive images show uniform conductivity of the membrane with isolated nanometric conduction defects. Current-voltage characteristics under different compression conditions show non-resonant tunneling electron transport properties, with two different conduction regimes as a function of the applied bias, in excellent agreement with theoretical predictions. This methodology opens the possibility for a detailed study of electron transport properties of supported biological membranes, and of soft materials in general.

  4. Improved methods for high resolution electron microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Taylor, J.R.

    1987-04-01

    Existing methods of making support films for high resolution transmission electron microscopy are investigated and novel methods are developed. Existing methods of fabricating fenestrated, metal reinforced specimen supports (microgrids) are evaluated for their potential to reduce beam induced movement of monolamellar crystals of C/sub 44/H/sub 90/ paraffin supported on thin carbon films. Improved methods of producing hydrophobic carbon films by vacuum evaporation, and improved methods of depositing well ordered monolamellar paraffin crystals on carbon films are developed. A novel technique for vacuum evaporation of metals is described which is used to reinforce microgrids. A technique is also developed to bond thin carbon films to microgrids with a polymer bonding agent. Unique biochemical methods are described to accomplish site specific covalent modification of membrane proteins. Protocols are given which covalently convert the carboxy terminus of papain cleaved bacteriorhodopsin to a free thiol. 53 refs., 19 figs., 1 tab.

  5. A Photoisomerizing Rhodopsin Mimic Observed at Atomic Resolution.

    Science.gov (United States)

    Nosrati, Meisam; Berbasova, Tetyana; Vasileiou, Chrysoula; Borhan, Babak; Geiger, James H

    2016-07-20

    The members of the rhodopsin family of proteins are involved in many essential light-dependent processes in biology. Specific photoisomerization of the protein-bound retinylidene PSB at a specified wavelength range of light is at the heart of all of these systems. Nonetheless, it has been difficult to reproduce in an engineered system. We have developed rhodopsin mimics, using intracellular lipid binding protein family members as scaffolds, to study fundamental aspects of protein/chromophore interactions. Herein we describe a system that specifically isomerizes the retinylidene protonated Schiff base both thermally and photochemically. This isomerization has been characterized at atomic resolution by quantitatively interconverting the isomers in the crystal both thermally and photochemically. This event is accompanied by a large pKa change of the imine similar to the pKa changes observed in bacteriorhodopsin and visual opsins during isomerization.

  6. Model Construction and Analysis of Respiration in Halobacterium salinarum.

    Directory of Open Access Journals (Sweden)

    Cherryl O Talaue

    Full Text Available The archaeon Halobacterium salinarum can produce energy using three different processes, namely photosynthesis, oxidative phosphorylation and fermentation of arginine, and is thus a model organism in bioenergetics. Compared to its bacteriorhodopsin-driven photosynthesis, less attention has been devoted to modeling its respiratory pathway. We created a system of ordinary differential equations that models its oxidative phosphorylation. The model consists of the electron transport chain, the ATP synthase, the potassium uniport and the sodium-proton antiport. By fitting the model parameters to experimental data, we show that the model can explain data on proton motive force generation, ATP production, and the charge balancing of ions between the sodium-proton antiporter and the potassium uniport. We performed sensitivity analysis of the model parameters to determine how the model will respond to perturbations in parameter values. The model and the parameters we derived provide a resource that can be used for analytical studies of the bioenergetics of H. salinarum.

  7. Towards local electromechanical probing of cellular and biomolecular systems in a liquid environment

    Energy Technology Data Exchange (ETDEWEB)

    Kalinin, Sergei V [Materials Sciences and Technology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37931 (United States); Rodriguez, Brian J [Materials Sciences and Technology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37931 (United States); Jesse, Stephen [Materials Sciences and Technology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37931 (United States); Seal, Katyayani [Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, Oak Ridge, TN 37931 (United States); Proksch, Roger [Asylum Research, Santa Barbara, CA 93117 (United States); Hohlbauch, Sophia [Asylum Research, Santa Barbara, CA 93117 (United States); Revenko, Irene [Asylum Research, Santa Barbara, CA 93117 (United States); Thompson, Gary Lee [Department of Bioengineering, Clemson University, Clemson, SC 29634 (United States); Vertegel, Alexey A [Department of Bioengineering, Clemson University, Clemson, SC 29634 (United States)

    2007-10-24

    Electromechanical coupling is ubiquitous in biological systems, with examples ranging from simple piezoelectricity in calcified and connective tissues to voltage-gated ion channels, energy storage in mitochondria, and electromechanical activity in cardiac myocytes and outer hair cell stereocilia. Piezoresponse force microscopy (PFM) originally emerged as a technique to study electromechanical phenomena in ferroelectric materials, and in recent years has been employed to study a broad range of non-ferroelectric polar materials, including piezoelectric biomaterials. At the same time, the technique has been extended from ambient to liquid imaging on model ferroelectric systems. Here, we present results on local electromechanical probing of several model cellular and biomolecular systems, including insulin and lysozyme amyloid fibrils, breast adenocarcinoma cells, and bacteriorhodopsin in a liquid environment. The specific features of PFM operation in liquid are delineated and bottlenecks on the route towards nanometre-resolution electromechanical imaging of biological systems are identified.

  8. Controlled in meso phase crystallization--a method for the structural investigation of membrane proteins.

    Directory of Open Access Journals (Sweden)

    Jan Kubicek

    Full Text Available We investigated in meso crystallization of membrane proteins to develop a fast screening technology which combines features of the well established classical vapor diffusion experiment with the batch meso phase crystallization, but without premixing of protein and monoolein. It inherits the advantages of both methods, namely (i the stabilization of membrane proteins in the meso phase, (ii the control of hydration level and additive concentration by vapor diffusion. The new technology (iii significantly simplifies in meso crystallization experiments and allows the use of standard liquid handling robots suitable for 96 well formats. CIMP crystallization furthermore allows (iv direct monitoring of phase transformation and crystallization events. Bacteriorhodopsin (BR crystals of high quality and diffraction up to 1.3 Å resolution have been obtained in this approach. CIMP and the developed consumables and protocols have been successfully applied to obtain crystals of sensory rhodopsin II (SRII from Halobacterium salinarum for the first time.

  9. In-Situ Observation of Membrane Protein Folding during Cell-Free Expression.

    Directory of Open Access Journals (Sweden)

    Axel Baumann

    Full Text Available Proper insertion, folding and assembly of functional proteins in biological membranes are key processes to warrant activity of a living cell. Here, we present a novel approach to trace folding and insertion of a nascent membrane protein leaving the ribosome and penetrating the bilayer. Surface Enhanced IR Absorption Spectroscopy selectively monitored insertion and folding of membrane proteins during cell-free expression in a label-free and non-invasive manner. Protein synthesis was performed in an optical cell containing a prism covered with a thin gold film with nanodiscs on top, providing an artificial lipid bilayer for folding. In a pilot experiment, the folding pathway of bacteriorhodopsin via various secondary and tertiary structures was visualized. Thus, a methodology is established with which the folding reaction of other more complex membrane proteins can be observed during protein biosynthesis (in situ and in operando at molecular resolution.

  10. Modelling small-angle scattering data from complex protein-lipid systems

    DEFF Research Database (Denmark)

    Kynde, Søren Andreas Røssell

    geometric objects and the discrete approach were models are build from a large number of points. It is the basic hypothesis of this thesis, that analysis of smallangle scattering data can be approached in a way that combines the continuous and the discrete modelling methods, and that such an approach can...... the techniques very well suited for the study of the nanodisc system. Chapter 3 explains two different modelling approaches that can be used in the analysis of small-angle scattering data from lipid-protein complexes. These are the continuous approach where the system of interest is modelled as a few regular...... of bacteriorhodopsin and a continuous model of the nanodisc. The position and orientation of the membrane protein relative to the nanodisc is determined as well as the structural changes of the nanodisc. Paper II describes the use of the same approach to determine the relative position of a nanodisc and the membrane...

  11. Fabrication of biomolecule copolymer hybrid nanovesicles as energy conversion systems

    Science.gov (United States)

    Ho, Dean; Chu, Benjamin; Lee, Hyeseung; Brooks, Evan K.; Kuo, Karen; Montemagno, Carlo D.

    2005-12-01

    This work demonstrates the integration of the energy-transducing proteins bacteriorhodopsin (BR) from Halobacterium halobium and cytochrome c oxidase (COX) from Rhodobacter sphaeroides into block copolymeric vesicles towards the demonstration of coupled protein functionality. An ABA triblock copolymer-based biomimetic membrane possessing UV-curable acrylate endgroups was synthesized to serve as a robust matrix for protein reconstitution. BR-functionalized polymers were shown to generate light-driven transmembrane pH gradients while pH gradient-induced electron release was observed from COX-functionalized polymers. Cooperative behaviour observed from composite membrane functionalized by both proteins revealed the generation of microamp-range currents with no applied voltage. As such, it has been shown that the fruition of technologies based upon bio-functionalizing abiotic materials may contribute to the realization of high power density devices inspired by nature.

  12. Mechanisms of photoisomerization of polyenes in confined media: from organic glasses to protein binding cavities.

    Science.gov (United States)

    Liu, Robert S H; Yang, Lan-Ying; Liu, Jin

    2007-01-01

    Photochemical reactivities of model organic systems (stilbene and diphenylbutadiene) in organic glasses were first examined and compared with those in solution and in organized media. These observations were in turn compared with reactivities of polyene chromophores in protein binding cavities (specifically PYP, rhodopsin and bacteriorhodopsin). The obvious conclusion is that the preference for the most volume-conserving Hula-twist mechanism isomerization in organic glasses is because of the close interaction between the guest and the host molecules. In organized media (zeolites, crystals and protein binding cavities), the residual empty space coupled with any specific guest-host interactions that are characteristic of a given system, could lead to involvement of the more volume-demanding one-bond-flip (i.e. torsional relaxation) or bicycle-pedal or an extended HT process in photoisomerization.

  13. H+-type and OH- -type biological protonic semiconductors and complementary devices.

    Science.gov (United States)

    Deng, Yingxin; Josberger, Erik; Jin, Jungho; Roudsari, Anita Fadavi; Rousdari, Anita Fadavi; Helms, Brett A; Zhong, Chao; Anantram, M P; Rolandi, Marco

    2013-10-03

    Proton conduction is essential in biological systems. Oxidative phosphorylation in mitochondria, proton pumping in bacteriorhodopsin, and uncoupling membrane potentials by the antibiotic Gramicidin are examples. In these systems, H(+) hop along chains of hydrogen bonds between water molecules and hydrophilic residues - proton wires. These wires also support the transport of OH(-) as proton holes. Discriminating between H(+) and OH(-) transport has been elusive. Here, H(+) and OH(-) transport is achieved in polysaccharide- based proton wires and devices. A H(+)- OH(-) junction with rectifying behaviour and H(+)-type and OH(-)-type complementary field effect transistors are demonstrated. We describe these devices with a model that relates H(+) and OH(-) to electron and hole transport in semiconductors. In turn, the model developed for these devices may provide additional insights into proton conduction in biological systems.

  14. Steady state emission of the fluorescent intermediate of Anabaena Sensory Rhodopsin as a function of light adaptation conditions

    Science.gov (United States)

    Cheminal, A.; Léonard, J.; Kim, S. Y.; Jung, K.-H.; Kandori, H.; Haacke, S.

    2013-11-01

    Steady-state fluorescence measurements of the first excited state of the anabaena sensory rhodopsin (ASR), and Bacteriorhodopsin are reported for different light stabilization conditions, including the dark-adapted state. We determine the fluorescence spectra of both all-trans (AT), and 13-cis (13C) protonated Schiff base of retinal, and compare the effect of the proteins. Referenced against the fluorescence quantum yield of AT-bR (2.5 × 10-4) we find for AT-ASR, 13C-ASR, and 13C-bR the values of 3.3 × 10-4, 0.8 × 10-4, and 1.7 × 10-4, respectively. Using reported excited state lifetimes, the radiative rates are deduced, and their differences discussed on the basis of a configuration-dependent oscillator strength.

  15. Photoinduced electron transfer pathways in hydrogen-evolving reduced graphene oxide-boosted hybrid nano-bio catalyst.

    Science.gov (United States)

    Wang, Peng; Dimitrijevic, Nada M; Chang, Angela Y; Schaller, Richard D; Liu, Yuzi; Rajh, Tijana; Rozhkova, Elena A

    2014-08-26

    Photocatalytic production of clean hydrogen fuels using water and sunlight has attracted remarkable attention due to the increasing global energy demand. Natural and synthetic dyes can be utilized to sensitize semiconductors for solar energy transformation using visible light. In this study, reduced graphene oxide (rGO) and a membrane protein bacteriorhodopsin (bR) were employed as building modules to harness visible light by a Pt/TiO2 nanocatalyst. Introduction of the rGO boosts the nano-bio catalyst performance that results in hydrogen production rates of approximately 11.24 mmol of H2 (μmol protein)(-1) h(-1). Photoelectrochemical measurements show a 9-fold increase in photocurrent density when TiO2 electrodes were modified with rGO and bR. Electron paramagnetic resonance and transient absorption spectroscopy demonstrate an interfacial charge transfer from the photoexcited rGO to the semiconductor under visible light.

  16. Protons and how they are transported by proton pumps

    DEFF Research Database (Denmark)

    Buch-Pedersen, Morten Jeppe; Pedersen, Bjørn Panyella; Nissen, Poul

    2008-01-01

    The very high mobility of protons in aqueous solutions demands special features of membrane proton transporters to sustain efficient yet regulated proton transport across biological membranes. By the use of the chemical energy of ATP, plasma-membrane-embedded ATPases extrude protons from cells...... of plants and fungi to generate electrochemical proton gradients. The recently published crystal structure of a plasma membrane H(+)-ATPase contributes to our knowledge about the mechanism of these essential enzymes. Taking the biochemical and structural data together, we are now able to describe the basic...... molecular components that allow the plasma membrane proton H(+)-ATPase to carry out proton transport against large membrane potentials. When divergent proton pumps such as the plasma membrane H(+)-ATPase, bacteriorhodopsin, and F(O)F(1) ATP synthase are compared, unifying mechanistic premises for biological...

  17. Genetic analysis of the br gene in halophilic archaea isolated from Xinjiang region, China

    Institute of Scientific and Technical Information of China (English)

    Xiaohong XU; Min WU; Huibin ZHANG; Zhihu LIU

    2008-01-01

    Some novel members of extremely halophilic archaea, strains AJ 11, AJ 12 and AJ 13, were isolated from the Aularz Lake located in the Altun Mountain National Nature Reserve of Xinjiang, Uygur Autonomous Region in China. Partial DNA fragments encoding a bacteriorho-dopsin (BR), as well as for 16S rRNA of isolated strains, were amplified by PCR and their DNA sequences were determined subsequently. On the basis of homology and phylogenetic analysis of the 16S rDNA, we thought that the isolated strains forming a microbiological population are the members of the genus Natrinema. The results of genetic analysis, such as GC content, transition/transver-sion (Ti/Tv) rate ratios and synonymous substitution rates (Ks) indicate that the br fragments, with a high level of genetic divergence, are faced with both purifying selection and bias mutation pressure. The study provides the basis for use of species and BR proteins resources.

  18. Reconstitution of halorhodopsin

    Energy Technology Data Exchange (ETDEWEB)

    Kong, T.

    1989-11-01

    Halobacterium halobium contains a family of retinal-bound proteins: bacteriorhodopsin (bR) which mediates phototrophic growth as a light-riven proton pump, halorhodopsin (hR) which is a light-driven chloride pump, and one or more sensory rhodopsins (sR) which mediate a phototactic response. Two-dimensional crystallization of halorhodopsin has been attempted though the reconstitution of purified halorhodopsin with purple membrane lipid for electron microscopy work. The first important step for crystallization is to get a homogeneous protein which is pure and not denatured. Homogeneous halorhodopsin has been obtained by a modification of existing purification methods. Some nice looking membrane patches which have the same density as purple membrane have been obtained. But unfortunately, they are not crystalline. The procedure of hR reconstitution is described in detail and some other strategies to induce the protein crystal in the reconstituted membrane are discussed in this dissertation. 76 refs., 20 figs., 6 tabs.

  19. Halorhodopsin and photosensory behaviour in Halobacterium halobium mutant strain L-33

    Energy Technology Data Exchange (ETDEWEB)

    Traulich, B.; Wagner, G. (Botanisches Institut l der Justus-Liebig-Universitat, Giessen (Germany, F.R.)); Hildebrand, E.; Schimz, A. (Kernforschungsanlage Juelich G.m.b.H. (Germany, F.R.). Inst. fuer Neurobiologie); Lanyi, J.K. (California Univ., Irvine (USA))

    1983-05-01

    Halobacterium halobium, strain L-33, which is deficient in bacteriorhodopsin (BR) but synthesizes increased amounts of halorhodopsin (HR), responds to changes in fluence rate with visible light or with UV light. The observations support an earlier report that BR is not essential for photosensing in H. halobium. In the UV-range, changes in light intensity elicit the maximal response at lambda = 370 nm. In the visible range, changes in light intensity show the maximal response at lambda = 565 nm and a secondary peak at lambda = 590 nm. The latter corresponds to the absorption maximum of HR (lambdasub(max) = 588 nm). This light-energy converting retinal pigment of H. halobium thus appears to contribute to photosensory behavior.

  20. A network model to investigate structural and electrical properties of proteins

    CERN Document Server

    Alfinito, E; Reggiani, L

    2007-01-01

    One of the main trend in to date research and development is the miniaturization of electronic devices. In this perspective, integrated nanodevices based on proteins or biomolecules are attracting a major interest. In fact, it has been shown that proteins like bacteriorhodopsin and azurin, manifest electrical properties which are promising for the development of active components in the field of molecular electronics. Here we focus on two relevant kinds of proteins: The bovine rhodopsin, prototype of GPCR protein, and the enzyme acetylcholinesterase (AChE), whose inhibition is one of the most qualified treatments of Alzheimer disease. Both these proteins exert their functioning starting with a conformational change of their native structure. Our guess is that such a change should be accompanied with a detectable variation of their electrical properties. To investigate this conjecture, we present an impedance network model of proteins, able to estimate the different electrical response associated with the diff...

  1. Dynamic Nuclear Polarization enhanced NMR at 187 GHz/284 MHz using an Extended Interaction Klystron amplifier

    Science.gov (United States)

    Kemp, Thomas F.; Dannatt, Hugh R. W.; Barrow, Nathan S.; Watts, Anthony; Brown, Steven P.; Newton, Mark E.; Dupree, Ray

    2016-04-01

    A Dynamic Nuclear Polarisation (DNP) enhanced solid-state Magic Angle Spinning (MAS) NMR spectrometer which uses a 187 GHz (corresponding to 1H NMR frequency of 284 MHz) Extended Interaction Klystron (EIK) amplifier as the microwave source is briefly described. Its performance is demonstrated for a biomolecule (bacteriorhodopsin), a pharmaceutical, and surface functionalised silica. The EIK is very compact and easily incorporated into an existing spectrometer. The bandwidth of the amplifier is sufficient that it obviates the need for a sweepable magnetic field, once set, for all commonly used radicals. The variable power (CW or pulsed) output from the EIK is transmitted to the DNP-NMR probe using a quasi-optic system with a high power isolator and a corrugated waveguide which feeds the microwaves into the DNP-NMR probe. Curved mirrors inside the probe project the microwaves down the axis of the MAS rotor, giving a very efficient system such that maximum DNP enhancement is achieved with less than 3 W output from the microwave source. The DNP-NMR probe operates with a sample temperature down to 90 K whilst spinning at 8 kHz. Significant enhancements, in excess of 100 for bacteriorhodopsin in purple membrane (bR in PM), are shown along with spectra which are enhanced by ≈25 with respect to room temperature, for both the pharmaceutical furosemide and surface functionalised silica. These enhancements allow hitherto prohibitively time consuming experiments to be undertaken. The power at which the DNP enhancement in bR in PM saturates does not change significantly between 90 K and 170 K even though the enhancement drops by a factor of ≈11. As the DNP build up time decreases by a factor 3 over this temperature range, the reduction in T1n is presumably a significant contribution to the drop in enhancement.

  2. New ultrarapid-scanning interferometer for FT-IR spectroscopy with microsecond time-resolution

    Science.gov (United States)

    Süss, B.; Ringleb, F.; Heberle, J.

    2016-06-01

    A novel Fourier-transform infrared (FT-IR) rapid-scan spectrometer has been developed (patent pending EP14194520.4) which yields 1000 times higher time resolution as compared to conventional rapid-scanning spectrometers. The central element to achieve faster scanning rates is based on a sonotrode whose front face represents the movable mirror of the interferometer. A prototype spectrometer with a time resolution of 13 μs was realized, capable of fully automated long-term measurements with a flow cell for liquid samples, here a photosynthetic membrane protein in solution. The performance of this novel spectrometer is demonstrated by recording the photoreaction of bacteriorhodopsin initiated by a short laser pulse that is synchronized to the data recording. The resulting data are critically compared to those obtained by step-scan spectroscopy and demonstrate the relevance of performing experiments on proteins in solution. The spectrometer allows for future investigations of fast, non-repetitive processes, whose investigation is challenging to step-scan FT-IR spectroscopy.

  3. Amphiphilic biopolymers (amphibiopols) as new surfactants for membrane protein solubilization

    Science.gov (United States)

    Duval-Terrié, Caroline; Cosette, Pascal; Molle, Gérard; Muller, Guy; Dé, Emmanuelle

    2003-01-01

    The aim of this study was to develop new surfactants for membrane protein solubilization, from a natural, biodegradable polymer: the polysaccharide pullulan. A set of amphiphilic pullulans (HMCMPs), differing in hydrophobic modification ratio, charge ratio, and the nature of the hydrophobic chains introduced, were synthesized and tested in solubilization experiments with outer membranes of Pseudomonas fluorescens. The membrane proteins were precipitated, and then resolubilized with various HMCMPs. The decyl alkyl chain (C10) was the hydrophobic graft that gave the highest level of solubilization. Decyl alkyl chain-bearing HMCMPs were also able to extract integral membrane proteins from their lipid environment. The best results were obtained with an amphiphilic pullulan bearing 18% decyl groups (18C10). Circular dichroism spectroscopy and membrane reconstitution experiments were used to test the structural and functional integrity of 18C10-solubilized proteins (OmpF from Escherichia coli and bacteriorhodopsin from Halobacterium halobium). Whatever their structure type (α or β), 18C10 did not alter either the structure or the function of the proteins analyzed. Thus, HMCMPs appear to constitute a promising new class of polymeric surfactants for membrane protein studies. PMID:12649425

  4. Study on the advanced orientation control technology of biopolymers; Seitai kobunshi zairyo no kodo haiko hairetsu seigyo gijutsu no kenkyu

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1996-03-01

    Creation of new functional organic materials for the medical application has been investigated under the microgravity. Facilities of the Japan Microgravity Center were used for this study. For the high-speed synthesis of uniform polymer particles under the microgravity condition for ten seconds, appropriately good results were obtained in the oxidation polymerization of pyrroles. For the synthesis of organic conductive thin films by the electrolytic polymerization, the resistance of electrolyte became larger in the microgravity field. It was required to set conditions so as to enhance the effects of microgravity environment. For the orientation control and thin film formation of proteins, the bacteriorhodopsin was examined. It was found that the microgravity improved the quality of electrocoatings. When the surface tension and viscosity of coating liquid were appropriately controlled under the microgravity, thin films were able to be prepared by utilizing a change from 1g to {mu}g. When the high viscosity fluid is placed in the artificial two-dimensional space composing of two parallel plates, and the low viscosity fluid, such as air, is injected into the above, the interface grows in the finger shaped pattern, namely, viscous fingering. The influence of gravity on this phenomenon was also investigated. 11 refs., 45 figs., 5 tabs.

  5. Archaebacterial rhodopsin sequences: Implications for evolution

    Science.gov (United States)

    Lanyi, J. K.

    1991-01-01

    It was proposed over 10 years ago that the archaebacteria represent a separate kingdom which diverged very early from the eubacteria and eukaryotes. It follows that investigations of archaebacterial characteristics might reveal features of early evolution. So far, two genes, one for bacteriorhodopsin and another for halorhodopsin, both from Halobacterium halobium, have been sequenced. We cloned and sequenced the gene coding for the polypeptide of another one of these rhodopsins, a halorhodopsin in Natronobacterium pharaonis. Peptide sequencing of cyanogen bromide fragments, and immuno-reactions of the protein and synthetic peptides derived from the C-terminal gene sequence, confirmed that the open reading frame was the structural gene for the pharaonis halorhodopsin polypeptide. The flanking DNA sequences of this gene, as well as those of other bacterial rhodopsins, were compared to previously proposed archaebacterial consensus sequences. In pairwise comparisons of the open reading frame with DNA sequences for bacterio-opsin and halo-opsin from Halobacterium halobium, silent divergences were calculated. These indicate very considerable evolutionary distance between each pair of genes, even in the dame organism. In spite of this, three protein sequences show extensive similarities, indicating strong selective pressures.

  6. Dynamic force microscopy imaging of native membranes

    Energy Technology Data Exchange (ETDEWEB)

    Kienberger, Ferry; Stroh, Cordula; Kada, Gerald; Moser, Rosita; Baumgartner, Werner; Pastushenko, Vassili; Rankl, Christian; Schmidt, Ute; Mueller, Harald; Orlova, Elena; LeGrimellec, Christian; Drenckhahn, Detlev; Blaas, Dieter; Hinterdorfer, Peter

    2003-10-15

    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.

  7. Comparison of prokaryotic community structure from Mediterranean and Atlantic saltern concentrator ponds by a metagenomic approach

    Directory of Open Access Journals (Sweden)

    Ana Beatriz eFernández

    2014-05-01

    Full Text Available We analyzed the prokaryotic community structure of a saltern pond with 21 % total salts located in Isla Cristina, Huelva, Southwest Spain, close to the Atlantic ocean coast. For this purpose, we constructed a metagenome (designated as IC21 obtained by pyrosequencing consisting of 486 Mb with an average read length of 397 bp and compared it with other metagenomic datasets obtained from ponds with 19, 33 and 37 % total salts acquired from Santa Pola marine saltern, located in Alicante, East Spain, on the Mediterranean coast. Although the salinity in IC21 is closer to the pond with 19 % total salts from Santa Pola saltern (designated as SS19, IC21 is more similar at higher taxonomic levels to the pond with 33 % total salts from Santa Pola saltern (designated as SS33, since both are predominated by the phylum Euryarchaeota. However, there are significant differences at lower taxonomic levels where most sequences were related to the genus Halorubrum in IC21 and to Haloquadratum in SS33. Within the Bacteroidetes, the genus Psychroflexus is the most abundant in IC21 while Salinibacter dominates in SS33. Sequences related to bacteriorhodopsins and halorhodopsins correlate with the abundance of Haloquadratum in Santa Pola SS19 to SS33 and of Halorubrum in Isla Cristina IC21 dataset, respectively. Differences in composition might be attributed to local ecological conditions since IC21 showed a decrease in the number of sequences related to the synthesis of compatible solutes and in the utilization of phosphonate.

  8. Super-Sensitive and Robust Biosensors from Supported Polymer Bilayers

    Energy Technology Data Exchange (ETDEWEB)

    Paxton, Walter F. [Sandia National Laboratories (SNL-NM), Albuquerque, NM (United States)

    2015-09-01

    Biological organisms are potentially the most sensitive and selective biological detection systems known, yet we are currently severely limited in our ability to exploit biological interactions in sensory devices, due in part to the limited stability of biological systems and derived materials. This proposal addresses an important aspect of integrating biological sensory materials in a solid state device. If successful, such technology could enable entirely new classes of robust biosensors that could be miniaturized and deployed in the field. The critical aims of the proposed work were 1) the calibration of a more versatile approach to measuring pH, 2) the use of this method to monitor pH changes caused by the light-induced pumping of protons across vesicles with bacteriorhodopsin integrated into the membranes (either polymer or lipid); 3) the preparation of bilayer assemblies on platinum surfaces; 4) the enhanced detection of lightinduced pH changes driven by bR-loaded supported bilayers. I have developed a methodology that may enable that at interfaces and developed a methodology to characterize the functionality of bilayer membranes with reconstituted membrane proteins. The integrity of the supported bilayer films however must be optimized prior to the full realization of the work originally envisioned in the original proposal. Nevertheless, the work performed on this project and the encouraging results it has produced has demonstrated that these goals are challenging yet within reach.

  9. Crystal Structure of Phosphatidylglycerophosphatase (PGPase), a Putative Membrane-Bound Lipid Phosphatase, Reveals a Novel Binuclear Metal Binding Site and Two Proton Wires

    Energy Technology Data Exchange (ETDEWEB)

    Kumaran,D.; Bonnano, J.; Burley, S.; Swaminathan, S.

    2006-01-01

    Phosphatidylglycerophosphatase (PGPase), an enzyme involved in lipid metabolism, catalyzes formation of phosphatidylglycerol from phosphatidylglycerophosphate. Phosphatidylglycerol is a multifunctional phospholipid, found in the biological membranes of many organisms. Here, we report the crystal structure of Listeria monocytogenes PGPase at 1.8 Angstroms resolution. PGPase, an all-helical molecule, forms a homotetramer. Each protomer contains an independent active site with two metal ions, Ca{sup 2+} and Mg{sup 2+}, forming a hetero-binuclear center located in a hydrophilic cavity near the surface of the molecule. The binuclear center, conserved ligands, metal-bound water molecules, and an Asp-His dyad form the active site. The catalytic mechanism of this enzyme is likely to proceed via binuclear metal activated nucleophilic water. The binuclear metal-binding active-site environment of this structure should provide insights into substrate binding and metal-dependent catalysis. A long channel with inter-linked linear water chains, termed 'proton wires', is observed at the tetramer interface. Comparison of similar water chain structures in photosynthetic reaction centers (RCs), Cytochrome f, gramicidin, and bacteriorhodopsin, suggests that PGPase may conduct protons via proton wires.

  10. Low-dose X-ray radiation induces structural alterations in proteins.

    Science.gov (United States)

    Borshchevskiy, Valentin; Round, Ekaterina; Erofeev, Ivan; Weik, Martin; Ishchenko, Andrii; Gushchin, Ivan; Mishin, Alexey; Willbold, Dieter; Büldt, Georg; Gordeliy, Valentin

    2014-10-01

    X-ray-radiation-induced alterations to protein structures are still a severe problem in macromolecular crystallography. One way to avoid the influence of radiation damage is to reduce the X-ray dose absorbed by the crystal during data collection. However, here it is demonstrated using the example of the membrane protein bacteriorhodopsin (bR) that even a low dose of less than 0.06 MGy may induce structural alterations in proteins. This dose is about 500 times smaller than the experimental dose limit which should ideally not be exceeded per data set (i.e. 30 MGy) and 20 times smaller than previously detected specific radiation damage at the bR active site. To date, it is the lowest dose at which radiation modification of a protein structure has been described. Complementary use was made of high-resolution X-ray crystallography and online microspectrophotometry to quantitatively study low-dose X-ray-induced changes. It is shown that structural changes of the protein correlate with the spectroscopically observed formation of the so-called bR orange species. Evidence is provided for structural modifications taking place at the protein active site that should be taken into account in crystallographic studies which aim to elucidate the molecular mechanisms of bR function.

  11. Amphipathic polymers: tools to fold integral membrane proteins to their active form.

    Science.gov (United States)

    Pocanschi, Cosmin L; Dahmane, Tassadite; Gohon, Yann; Rappaport, Fabrice; Apell, Hans-Jürgen; Kleinschmidt, Jörg H; Popot, Jean-Luc

    2006-11-28

    Among the major obstacles to pharmacological and structural studies of integral membrane proteins (MPs) are their natural scarcity and the difficulty in overproducing them in their native form. MPs can be overexpressed in the non-native state as inclusion bodies, but inducing them to achieve their functional three-dimensional structure has proven to be a major challenge. We describe here the use of an amphipathic polymer, amphipol A8-35, as a novel environment that allows both beta-barrel and alpha-helical MPs to fold to their native state, in the absence of detergents or lipids. Amphipols, which are extremely mild surfactants, appear to favor the formation of native intramolecular protein-protein interactions over intermolecular or protein-surfactant ones. The feasibility of the approach is demonstrated using as models OmpA and FomA, two outer membrane proteins from the eubacteria Escherichia coli and Fusobacterium nucleatum, respectively, and bacteriorhodopsin, a light-driven proton pump from the plasma membrane of the archaebacterium Halobacterium salinarium.

  12. Predictive energy landscapes for folding membrane protein assemblies

    Science.gov (United States)

    Truong, Ha H.; Kim, Bobby L.; Schafer, Nicholas P.; Wolynes, Peter G.

    2015-12-01

    We study the energy landscapes for membrane protein oligomerization using the Associative memory, Water mediated, Structure and Energy Model with an implicit membrane potential (AWSEM-membrane), a coarse-grained molecular dynamics model previously optimized under the assumption that the energy landscapes for folding α-helical membrane protein monomers are funneled once their native topology within the membrane is established. In this study we show that the AWSEM-membrane force field is able to sample near native binding interfaces of several oligomeric systems. By predicting candidate structures using simulated annealing, we further show that degeneracies in predicting structures of membrane protein monomers are generally resolved in the folding of the higher order assemblies as is the case in the assemblies of both nicotinic acetylcholine receptor and V-type Na+-ATPase dimers. The physics of the phenomenon resembles domain swapping, which is consistent with the landscape following the principle of minimal frustration. We revisit also the classic Khorana study of the reconstitution of bacteriorhodopsin from its fragments, which is the close analogue of the early Anfinsen experiment on globular proteins. Here, we show the retinal cofactor likely plays a major role in selecting the final functional assembly.

  13. Time-resolved structural studies with serial crystallography: A new light on retinal proteins

    Directory of Open Access Journals (Sweden)

    Valérie Panneels

    2015-07-01

    Full Text Available Structural information of the different conformational states of the two prototypical light-sensitive membrane proteins, bacteriorhodopsin and rhodopsin, has been obtained in the past by X-ray cryo-crystallography and cryo-electron microscopy. However, these methods do not allow for the structure determination of most intermediate conformations. Recently, the potential of X-Ray Free Electron Lasers (X-FELs for tracking the dynamics of light-triggered processes by pump-probe serial femtosecond crystallography has been demonstrated using 3D-micron-sized crystals. In addition, X-FELs provide new opportunities for protein 2D-crystal diffraction, which would allow to observe the course of conformational changes of membrane proteins in a close-to-physiological lipid bilayer environment. Here, we describe the strategies towards structural dynamic studies of retinal proteins at room temperature, using injector or fixed-target based serial femtosecond crystallography at X-FELs. Thanks to recent progress especially in sample delivery methods, serial crystallography is now also feasible at synchrotron X-ray sources, thus expanding the possibilities for time-resolved structure determination.

  14. Resonance Raman spectroscopy of chemically modified and isotopically labelled purple membranes: I. A critical examination of the carbon-nitrogen vibrational modes

    Energy Technology Data Exchange (ETDEWEB)

    Ehrenberg, B. (Cornell Univ., Ithaca, NY); Lemley, A.T.; Lewis, A.; Zastrow, M.V.; Crespi, H.L.

    1980-01-01

    Resonance Raman spectra of bacteriorhodopsin are compared to the spectra of this protein modified in the following ways: (1) selective deuteration at the C-15 carbon atom of retinal, (2) full deuteration of the retinal, (3) the addition of a conjugated double bond in the ..beta..-ionone ring (3-dehydroretinal), (4) full deuteration of the protein and lipid components, (5) /sup 15/N enrichment of the entire membrane and (6) deuteration of the entire membrane. A detailed comparison of the /sup 15/N-enriched membrane and naturally occurring purple membrane from 800 cm/sup -1/ to 1700 cm/sup -1/ reveals that /sup 15/N enrichment affects the frequency of only two vibrational modes. These occur at 1642 cm/sup -1/ and 1620 cm/sup -1/ in naturally occurring purple membrane and at 1628 cm/sup -1/ and 1615 cm/sup -1/ in the /sup 15/N-enriched samples. Therefore, this pair of bands reflects the states of protonation of the Schiff base.

  15. Electron crystallography for structural and functional studies of membrane proteins.

    Science.gov (United States)

    Fujiyoshi, Yoshinori

    2011-01-01

    Membrane proteins are important research targets for basic biological sciences and drug design, but studies of their structure and function are considered difficult to perform. Studies of membrane structures have been greatly facilitated by technological and instrumental advancements in electron microscopy together with methodological advancements in biology. Electron crystallography is especially useful in studying the structure and function of membrane proteins. Electron crystallography is now an established method of analyzing the structures of membrane proteins in lipid bilayers, which resembles their natural biological environment. To better understand the neural system function from a structural point of view, we developed the cryo-electron microscope with a helium-cooled specimen stage, which allows for analysis of the structures of membrane proteins at a resolution higher than 3 Å. This review introduces recent instrumental advances in cryo-electron microscopy and presents some examples of structure analyses of membrane proteins, such as bacteriorhodopsin, water channels and gap junction channels. This review has two objectives: first, to provide a personal historical background to describe how we came to develop the cryo-electron microscope and second, to discuss some of the technology required for the structural analysis of membrane proteins based on cryo-electron microscopy.

  16. Bioinformatic analysis of the distribution of inorganic carbon transporters and prospective targets for bioengineering to increase Ci uptake by cyanobacteria.

    Science.gov (United States)

    Gaudana, Sandeep B; Zarzycki, Jan; Moparthi, Vamsi K; Kerfeld, Cheryl A

    2015-10-01

    Cyanobacteria have evolved a carbon-concentrating mechanism (CCM) which has enabled them to inhabit diverse environments encompassing a range of inorganic carbon (Ci: [Formula: see text] and CO2) concentrations. Several uptake systems facilitate inorganic carbon accumulation in the cell, which can in turn be fixed by ribulose 1,5-bisphosphate carboxylase/oxygenase. Here we survey the distribution of genes encoding known Ci uptake systems in cyanobacterial genomes and, using a pfam- and gene context-based approach, identify in the marine (alpha) cyanobacteria a heretofore unrecognized number of putative counterparts to the well-known Ci transporters of beta cyanobacteria. In addition, our analysis shows that there is a huge repertoire of transport systems in cyanobacteria of unknown function, many with homology to characterized Ci transporters. These can be viewed as prospective targets for conversion into ancillary Ci transporters through bioengineering. Increasing intracellular Ci concentration coupled with efforts to increase carbon fixation will be beneficial for the downstream conversion of fixed carbon into value-added products including biofuels. In addition to CCM transporter homologs, we also survey the occurrence of rhodopsin homologs in cyanobacteria, including bacteriorhodopsin, a class of retinal-binding, light-activated proton pumps. Because they are light driven and because of the apparent ease of altering their ion selectivity, we use this as an example of re-purposing an endogenous transporter for the augmentation of Ci uptake by cyanobacteria and potentially chloroplasts.

  17. Effect of the compatible solute ectoine on the stability of the membrane proteins.

    Science.gov (United States)

    Roychoudhury, Arpita; Haussinger, Dieter; Oesterhelt, Filipp

    2012-08-01

    Mechanical single molecule techniques offer exciting possibilities for investigating protein folding and stability in native environments at sub-nanometer resolutions. Compatible solutes show osmotic activity which even at molar concentrations do not interfere with cell metabolism. They are known to protect proteins against external stress like temperature, high salt concentrations and dehydrating conditions. We studied the impact of the compatible solute ectoine (1M) on membrane proteins by analyzing the mechanical properties of Bacteriorhodopsin (BR) in its presence and absence by single molecule force spectroscopy. The unfolding experiments on BR revealed that ectoine decreases the persistence length of its polypeptide chain thereby increasing its tendency to coil up. In addition, we found higher unfolding forces indicating strengthening of those intra molecular interactions which are crucial for stability. This shows that force spectroscopy is well suited to study the effect of compatible solutes to stabilize membrane proteins against unfolding. In addition, it may lead to a better understanding of their detailed mechanism of action.

  18. A phylogenetically distinctive and extremely heat stable light-driven proton pump from the eubacterium Rubrobacter xylanophilus DSM 9941T

    Science.gov (United States)

    Kanehara, Kanae; Yoshizawa, Susumu; Tsukamoto, Takashi; Sudo, Yuki

    2017-01-01

    Rhodopsins are proteins that contain seven transmembrane domains with a chromophore retinal and that function as photoreceptors for light-energy conversion and light-signal transduction in a wide variety of organisms. Here we characterized a phylogenetically distinctive new rhodopsin from the thermophilic eubacterium Rubrobacter xylanophilus DSM 9941T that was isolated from thermally polluted water. Although R. xylanophilus rhodopsin (RxR) is from Actinobacteria, it is located between eukaryotic and archaeal rhodopsins in the phylogenetic tree. Escherichia coli cells expressing RxR showed a light-induced decrease in environmental pH and inhibition by a protonophore, indicating that it works as a light-driven outward proton pump. We characterized purified RxR spectroscopically, and showed that it has an absorption maximum at 541 nm and binds nearly 100% all-trans retinal. The pKa values for the protonated retinal Schiff base and its counterion were estimated to be 10.7 and 1.3, respectively. Time-resolved flash-photolysis experiments revealed the formation of a red-shifted intermediate. Of note, RxR showed an extremely high thermal stability in comparison with other proton pumping rhodopsins such as thermophilic rhodopsin TR (by 16-times) and bacteriorhodopsin from Halobacterium salinarum (HsBR, by 4-times). PMID:28290523

  19. A phylogenetically distinctive and extremely heat stable light-driven proton pump from the eubacterium Rubrobacter xylanophilus DSM 9941(T).

    Science.gov (United States)

    Kanehara, Kanae; Yoshizawa, Susumu; Tsukamoto, Takashi; Sudo, Yuki

    2017-03-14

    Rhodopsins are proteins that contain seven transmembrane domains with a chromophore retinal and that function as photoreceptors for light-energy conversion and light-signal transduction in a wide variety of organisms. Here we characterized a phylogenetically distinctive new rhodopsin from the thermophilic eubacterium Rubrobacter xylanophilus DSM 9941(T) that was isolated from thermally polluted water. Although R. xylanophilus rhodopsin (RxR) is from Actinobacteria, it is located between eukaryotic and archaeal rhodopsins in the phylogenetic tree. Escherichia coli cells expressing RxR showed a light-induced decrease in environmental pH and inhibition by a protonophore, indicating that it works as a light-driven outward proton pump. We characterized purified RxR spectroscopically, and showed that it has an absorption maximum at 541 nm and binds nearly 100% all-trans retinal. The pKa values for the protonated retinal Schiff base and its counterion were estimated to be 10.7 and 1.3, respectively. Time-resolved flash-photolysis experiments revealed the formation of a red-shifted intermediate. Of note, RxR showed an extremely high thermal stability in comparison with other proton pumping rhodopsins such as thermophilic rhodopsin TR (by 16-times) and bacteriorhodopsin from Halobacterium salinarum (HsBR, by 4-times).

  20. Ion-Pumping Microbial Rhodopsins

    Directory of Open Access Journals (Sweden)

    Hideki eKandori

    2015-09-01

    Full Text Available Rhodopsins are light-sensing proteins used in optogenetics. The word rhodopsin originates from the Greek words rhodo and opsis, indicating rose and sight, respectively. Although the classical meaning of rhodopsin is the red-colored pigment in our eyes, the modern meaning of rhodopsin encompasses photoactive proteins containing a retinal chromophore in animals and microbes. Animal and microbial rhodopsins possess 11-cis and all-trans retinal, respectively, to capture light in seven transmembrane α-helices, and photoisomerizations into all-trans and 13-cis forms, respectively, initiate each function. Ion-transporting proteins can be found in microbial rhodopsins, such as light-gated channels and light-driven pumps, which are the main tools in optogenetics. Light-driven pumps, such as archaeal H+ pump bacteriorhodopsin (BR and Cl- pump halorhodopsin (HR, were discovered in the 1970s, and their mechanism has been extensively studied. On the other hand, different kinds of H+ and Cl- pumps have been found in marine bacteria, such as proteorhodopsin (PR and Fulvimarina pelagi rhodopsin (FR, respectively. In addition, a light-driven Na+ pump was found, Krokinobacter eikastus rhodopsin 2 (KR2. These light-driven ion-pumping microbial rhodopsins are classified as DTD, TSA, DTE, NTQ and NDQ rhodopsins for BR, HR, PR, FR and KR2, respectively. Recent understanding of ion-pumping microbial rhodopsins is reviewed in this paper.

  1. Surface proton transport of fully protonated poly(aspartic acid) thin films on quartz substrates

    Energy Technology Data Exchange (ETDEWEB)

    Nagao, Yuki, E-mail: ynagao@jaist.ac.jp; Kubo, Takahiro

    2014-12-30

    Graphical abstract: - Highlights: • Proton transport of fully protonated poly(aspartic acid) thin film was investigated. • The thin film structure differed greatly from the partially protonated one. • Proton transport occurs on the surface, not inside of the thin film. • This result contributes to biological transport systems such as bacteriorhodopsin. - Abstract: Thin film structure and the proton transport property of fully protonated poly(aspartic acid) (P-Asp100) have been investigated. An earlier study assessed partially protonated poly(aspartic acid), highly oriented thin film structure and enhancement of the internal proton transport. In this study of P-Asp100, IR p-polarized multiple-angle incidence resolution (P-MAIR) spectra were measured to investigate the thin film structure. The obtained thin films, with thicknesses of 120–670 nm, had no oriented structure. Relative humidity dependence of the resistance, proton conductivity, and normalized resistance were examined to ascertain the proton transport property of P-Asp100 thin films. The obtained data showed that the proton transport of P-Asp100 thin films might occur on the surface, not inside of the thin film. This phenomenon might be related with the proton transport of the biological system.

  2. Fabrication of a Retinal-protein Photoreceptor Array

    Institute of Scientific and Technical Information of China (English)

    赵迎春; 吴佳; 黄伟达; 丁建东

    2012-01-01

    A pixel-architecture film of retinal proteins was prepared by an approach combining chemical, physical and biological technologies. Oriented multilayers of purple membrane composed of bacteriorhodopsin (BR) and lipids were patterned on an array of gold electrode pixels. In order to improve stability and resolution, the gene engineer- ing technique was employed to make a mutant of the protein BR by replacing the 36th amino acid residue from as- partic acid to cysteine with a thiol end group ready to react with gold; electric sedimentation was used to guarantee the high probability of formation of the Au-S bond and meanwhile to orient BR; further chemical crosslinking was introduced among layers of purple membranes to significantly enhance photoelectrical signals while keeping high stability. The non-bound BR region was eventually washed out by detergent, and the remaining BR pixels were thus detergent resistant due to chemical crosslinking among BR layers and covalent binding between the multilayer and the substrate. The protein array was confirmed to keep photoelectrical activity.

  3. Glass is a Viable Substrate for Precision Force Microscopy of Membrane Proteins.

    Science.gov (United States)

    Chada, Nagaraju; Sigdel, Krishna P; Gari, Raghavendar Reddy Sanganna; Matin, Tina Rezaie; Randall, Linda L; King, Gavin M

    2015-07-31

    Though ubiquitous in optical microscopy, glass has long been overlooked as a specimen supporting surface for high resolution atomic force microscopy (AFM) investigations due to its roughness. Using bacteriorhodopsin from Halobacterium salinarum and the translocon SecYEG from Escherichia coli, we demonstrate that faithful images of 2D crystalline and non-crystalline membrane proteins in lipid bilayers can be obtained on microscope cover glass following a straight-forward cleaning procedure. Direct comparison between AFM data obtained on glass and on mica substrates show no major differences in image fidelity. Repeated association of the ATPase SecA with the cytoplasmic protrusion of SecYEG demonstrates that the translocon remains competent for binding after tens of minutes of continuous AFM imaging. This opens the door for precision long-timescale investigations of the active translocase in near-native conditions and, more generally, for integration of high resolution biological AFM with many powerful optical techniques that require non-birefringent substrates.

  4. Anharmonic transitions in nearly dry L-cysteine I.

    Science.gov (United States)

    Lima, T A; Sato, E T; Martins, E T; Homem-de-Mello, P; Lago, A F; Coutinho-Neto, M D; Ferreira, F F; Giles, C; Pires, M O C; Martinho, H

    2012-05-16

    Two special dynamical transitions of universal character have recently been observed in macromolecules (lysozyme, myoglobin, bacteriorhodopsin, DNA and RNA) at T* ~100-150 K and T(D) ~180-220 K. The underlying mechanisms governing these transitions have been the subject of debate. In the present work, a survey is reported on the temperature dependence of structural, vibrational and thermodynamical properties of a nearly anhydrous amino acid (orthorhombic polymorph of the amino acid l-cysteine at a hydration level of 3.5%). The temperature dependence of x-ray powder diffraction patterns, Raman spectra and specific heat revealed these two transitions at T* = 70 K and T(D) = 230 K for this sample. The data were analyzed considering amino acid-amino acid, amino acid-water, water-water phonon-phonon interactions and molecular rotor activation. Our results indicated that the two referred temperatures define the triggering of very simple and particular events that govern all the interactions of the biomolecular: activation of CH(2) rigid rotors (T T(D)).

  5. Driving electrocatalytic activity by interface electronic structure control in a metalloprotein hybrid catalyst for efficient hydrogen evolution.

    Science.gov (United States)

    Behera, Sushant Kumar; Deb, Pritam; Ghosh, Arghya

    2016-08-17

    The rational design of metalloprotein hybrid structures and precise calculations for understanding the role of the interfacial electronic structure in regulating the HER activity of water splitting sites and their microscopic effect for obtaining robust hydrogen evolution possess great promise for developing highly efficient nano-bio hybrid HER catalysts. Here, we employ high-accuracy linear-scaling density functional theory calculations using a near-complete basis set and a minimal parameter implicit solvent model within the self-consistent calculations, on silver (Ag) ions assimilated on bacteriorhodopsin (bR) at specific binding sites. Geometry optimization indicates the formation of active sites at the interface of the metalloprotein complex and the density of states reflects the metallic nature of the active sites. The reduced value of the canonical orbital gap indicates the state of dynamic nature after Ag ion assimilation on active sites and smooth electron transfer. These incorporated active protein sites are more efficient in electrolytic splitting of water than pristine sites due to their low value of Gibbs free energy for the HER in terms of hydrogen coverages. Volcano plot analysis and the free energy diagram are compared for understanding the hydrogen evolution efficiency. Moreover, the essential role of the interfacial electronic properties in regulating the HER catalytic activity of water splitting sites and enhancing the efficiency is elucidated.

  6. Control of surface plasmon resonance in out-diffused silver nanoislands for surface-enhanced Raman scattering

    Science.gov (United States)

    Piliugina, E. S.; Heisler, F.; Chervinskii, S. D.; Samusev, A. K.; Lipovskii, A. A.

    2015-12-01

    We present the studies of self-assembled silver nanoislands on the surface of silver ion-exchanged glasses. The nanoislands were formed by out-diffusion of reduced silver atoms from the bulk of the glass to its surface. Control of silver ions distribution in the glass by thermal poling after the ion exchange allowed formation of relatively big, up to 250 nm, isolated silver nanoislands while without the poling an ensemble of silver nanoislands with average size from several to tens of nanometers with random size distribution was formed. The nanoislands were characterized using atomic force microscopy and spectral measurements. We used optical absorption spectroscopy for “random” nanoislands and dark field scattering spectroscopy for isolated ones, corresponding spectra showed peaks in the vicinity of 450 nm and 600 nm, respectively. The “random” nanoislands significantly enhanced Raman scattering from Rhodamine 6G, also the modification of Raman signal from deposited on the surface of the samples bacteriorhodopsin in purple membranes was registered.

  7. Low-Z polymer sample supports for fixed-target serial femtosecond X-ray crystallography

    Energy Technology Data Exchange (ETDEWEB)

    Feld, Geoffrey K. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); National Institute of Environmental Health Science, Research Triangle Park, NC (United States); Heymann, Michael [Brandeis Univ., Waltham, MA (United States); Univ. of Hamburg and DESY, Hamburg (Germany); Benner, W. Henry [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Pardini, Tommaso [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Tsai, Ching -Ju [Paul Scherrer Inst. (PSI), Villigen (Switzerland); Boutet, Sebastien [SLAC National Accelerator Lab., Menlo Park, CA (United States); Coleman, Matthew A. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Hunter, Mark S. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); SLAC National Accelerator Lab., Menlo Park, CA (United States); Li, Xiaodan [Paul Scherrer Inst. (PSI), Villigen (Switzerland); Messerschmidt, Marc [SLAC National Accelerator Lab., Menlo Park, CA (United States); BioXFEL Science and Technology Center, Buffalo, NY (United States); Opathalage, Achini [Brandeis Univ., Waltham, MA (United States); Pedrini, Bill [Paul Scherrer Inst. (PSI), Villigen (Switzerland); Williams, Garth J. [SLAC National Accelerator Lab., Menlo Park, CA (United States); Krantz, Bryan A. [Univ. of California, Berkeley, CA (United States); Fraden, Seth [Brandeis Univ., Waltham, MA (United States); Hau-Riege, Stefan [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Evans, James E. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Segelke, Brent W. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Frank, Matthias [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2015-06-27

    X-ray free-electron lasers (XFELs) offer a new avenue to the structural probing of complex materials, including biomolecules. Delivery of precious sample to the XFEL beam is a key consideration, as the sample of interest must be serially replaced after each destructive pulse. The fixed-target approach to sample delivery involves depositing samples on a thin-film support and subsequent serial introduction via a translating stage. Some classes of biological materials, including two-dimensional protein crystals, must be introduced on fixed-target supports, as they require a flat surface to prevent sample wrinkling. A series of wafer and transmission electron microscopy (TEM)-style grid supports constructed of low-Z plastic have been custom-designed and produced. Aluminium TEM grid holders were engineered, capable of delivering up to 20 different conventional or plastic TEM grids using fixed-target stages available at the Linac Coherent Light Source (LCLS). As proof-of-principle, X-ray diffraction has been demonstrated from two-dimensional crystals of bacteriorhodopsin and three-dimensional crystals of anthrax toxin protective antigen mounted on these supports at the LCLS. In conclusion, the benefits and limitations of these low-Z fixed-target supports are discussed; it is the authors' belief that they represent a viable and efficient alternative to previously reported fixed-target supports for conducting diffraction studies with XFELs.

  8. Probing Saltern Brines with an Oxygen Electrode: What Can We Learn about the Community Metabolism in Hypersaline Systems?

    Directory of Open Access Journals (Sweden)

    Aharon Oren

    2016-06-01

    Full Text Available We have explored the use of optical oxygen electrodes to study oxygenic photosynthesis and heterotrophic activities in crystallizer brines of the salterns in Eilat, Israel. Monitoring oxygen uptake rates in the dark enables the identification of organic substrates that are preferentially used by the community. Addition of glycerol (the osmotic solute synthesized by Dunaliella or dihydroxyacetone (produced from glycerol by Salinibacter enhanced respiration rates. Pyruvate, produced from glycerol or from some sugars by certain halophilic Archaea also stimulated community respiration. Fumarate had a sparing effect on respiration, possibly as many halophilic Archaea can use fumarate as a terminal electron acceptor in respiration. Calculating the photosynthetic activity of Dunaliella by monitoring oxygen concentration changes during light/dark incubations is not straightforward as light also affects respiration of some halophilic Archaea and Bacteria due to action of light-driven proton pumps. When illuminated, community respiration of brine samples in which oxygenic photosynthesis was inhibited by DCMU decreased by ~40%. This effect was interpreted as the result of competition between two energy yielding systems: the bacteriorhodopsin proton pump and the respiratory chain of the prokaryotes. These findings have important implications for the interpretation of other published data on photosynthetic and respiratory activities in hypersaline environments.

  9. Nanoscale Electric Characteristics and Oriented Assembly of Halobacterium salinarum Membrane Revealed by Electric Force Microscopy

    Directory of Open Access Journals (Sweden)

    Denghua Li

    2016-11-01

    Full Text Available Purple membranes (PM of the bacteria Halobacterium salinarum are a unique natural membrane where bacteriorhodopsin (BR can convert photon energy and pump protons. Elucidating the electronic properties of biomembranes is critical for revealing biological mechanisms and developing new devices. We report here the electric properties of PMs studied by using multi-functional electric force microscopy (EFM at the nanoscale. The topography, surface potential, and dielectric capacity of PMs were imaged and quantitatively measured in parallel. Two orientations of PMs were identified by EFM because of its high resolution in differentiating electrical characteristics. The extracellular (EC sides were more negative than the cytoplasmic (CP side by 8 mV. The direction of potential difference may facilitate movement of protons across the membrane and thus play important roles in proton pumping. Unlike the side-dependent surface potentials observed in PM, the EFM capacitive response was independent of the side and was measured to be at a dC/dz value of ~5.25 nF/m. Furthermore, by modification of PM with de novo peptides based on peptide-protein interaction, directional oriented PM assembly on silicon substrate was obtained for technical devices. This work develops a new method for studying membrane nanoelectronics and exploring the bioelectric application at the nanoscale.

  10. Glucose Synthesis in a Protein-Based Artificial Photosynthesis System.

    Science.gov (United States)

    Lu, Hao; Yuan, Wenqiao; Zhou, Jack; Chong, Parkson Lee-Gau

    2015-09-01

    The objective of this study was to understand glucose synthesis of a protein-based artificial photosynthesis system affected by operating conditions, including the concentrations of reactants, reaction temperature, and illumination. Results from non-vesicle-based glyceraldehyde-3-phosphate (GAP) and glucose synthesis showed that the initial concentrations of ribulose-1,5-bisphosphate (RuBP) and adenosine triphosphate (ATP), lighting source, and temperature significantly affected glucose synthesis. Higher initial concentrations of RuBP and ATP significantly enhanced GAP synthesis, which was linearly correlated to glucose synthesis, confirming the proper functions of all catalyzing enzymes in the system. White fluorescent light inhibited artificial photosynthesis and reduced glucose synthesis by 79.2 % compared to in the dark. The reaction temperature of 40 °C was optimum, whereas lower or higher temperature reduced glucose synthesis. Glucose synthesis in the vesicle-based artificial photosynthesis system reconstituted with bacteriorhodopsin, F 0 F 1 ATP synthase, and polydimethylsiloxane-methyloxazoline-polydimethylsiloxane triblock copolymer was successfully demonstrated. This system efficiently utilized light-induced ATP to drive glucose synthesis, and 5.2 μg ml(-1) glucose was synthesized in 0.78-ml reaction buffer in 7 h. Light-dependent reactions were found to be the bottleneck of the studied artificial photosynthesis system.

  11. Microbial weeds in hypersaline habitats: the enigma of the weed-like Haloferax mediterranei

    Science.gov (United States)

    Oren, Aharon; Hallsworth, John E.

    2014-10-01

    Heterotrophic prokaryotic communities that inhabit saltern crystallizer ponds are typically dominated by two species, the archaeon Haloquadratum walsbyi and the bacterium Salinibacter ruber, regardless of location. These organisms behave as 'microbial weeds' as defined by Cray et al. (Microb Biotechnol6: 453–492, 2013) that possess the biological traits required to dominate the microbiology of these open habitats. Here, we discuss the enigma of the less abundant Haloferax mediterranei, an archaeon that grows faster than any other, comparable extreme halophile. It has a wide window for salt tolerance, can grow on simple as well as on complex substrates and degrade polymeric substances, has different modes of anaerobic growth, can accumulate storage polymers, produces gas vesicles, and excretes halocins capable of killing other Archaea. Therefore, Hfx. mediterranei is apparently more qualified as a 'microbial weed' than Haloquadratum and Salinibacter. However, the former differs because it produces carotenoid pigments only in the lower salinity range and lacks energy-generating retinal-based, light-driven ion pumps such as bacteriorhodopsin and halorhodopsin. We discuss these observations in relation to microbial weed biology in, and the open-habitat ecology of, hypersaline systems.

  12. Protons and how they are transported by proton pumps.

    Science.gov (United States)

    Buch-Pedersen, M J; Pedersen, B P; Veierskov, B; Nissen, P; Palmgren, M G

    2009-01-01

    The very high mobility of protons in aqueous solutions demands special features of membrane proton transporters to sustain efficient yet regulated proton transport across biological membranes. By the use of the chemical energy of ATP, plasma-membrane-embedded ATPases extrude protons from cells of plants and fungi to generate electrochemical proton gradients. The recently published crystal structure of a plasma membrane H(+)-ATPase contributes to our knowledge about the mechanism of these essential enzymes. Taking the biochemical and structural data together, we are now able to describe the basic molecular components that allow the plasma membrane proton H(+)-ATPase to carry out proton transport against large membrane potentials. When divergent proton pumps such as the plasma membrane H(+)-ATPase, bacteriorhodopsin, and F(O)F(1) ATP synthase are compared, unifying mechanistic premises for biological proton pumps emerge. Most notably, the minimal pumping apparatus of all pumps consists of a central proton acceptor/donor, a positively charged residue to control pK(a) changes of the proton acceptor/donor, and bound water molecules to facilitate rapid proton transport along proton wires.

  13. The lifetimes of Pharaonis phoborhodopsin signaling states depend on the rates of proton transfers--effects of hydrostatic pressure and stopped flow experiments.

    Science.gov (United States)

    Kikukawa, Takashi; Saha, Chabita K; Balashov, Sergei P; Imasheva, Eleonora S; Zaslavsky, Dmitry; Gennis, Robert B; Abe, Takayuki; Kamo, Naoki

    2008-01-01

    Pharaonis phoborhodopsin (ppR), a negative phototaxis receptor of Natronomonas pharaonis, undergoes photocycle similar to the light-driven proton pump bacteriorhodopsin (BR), but the turnover rate is much slower due to much longer lifetimes of the M and O intermediates. The M decay was shown to become as fast as it is in BR in the L40T/F86D mutant. We examined the effects of hydrostatic pressure on the decay of these intermediates. For BR, pressure decelerated M decay but slightly affected O decay. In contrast, with ppR and with its L40T/F86D mutant, pressure slightly affected M decay but accelerated O decay. Clearly, the pressure-dependent factors for M and O decay are different in BR and ppR. In order to examine the deprotonation of Asp75 in unphotolyzed ppR we performed stopped flow experiments. The pH jump-induced deprotonation of Asp75 occurred with 60 ms, which is at least 20 times slower than deprotonation of the equivalent Asp85 in BR and about 10-fold faster than the O decay of ppR. These data suggest that proton transfer is slowed not only in the cytoplasmic channel but also in the extracellular channel of ppR and that the light-induced structural changes in the O intermediate of ppR additionally decrease this rate.

  14. The Function of Gas Vesicles in Halophilic Archaea and Bacteria: Theories and Experimental Evidence

    Directory of Open Access Journals (Sweden)

    Aharon Oren

    2012-12-01

    Full Text Available A few extremely halophilic Archaea (Halobacterium salinarum, Haloquadratum walsbyi, Haloferax mediterranei, Halorubrum vacuolatum, Halogeometricum borinquense, Haloplanus spp. possess gas vesicles that bestow buoyancy on the cells. Gas vesicles are also produced by the anaerobic endospore-forming halophilic Bacteria Sporohalobacter lortetii and Orenia sivashensis. We have extensive information on the properties of gas vesicles in Hbt. salinarum and Hfx. mediterranei and the regulation of their formation. Different functions were suggested for gas vesicle synthesis: buoying cells towards oxygen-rich surface layers in hypersaline water bodies to prevent oxygen limitation, reaching higher light intensities for the light-driven proton pump bacteriorhodopsin, positioning the cells optimally for light absorption, light shielding, reducing the cytoplasmic volume leading to a higher surface-area-to-volume ratio (for the Archaea and dispersal of endospores (for the anaerobic spore-forming Bacteria. Except for Hqr. walsbyi which abounds in saltern crystallizer brines, gas-vacuolate halophiles are not among the dominant life forms in hypersaline environments. There only has been little research on gas vesicles in natural communities of halophilic microorganisms, and the few existing studies failed to provide clear evidence for their possible function. This paper summarizes the current status of the different theories why gas vesicles may provide a selective advantage to some halophilic microorganisms.

  15. High-throughput single-molecule force spectroscopy for membrane proteins

    Energy Technology Data Exchange (ETDEWEB)

    Bosshart, Patrick D; Casagrande, Fabio; Frederix, Patrick L T M; Engel, Andreas; Fotiadis, Dimitrios [M E Mueller Institute for Structural Biology, Biozentrum of the University of Basel, CH-4056 Basel (Switzerland); Ratera, Merce; Palacin, Manuel [Institute for Research in Biomedicine, Barcelona Science Park, Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona and Centro de Investigacion Biomedica en Red de Enfermedades Raras, E-08028 Barcelona (Spain); Bippes, Christian A; Mueller, Daniel J [BioTechnology Center, Technical University, Tatzberg 47, D-01307 Dresden (Germany)], E-mail: andreas.engel@unibas.ch, E-mail: dimitrios.fotiadis@mci.unibe.ch

    2008-09-24

    Atomic force microscopy-based single-molecule force spectroscopy (SMFS) is a powerful tool for studying the mechanical properties, intermolecular and intramolecular interactions, unfolding pathways, and energy landscapes of membrane proteins. One limiting factor for the large-scale applicability of SMFS on membrane proteins is its low efficiency in data acquisition. We have developed a semi-automated high-throughput SMFS (HT-SMFS) procedure for efficient data acquisition. In addition, we present a coarse filter to efficiently extract protein unfolding events from large data sets. The HT-SMFS procedure and the coarse filter were validated using the proton pump bacteriorhodopsin (BR) from Halobacterium salinarum and the L-arginine/agmatine antiporter AdiC from the bacterium Escherichia coli. To screen for molecular interactions between AdiC and its substrates, we recorded data sets in the absence and in the presence of L-arginine, D-arginine, and agmatine. Altogether {approx}400 000 force-distance curves were recorded. Application of coarse filtering to this wealth of data yielded six data sets with {approx}200 (AdiC) and {approx}400 (BR) force-distance spectra in each. Importantly, the raw data for most of these data sets were acquired in one to two days, opening new perspectives for HT-SMFS applications.

  16. Photochromic kinetic spectra and intermediates of BR-D96N

    Institute of Scientific and Technical Information of China (English)

    ZHENG; Yuan(郑媛); YAO; Baoli(姚保利); WANG; Yingli(王英利); LEI; Ming(雷铭); CHEN; Guofu(陈国夫); Norbert; Hampp

    2003-01-01

    BR-D96N is a kind of genetically site-specific mutant of bacteriorhodopsin (BR) with obvious photochromic effect. Compared to the wild type BR, the lifetime of M state of BR-D96N is prolonged to several minutes so that the photochromic kinetics and the intermediates formation can be studied by the conventional spectra analysis. In the experiment, the absorption spectra of the sample at different time after light illumination are measured with spectrophotometer. By fitting and analyzing the variation of the spectra, we suppose there are three main states in the photochromic process, i.e. B state (light-adapted state), M state and D state (dark-adapted state). The absorption spectra of the B state, M state and D state are extracted from the experimental data based on this three-state model and the spectra at various time are fitted with the least square method. So, the variations of population percentages of the M state, B state and D state are obtained and the M state and B state lifetimes are estimated. In another way, from measuring the absorption dynamics at 407 nm and 568 nm, the M state and B state lifetimes are also obtained by two exponential data fitting, which give coincident results with those of the spectra analysis.

  17. Photoreactions and Structural Changes of Anabaena Sensory Rhodopsin

    Directory of Open Access Journals (Sweden)

    Akira Kawanabe

    2009-12-01

    Full Text Available Anabaena sensory rhodopsin (ASR is an archaeal-type rhodopsin found in eubacteria. The gene encoding ASR forms a single operon with ASRT (ASR transducer which is a 14 kDa soluble protein, suggesting that ASR functions as a photochromic sensor by activating the soluble transducer. This article reviews the detailed photoreaction processes of ASR, which were studied by low-temperature Fourier-transform infrared (FTIR and UV-visible spectroscopy. The former research reveals that the retinal isomerization is similar to bacteriorhodopsin (BR, but the hydrogen-bonding network around the Schiff base and cytoplasmic region is different. The latter study shows the stable photoproduct of the all-trans form is 100% 13-cis, and that of the 13-cis form is 100% all-trans. These results suggest that the structural changes of ASR in the cytoplasmic domain play important roles in the activation of the transducer protein, and photochromic reaction is optimized for its sensor function.

  18. The future of MEBC: panel discussion.

    Science.gov (United States)

    Biczó, G

    1995-01-01

    The expected developments in the not too distant future (5-10 years) of molecular electronics and biocomputing (MEBC) are discussed. In the short-term, the study of very specific basic phenomena is expected (e.g. conducting polymers, strange electronic states of insulating polymers, bacteriorhodopsin (BR), arrays of molecules, self-organization of biomaterials, very specific biological systems, quantum coherence in cytoskeletal microtubules, optoelectronic information storage, associative memories, pattern recognition, hierarchical nature of biological information). New application fields outside the range of conventional technology (e.g. randomized algorithms, optoelectronic devices, chemical and biosensors, as well as a certain extent of commercialisation) have also been predicted. In the long-term, the study and solution of much deeper (sometimes scientific fiction-like) problems were foreseen, such as the self-organization of biomaterials, artificial self-reproduction, implementation of artificial cell dynamic control structures based on molecular devices for medical and environmental applications and the construction of neuronal computers as aids to the human brain.

  19. The study of terbium regenerated bacterirhodopsin

    Institute of Scientific and Technical Information of China (English)

    XU Bing; ZHANG Yue; HU Kunsheng

    2005-01-01

    The localization of Terbium (Tb3+) cations binding to deionized bacteriorhodopsin (bR) has been studied by using spectroscopic methods. It was found that adding Tb3+ cations to deionized bR affects the fluorescence lifetimes of tryptophan (Trp) in bR, the wavelength of fluorescence peak shifts "blue" and the peak value of fluorescence decreases. It was also found that adding one Tb3+ cation to deionized bR can restore the purple state from its blue state obviously. The measurements of absorbance, fluorescence and lifetime of fluorescence also show that when more than three Tb3+ cations are added, no further changes can be found. It is suggested that one Tb3+ specific binding site for the color-controlling is located on the exterior of the bR trimer structure to negatively charged lipids near Trp-10 and Trp-12. Three Tb3+ cations binding per bR is needed for the regenerated bR.

  20. Nanoporous microbead supported bilayers: stability, physical characterization, and incorporation of functional transmembrane proteins.

    Energy Technology Data Exchange (ETDEWEB)

    Davis, Ryan W. (University of New Mexico, Albuquerque, NM); Brozik, James A. (University of New Mexico, Albuquerque, NM); Brozik, Susan Marie; Cox, Jason M. (University of New Mexico, Albuquerque, NM); Lopez, Gabriel P. (University of New Mexico, Albuquerque, NM); Barrick, Todd A. (University of New Mexico, Albuquerque, NM); Flores, Adrean (University of New Mexico, Albuquerque, NM)

    2007-03-01

    The introduction of functional transmembrane proteins into supported bilayer-based biomimetic systems presents a significant challenge for biophysics. Among the various methods for producing supported bilayers, liposomal fusion offers a versatile method for the introduction of membrane proteins into supported bilayers on a variety of substrates. In this study, the properties of protein containing unilamellar phosphocholine lipid bilayers on nanoporous silica microspheres are investigated. The effects of the silica substrate, pore structure, and the substrate curvature on the stability of the membrane and the functionality of the membrane protein are determined. Supported bilayers on porous silica microspheres show a significant increase in surface area on surfaces with structures in excess of 10 nm as well as an overall decrease in stability resulting from increasing pore size and curvature. Comparison of the liposomal and detergent-mediated introduction of purified bacteriorhodopsin (bR) and the human type 3 serotonin receptor (5HT3R) are investigated focusing on the resulting protein function, diffusion, orientation, and incorporation efficiency. In both cases, functional proteins are observed; however, the reconstitution efficiency and orientation selectivity are significantly enhanced through detergent-mediated protein reconstitution. The results of these experiments provide a basis for bulk ionic and fluorescent dye-based compartmentalization assays as well as single-molecule optical and single-channel electrochemical interrogation of transmembrane proteins in a biomimetic platform.

  1. Sugar-induced blue membrane: release of divalent cations during phase transition of purple membranes observed in sugar-derived glasses.

    Science.gov (United States)

    Rhinow, Daniel; Hampp, Norbert A

    2008-04-17

    The formation of blue membrane from purple membranes (PM) has been observed in glassy films made from PM and various sugars. The phase transition of PM at about 70 degrees C causes the complexation of divalent cations to be weakened. The vicinal diol structures in sugars are capable to complex divalent cations and delocalize them throughout the matrix as long as its glass transition temperature is lower than the phase transition temperature of PM. The loss of divalent cations from bacteriorhodopsin (BR), the only protein in PM, causes the formation of blue membrane (BM), which is accompanied by a loss of beta-sheet structure observable in the infrared spectrum. Glassy sugars are particular useful to observe this transition, as sugar entrapment does not restrict conformational changes of BR but rather retards them. The material obtained was named sugar-induced blue membrane (SIBM). The formation of SIBM is inhibited by the addition of divalent cations. Furthermore, SIBM is reverted immediately to PM by addition of water. A characteristic time dependence of the thermal reversion of SIBM to PM proves that the phase transition of PM triggers the release and uptake of divalent cations and the corresponding color change.

  2. Lack of evidence for phase-only control of retinal photoisomerization in the strict one-photon limit

    Science.gov (United States)

    Liebel, M.; Kukura, P.

    2017-01-01

    The concept of shaping electric fields to steer light-induced processes coherently has fascinated scientists for decades. Despite early theoretical considerations that ruled out one-photon coherent control (CC), several experimental studies reported that molecular responses are sensitive to the shape of the excitation field in the weak-field limit. These observations were largely attributed to the presence of rapid-decay channels, but experimental verification is lacking. Here, we test this hypothesis by investigating the degree of achievable control over the photoisomerization of the retinal protonated Schiff-base in bacteriorhodopsin, isorhodopsin and rhodopsin, all of which exhibit similar chromophores but different isomerization yields and excited-state lifetimes. Irrespective of the system studied, we find no evidence for dissipation-dependent behaviour, nor for any CC in the strict one-photon limit. Our results question the extent to which a photochemical process at ambient conditions can be controlled at the amplitude level, and how the underlying molecular potential-energy surfaces and dynamics may influence this controllability.

  3. Automatic recovery of missing amplitudes and phases in tilt-limited electron crystallography of two-dimensional crystals

    Science.gov (United States)

    Gipson, Bryant R.; Masiel, Daniel J.; Browning, Nigel D.; Spence, John; Mitsuoka, Kaoru; Stahlberg, Henning

    2011-07-01

    Electron crystallography of 2D protein crystals provides a powerful tool for the determination of membrane protein structure. In this method, data is acquired in the Fourier domain as randomly sampled, uncoupled, amplitudes and phases. Due to physical constraints on specimen tilting, those Fourier data show a vast un-sampled “missing cone” of information, producing resolution loss in the direction perpendicular to the membrane plane. Based on the flexible language of projection onto sets, we provide a full solution for these problems with a projective constraint optimization algorithm that, for sufficiently oversampled data, produces complete recovery of unmeasured data in the missing cone. We apply this method to an experimental data set of Bacteriorhodopsin and show that, in addition to producing superior results compared to traditional reconstruction methods, full, reproducible, recovery of the missing cone from noisy data is possible. Finally, we present an automatic implementation of the refinement routine as open source, freely distributed, software that will be included in our 2dx software package.

  4. N-terminal fusion tags for effective production of g-protein-coupled receptors in bacterial cell-free systems.

    Science.gov (United States)

    Lyukmanova, E N; Shenkarev, Z O; Khabibullina, N F; Kulbatskiy, D S; Shulepko, M A; Petrovskaya, L E; Arseniev, A S; Dolgikh, D A; Kirpichnikov, M P

    2012-10-01

    G-protein-coupled receptors (GPCR) constitute one of the biggest families of membrane proteins. In spite of the fact that they are highly relevant to pharmacy, they have remained poorly explored. One of the main bottlenecks encountered in structural-functional studies of GPCRs is the difficulty to produce sufficient amounts of the proteins. Cell-free systems based on bacterial extracts fromE. colicells attract much attention as an effective tool for recombinant production of membrane proteins. GPCR production in bacterial cell-free expression systems is often inefficient because of the problems associated with the low efficiency of the translation initiation process. This problem could be resolved if GPCRs were expressed in the form of hybrid proteins with N-terminal polypeptide fusion tags. In the present work, three new N-terminal fusion tags are proposed for cell-free production of the human β2-adrenergic receptor, human M1 muscarinic acetylcholine receptor, and human somatostatin receptor type 5. It is demonstrated that the application of an N-terminal fragment (6 a.a.) of bacteriorhodopsin fromExiguobacterium sibiricum(ESR-tag), N-terminal fragment (16 а.о.) of RNAse A (S-tag), and Mistic protein fromB. subtilisallows to increase the CF synthesis of the target GPCRs by 5-38 times, resulting in yields of 0.6-3.8 mg from 1 ml of the reaction mixture, which is sufficient for structural-functional studies.

  5. Synthesis of an oligonucleotide-derivatized amphipol and its use to trap and immobilize membrane proteins.

    Science.gov (United States)

    Le Bon, Christel; Della Pia, Eduardo Antonio; Giusti, Fabrice; Lloret, Noémie; Zoonens, Manuela; Martinez, Karen L; Popot, Jean-Luc

    2014-06-01

    Amphipols (APols) are specially designed amphipathic polymers that stabilize membrane proteins (MPs) in aqueous solutions in the absence of detergent. A8-35, a polyacrylate-based APol, has been grafted with an oligodeoxynucleotide (ODN). The synthesis, purification and properties of the resulting 'OligAPol' have been investigated. Grafting was performed by reacting an ODN carrying an amine-terminated arm with the carboxylates of A8-35. The use of OligAPol for trapping MPs and immobilizing them onto solid supports was tested using bacteriorhodopsin (BR) and the transmembrane domain of Escherichia coli outer membrane protein A (tOmpA) as model proteins. BR and OligAPol form water-soluble complexes in which BR remains in its native conformation. Hybridization of the ODN arm with a complementary ODN was not hindered by the assembly of OligAPol into particles, nor by its association with BR. BR/OligAPol and tOmpA/OligAPol complexes could be immobilized onto either magnetic beads or gold nanoparticles grafted with the complementary ODN, as shown by spectroscopic measurements, fluorescence microscopy and the binding of anti-BR and anti-tOmpA antibodies. OligAPols provide a novel, highly versatile approach to tagging MPs, without modifying them chemically nor genetically, for specific, reversible and targetable immobilization, e.g. for nanoscale applications.

  6. Evolutionary Competition Between Primitive Photosynthetic Systems: Existence of an early purple Earth?

    Science.gov (United States)

    Sparks, William B.; DasSarma, S.; Reid, I. N.

    2006-12-01

    The onset of photosynthesis in primitive cyanobacteria is thought to have profoundly altered the Earth’s atmosphere by producing an oxygen-rich atmosphere some 2 billion years ago. However, the pigments used by chlorophyll-based photosynthesis absorb at a variety of wavelengths, curiously except those centered around the peak of the Solar spectrum, 550nm. By contrast, simpler retinal-based light harvesting systems such as the haloarchaeal purple membrane bacteriorhodopsin and halorhodopsin show a strong well-defined peak of absorbance centered at 550nm. The spectroscopic complementarity for retinal pigments with chlorophyll-based pigments suggests an intriguing possibility of their co-evolution. This hypothesis argues that simpler retinal-based phototrophic capability may have evolved earlier, in microorganisms that dominated during the anaerobic and purple phase of the planet. Later, the more complex chlorophyll-based photosystem pigments could have evolved to harvest light in regions of the spectrum not absorbed by preexisting species. This would have led to the greening and oxidation of our planet and displacement of most of the retinal-based microorganisms. Not surprisingly, evidence for retinal chromoproteins have recently turned up in a variety of planktonic microorganisms. Although speculative, such a scenario would indicate that retinal-based phototrophy may be one of the oldest metabolic capabilities on Earth. Moreover, if the chlorophyll absorption spectrum is simply a product of adaptation, then its utility as a potential biomarker is likely to be limited.

  7. Lipidic cubic phase injector is a viable crystal delivery system for time-resolved serial crystallography.

    Science.gov (United States)

    Nogly, Przemyslaw; Panneels, Valerie; Nelson, Garrett; Gati, Cornelius; Kimura, Tetsunari; Milne, Christopher; Milathianaki, Despina; Kubo, Minoru; Wu, Wenting; Conrad, Chelsie; Coe, Jesse; Bean, Richard; Zhao, Yun; Båth, Petra; Dods, Robert; Harimoorthy, Rajiv; Beyerlein, Kenneth R; Rheinberger, Jan; James, Daniel; DePonte, Daniel; Li, Chufeng; Sala, Leonardo; Williams, Garth J; Hunter, Mark S; Koglin, Jason E; Berntsen, Peter; Nango, Eriko; Iwata, So; Chapman, Henry N; Fromme, Petra; Frank, Matthias; Abela, Rafael; Boutet, Sébastien; Barty, Anton; White, Thomas A; Weierstall, Uwe; Spence, John; Neutze, Richard; Schertler, Gebhard; Standfuss, Jörg

    2016-08-22

    Serial femtosecond crystallography (SFX) using X-ray free-electron laser sources is an emerging method with considerable potential for time-resolved pump-probe experiments. Here we present a lipidic cubic phase SFX structure of the light-driven proton pump bacteriorhodopsin (bR) to 2.3 Å resolution and a method to investigate protein dynamics with modest sample requirement. Time-resolved SFX (TR-SFX) with a pump-probe delay of 1 ms yields difference Fourier maps compatible with the dark to M state transition of bR. Importantly, the method is very sample efficient and reduces sample consumption to about 1 mg per collected time point. Accumulation of M intermediate within the crystal lattice is confirmed by time-resolved visible absorption spectroscopy. This study provides an important step towards characterizing the complete photocycle dynamics of retinal proteins and demonstrates the feasibility of a sample efficient viscous medium jet for TR-SFX.

  8. Controlled ionic condensation at the surface of a native extremophile membrane

    Science.gov (United States)

    Contera, Sonia Antoranz; Voïtchovsky, Kislon; Ryan, John F.

    2010-02-01

    At the nanoscale level biological membranes present a complex interface with the solvent. The functional dynamics and relative flexibility of membrane components together with the presence of specific ionic effects can combine to create exciting new phenomena that challenge traditional theories such as the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory or models interpreting the role of ions in terms of their ability to structure water (structure making/breaking). Here we investigate ionic effects at the surface of a highly charged extremophile membrane composed of a proton pump (bacteriorhodopsin) and archaeal lipids naturally assembled into a 2D crystal. Using amplitude-modulation atomic force microscopy (AM-AFM) in solution, we obtained sub-molecular resolution images of ion-induced surface restructuring of the membrane. We demonstrate the presence of a stiff cationic layer condensed at its extracellular surface. This layer cannot be explained by traditional continuum theories. Dynamic force spectroscopy experiments suggest that it is produced by electrostatic correlation mediated by a Manning-type condensation of ions. In contrast, the cytoplasmic surface is dominated by short-range repulsive hydration forces. These findings are relevant to archaeal bioenergetics and halophilic adaptation. Importantly, they present experimental evidence of a natural system that locally controls its interactions with the surrounding medium and challenges our current understanding of biological interfaces.At the nanoscale level biological membranes present a complex interface with the solvent. The functional dynamics and relative flexibility of membrane components together with the presence of specific ionic effects can combine to create exciting new phenomena that challenge traditional theories such as the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory or models interpreting the role of ions in terms of their ability to structure water (structure making/breaking). Here we

  9. Proteins as "dopable" bio-electronic materials

    Science.gov (United States)

    Cahen, David

    2013-02-01

    Proteins are surprisingly good solid-state electronic conductors. This holds also for proteins without any known biological electron transfer function. How do they do it? To answer this question we measure solid-state electron transport (ETp) across proteins that are "dry" (only tightly bound water, to retain the conformation, still present). We compare results for the electron transfer (ET) protein, Azurin (Az), the proton-pumping membrane protein Bacteriorhodopsin (bR), and for Human and Bovine Serum Albumin (HSA and BSA). Clear differences between these proteins are seen, which preserve their structure in the solid state measurement configuration. Importantly for future bioelectronics, the results are sensitive to protein modification, e.g., removing or disconnecting the retinal in bR and removing or replacing the Cu redox centre in Az. These cofactors can thus be viewed as natural dopants for proteins. Insight in the ETp mechanism comes from temperature-dependent studies. Az shows 40-360K temperature-independent ETp across its 3.5 nm long axis, until its denaturation temperature, indicative of tunneling. Cu removal, replacement (by Zn) or deuteration changes this to thermally activated ETp. This suggests hopping and involvement of the amide backbone in the ETp. The latter, which rhymes with indications from ETp experiments on oligopeptide and simulations of ET in proteins, opens the way for modeling what otherwise is an awfully complex system. Below 200K all proteins and their variants show temperature-independent ETp. We can furthermore make a totally electrically inactive protein, HSA, into an efficient ETp medium by doping it with natural poly-ene. Putting our data in perspective by comparing them to all known protein ETp data in the literature, we conclude that, in general, proteins are well described as dopable molecular wires.

  10. Detection of fast light-activated H+ release and M intermediate formation from proteorhodopsin.

    Directory of Open Access Journals (Sweden)

    DeVita Anne

    2002-04-01

    Full Text Available Abstract Background Proteorhodopsin (pR is a light-activated proton pump homologous to bacteriorhodopsin and recently discovered in oceanic γ-proteobacteria. One perplexing difference between these two proteins is the absence in pR of homologues of bR residues Glu-194 and Glu-204. These two residues, along with Arg-82, have been implicated in light-activated fast H+ release to the extracellular medium in bR. It is therefore uncertain that pR carries out its physiological activity using a mechanism that is completely homologous to that of bR. Results A pR purification procedure is described that utilizes Phenylsepharose™ and hydroxylapatite columns and yields 85% (w/w purity. Through SDS-PAGE of the pure protein, the molecular weight of E.-coli-produced pR was determined to be 36,000, approximately 9,000 more than the 27,000 predicted by the DNA sequence. Post-translational modification of one or more of the cysteine residues accounts for 5 kDa of the weight difference as measured on a cys-less pR mutant. At pH 9.5 and in the presence of octylglucoside and diheptanoylphosphotidylcholine, flash photolysis results in fast H+ release and a 400-nm absorbing (M-like photoproduct. Both of these occur with a similar rise time (4–10 μs as reported for monomeric bR in detergent. Conclusions The presence of fast H+ release in pR indicates that either different groups are responsible for fast H+ release in pR and bR (i.e. that the H+ release group is not highly conserved; or, that the H+ release group is conserved and is therefore likely Arg-94 itself in pR (and Arg-82 in bR, correspondingly.

  11. Assembly of transmembrane helices of simple polytopic membrane proteins from sequence conservation patterns.

    Science.gov (United States)

    Park, Yungki; Helms, Volkhard

    2006-09-01

    The transmembrane (TM) domains of most membrane proteins consist of helix bundles. The seemingly simple task of TM helix bundle assembly has turned out to be extremely difficult. This is true even for simple TM helix bundle proteins, i.e., those that have the simple form of compact TM helix bundles. Herein, we present a computational method that is capable of generating native-like structural models for simple TM helix bundle proteins having modest numbers of TM helices based on sequence conservation patterns. Thus, the only requirement for our method is the presence of more than 30 homologous sequences for an accurate extraction of sequence conservation patterns. The prediction method first computes a number of representative well-packed conformations for each pair of contacting TM helices, and then a library of tertiary folds is generated by overlaying overlapping TM helices of the representative conformations. This library is scored using sequence conservation patterns, and a subsequent clustering analysis yields five final models. Assuming that neighboring TM helices in the sequence contact each other (but not that TM helices A and G contact each other), the method produced structural models of Calpha atom root-mean-square deviation (CA RMSD) of 3-5 A from corresponding crystal structures for bacteriorhodopsin, halorhodopsin, sensory rhodopsin II, and rhodopsin. In blind predictions, this type of contact knowledge is not available. Mimicking this, predictions were made for the rotor of the V-type Na(+)-adenosine triphosphatase without such knowledge. The CA RMSD between the best model and its crystal structure is only 3.4 A, and its contact accuracy reaches 55%. Furthermore, the model correctly identifies the binding pocket for sodium ion. These results demonstrate that the method can be readily applied to ab initio structure prediction of simple TM helix bundle proteins having modest numbers of TM helices.

  12. Cation-Specific Conformations in a Dual-Function Ion-Pumping Microbial Rhodopsin.

    Science.gov (United States)

    da Silva, Giordano F Z; Goblirsch, Brandon R; Tsai, Ah-Lim; Spudich, John L

    2015-06-30

    A recently discovered rhodopsin ion pump (DeNaR, also known as KR2) in the marine bacterium Dokdonia eikasta uses light to pump protons or sodium ions from the cell depending on the ionic composition of the medium. In cells suspended in a KCl solution, DeNaR functions as a light-driven proton pump, whereas in a NaCl solution, DeNaR conducts light-driven sodium ion pumping, a novel activity within the rhodopsin family. These two distinct functions raise the questions of whether the conformations of the protein differ in the presence of K(+) or Na(+) and whether the helical movements that result in the canonical E → C conformational change in other microbial rhodopsins are conserved in DeNaR. Visible absorption maxima of DeNaR in its unphotolyzed (dark) state show an 8 nm difference between Na(+) and K(+) in decyl maltopyranoside micelles, indicating an influence of the cations on the retinylidene photoactive site. In addition, electronic paramagnetic resonance (EPR) spectra of the dark states reveal repositioning of helices F and G when K(+) is replaced with Na(+). Furthermore, the conformational changes assessed by EPR spin-spin dipolar coupling show that the light-induced transmembrane helix movements are very similar to those found in bacteriorhodopsin but are altered by the presence of Na(+), resulting in a new feature, the clockwise rotation of helix F. The results establish the first observation of a cation switch controlling the conformations of a microbial rhodopsin and indicate specific interactions of Na(+) with the half-channels of DeNaR to open an appropriate path for ion translocation.

  13. Exploring the active site structure of photoreceptor proteins by Raman optical activity

    Science.gov (United States)

    Unno, Masashi

    2015-03-01

    Understanding protein function at the atomic level is a major challenge in a field of biophysics and requires the combined efforts of structural and functional methods. We use photoreceptor proteins as a model system to understand in atomic detail how a chromophore and a protein interact to sense light and send a biological signal. A potential technique for investigating molecular structures is Raman optical activity (ROA), which is a spectroscopic method with a high sensitivity to the structural details of chiral molecules. However, its application to photoreceptor proteins has not been reported. Thus we have constructed ROA spectrometer using near-infrared (NIR) laser excitation at 785 nm. The NIR excitation enables us to measure ROA spectra for a variety of biological samples, including photoreceptor proteins, without fluorescence from the samples. In the present study, we have applied the NIR-ROA to bacteriorhodopsin (BR) and photoactive yellow protein (PYP). BR is a light-driven proton pump and contains a protonated Schiff base of retinal as a chromophore. PYP is a blue light receptor, and this protein has the 4-hydroxycinnamyl chromophore, which is covalently linked to Cys69 through a thiolester bond. We have successfully obtained the ROA spectra of the chromophore within a protein environment. Furthermore, calculations of the ROA spectra utilizing density functional theory provide detailed structural information, such as data on out-of-plane distortions of the chromophore. The structural information obtained from the ROA spectra includes the positions of hydrogen atoms, which are usually not detected in the crystal structures of biological samples.

  14. A molecular dynamics approach to receptor mapping: application to the 5HT3 and beta 2-adrenergic receptors.

    Science.gov (United States)

    Gouldson, P R; Winn, P J; Reynolds, C A

    1995-09-29

    A molecular dynamics-based approach to receptor mapping is proposed, based on the method of Rizzi (Rizzi, J. P.; et al. J. Med. Chem. 1990, 33, 2721). In Rizzi's method, the interaction energy between a series of drug molecules and probe atoms (which mimic functional groups on the receptor, such as hydrogen bond donors) was calculated. These interactions were calculated on a three-dimensional grid within a molecular mechanics parameters, were placed at these minima. The distances between the dummy atom sites were monitored during molecular dynamics simulations and plotted as distance distribution functions. Important distances within the receptor became apparent, as drugs with a common mode of binding share similar peaks in the distance distribution functions. In the case of specific 5HT3 ligands, the important donor--acceptor distance within the receptor has a range of ca. 7.9--8.9 A. In the case of specific beta 2-adrenergic ligands, the important donor--acceptor distances within the receptor lie between ca. 7--9 A and between 8 and 10 A. These distances distribution functions were used to assess three different models of the beta 2-adrenergic G-protein-coupled receptor. The comparison of the distance distribution functions for the simulation with the actual donor--acceptor distances in the receptor models suggested that two of the three receptor models were much more consistent with the receptor-mapping studies. These receptor-mapping studies gave support for the use of rhodopsin, rather than the bacteriorhodopsin template, for modeling G-protein-coupled receptors but also sounded a warning that agreement with binding data from site-directed mutagenesis experiments does not necessarily validate a receptor model.

  15. Dynamical Coupling of Intrinsically Disordered Proteins and Their Hydration Water: Comparison with Folded Soluble and Membrane Proteins

    Science.gov (United States)

    Gallat, F.-X.; Laganowsky, A.; Wood, K.; Gabel, F.; van Eijck, L.; Wuttke, J.; Moulin, M.; Härtlein, M.; Eisenberg, D.; Colletier, J.-P.; Zaccai, G.; Weik, M.

    2012-01-01

    Hydration water is vital for various macromolecular biological activities, such as specific ligand recognition, enzyme activity, response to receptor binding, and energy transduction. Without hydration water, proteins would not fold correctly and would lack the conformational flexibility that animates their three-dimensional structures. Motions in globular, soluble proteins are thought to be governed to a certain extent by hydration-water dynamics, yet it is not known whether this relationship holds true for other protein classes in general and whether, in turn, the structural nature of a protein also influences water motions. Here, we provide insight into the coupling between hydration-water dynamics and atomic motions in intrinsically disordered proteins (IDP), a largely unexplored class of proteins that, in contrast to folded proteins, lack a well-defined three-dimensional structure. We investigated the human IDP tau, which is involved in the pathogenic processes accompanying Alzheimer disease. Combining neutron scattering and protein perdeuteration, we found similar atomic mean-square displacements over a large temperature range for the tau protein and its hydration water, indicating intimate coupling between them. This is in contrast to the behavior of folded proteins of similar molecular weight, such as the globular, soluble maltose-binding protein and the membrane protein bacteriorhodopsin, which display moderate to weak coupling, respectively. The extracted mean square displacements also reveal a greater motional flexibility of IDP compared with globular, folded proteins and more restricted water motions on the IDP surface. The results provide evidence that protein and hydration-water motions mutually affect and shape each other, and that there is a gradient of coupling across different protein classes that may play a functional role in macromolecular activity in a cellular context. PMID:22828339

  16. Optical protein modulation via quantum dot coupling and use of a hybrid sensor protein.

    Science.gov (United States)

    Griep, Mark; Winder, Eric; Lueking, Donald; Friedrich, Craig; Mallick, Govind; Karna, Shashi

    2010-09-01

    Harnessing the energy transfer interactions between the optical protein bacteriorhodopsin (bR) and CdSe/ZnS quantum dots (QDs) could provide a novel bio-nano electronics substrate with a variety of applications. In the present study, a polydimethyldiallyammonium chloride based I-SAM technique has been utilized to produce bilayers, trilayers and multilayers of alternating monolayers of bR, PDAC and QD's on a conductive ITO substrate. The construction of multilayer systems was directly monitored by measuring the unique A570 nm absorbance of bR, as well as QD fluorescence emission. Both of these parameters displayed a linear relationship to the number of monolayers present on the ITO substrate. The photovoltaic response of bilayers of bR/PDAC was observed over a range of 3 to 12 bilayers and the ability to efficiently create an electrically active multilayered substrate composed of bR and QDs has been demonstrated for the first time. Evaluation of QD fluorescence emission in the multilayer system strongly suggests that FRET coupling is occurring and, since the I-SAM technique provide a means to control the bR/QD separation distance on the nanometer scale, this technique may prove highly valuable for optimizing the distance dependent energy transfer effects for maximum sensitivity to target molecule binding by a biosensor. Finally, preliminary studies on the production of a sensor protein/bR hybrid gene construct are presented. It is proposed that the energy associated with target molecule binding to a hybrid sensor protein would provide a means to directly modulate the electrical output from a sensor protein/bR biosensor platform.

  17. Proton transfer from the bulk to the bound ubiquinone QB of the reaction center in chromatophores of Rhodobacter sphaeroides: Retarded conveyance by neutral water

    Science.gov (United States)

    Gopta, Oksana A.; Cherepanov, Dmitry A.; Junge, Wolfgang; Mulkidjanian, Armen Y.

    1999-01-01

    The mechanism of proton transfer from the bulk into the membrane protein interior was studied. The light-induced reduction of a bound ubiquinone molecule QB by the photosynthetic reaction center is accompanied by proton trapping. We used kinetic spectroscopy to measure (i) the electron transfer to QB (at 450 nm), (ii) the electrogenic proton delivery from the surface to the QB site (by electrochromic carotenoid response at 524 nm), and (iii) the disappearance of protons from the bulk solution (by pH indicators). The electron transfer to QB− and the proton-related electrogenesis proceeded with the same time constant of ≈100 μs (at pH 6.2), whereas the alkalinization in the bulk was distinctly delayed (τ ≈ 400 μs). We investigated the latter reaction as a function of the pH indicator concentration, the added pH buffers, and the temperature. The results led us to the following conclusions: (i) proton transfer from the surface-located acidic groups into the QB site followed the reduction of QB without measurable delay; (ii) the reprotonation of these surface groups by pH indicators and hydronium ions was impeded, supposedly, because of their slow diffusion in the surface water layer; and (iii) as a result, the protons were slowly donated by neutral water to refill the proton vacancies at the surface. It is conceivable that the same mechanism accounts for the delayed relaxation of the surface pH changes into the bulk observed previously with bacteriorhodopsin membranes and thylakoids. Concerning the coupling between proton pumps in bioenergetic membranes, our results imply a tendency for the transient confinement of protons at the membrane surface. PMID:10557290

  18. Calculating absorption shifts for retinal proteins: computational challenges.

    Science.gov (United States)

    Wanko, M; Hoffmann, M; Strodel, P; Koslowski, A; Thiel, W; Neese, F; Frauenheim, T; Elstner, M

    2005-03-01

    Rhodopsins can modulate the optical properties of their chromophores over a wide range of wavelengths. The mechanism for this spectral tuning is based on the response of the retinal chromophore to external stress and the interaction with the charged, polar, and polarizable amino acids of the protein environment and is connected to its large change in dipole moment upon excitation, its large electronic polarizability, and its structural flexibility. In this work, we investigate the accuracy of computational approaches for modeling changes in absorption energies with respect to changes in geometry and applied external electric fields. We illustrate the high sensitivity of absorption energies on the ground-state structure of retinal, which varies significantly with the computational method used for geometry optimization. The response to external fields, in particular to point charges which model the protein environment in combined quantum mechanical/molecular mechanical (QM/MM) applications, is a crucial feature, which is not properly represented by previously used methods, such as time-dependent density functional theory (TDDFT), complete active space self-consistent field (CASSCF), and Hartree-Fock (HF) or semiempirical configuration interaction singles (CIS). This is discussed in detail for bacteriorhodopsin (bR), a protein which blue-shifts retinal gas-phase excitation energy by about 0.5 eV. As a result of this study, we propose a procedure which combines structure optimization or molecular dynamics simulation using DFT methods with a semiempirical or ab initio multireference configuration interaction treatment of the excitation energies. Using a conventional QM/MM point charge representation of the protein environment, we obtain an absorption energy for bR of 2.34 eV. This result is already close to the experimental value of 2.18 eV, even without considering the effects of protein polarization, differential dispersion, and conformational sampling.

  19. Modeling the endosomal escape of cell-penetrating peptides using a transmembrane pH gradient.

    Science.gov (United States)

    Madani, Fatemeh; Abdo, Rania; Lindberg, Staffan; Hirose, Hisaaki; Futaki, Shiroh; Langel, Ulo; Gräslund, Astrid

    2013-04-01

    Cell-penetrating peptides (CPPs) can internalize into cells with covalently or non-covalently bound biologically active cargo molecules, which by themselves are not able to pass the cell membrane. Direct penetration and endocytosis are two main pathways suggested for the cellular uptake of CPPs. Cargo molecules which have entered the cell via an endocytotic pathway must be released from the endosome before degradation by enzymatic processes and endosomal acidification. Endosomal entrapment seems to be a major limitation in delivery of these molecules into the cytoplasm. Bacteriorhodopsin (BR) asymmetrically introduced into large unilamellar vesicles (LUVs) was used to induce a pH gradient across the lipid bilayer. By measuring pH outside the LUVs, we observed light-induced proton pumping mediated by BR from the outside to the inside of the LUVs, creating an acidic pH inside the LUVs, similar to the late endosomes in vivo. Here we studied the background mechanism(s) of endosomal escape. 20% negatively charged LUVs were used as model endosomes with incorporated BR into the membrane and fluorescein-labeled CPPs entrapped inside the LUVs, together with a fluorescence quencher. The translocation of different CPPs in the presence of a pH gradient across the membrane was studied. The results show that the light-induced pH gradient induced by BR facilitates vesicle membrane translocation, particularly for the intermediately hydrophobic CPPs, and much less for hydrophilic CPPs. The presence of chloroquine inside the LUVs or addition of pyrenebutyrate outside the LUVs destabilizes the vesicle membrane, resulting in significant changes of the pH gradient across the membrane.

  20. Microarray analysis in the archaeon Halobacterium salinarum strain R1.

    Directory of Open Access Journals (Sweden)

    Jens Twellmeyer

    Full Text Available BACKGROUND: Phototrophy of the extremely halophilic archaeon Halobacterium salinarum was explored for decades. The research was mainly focused on the expression of bacteriorhodopsin and its functional properties. In contrast, less is known about genome wide transcriptional changes and their impact on the physiological adaptation to phototrophy. The tool of choice to record transcriptional profiles is the DNA microarray technique. However, the technique is still rarely used for transcriptome analysis in archaea. METHODOLOGY/PRINCIPAL FINDINGS: We developed a whole-genome DNA microarray based on our sequence data of the Hbt. salinarum strain R1 genome. The potential of our tool is exemplified by the comparison of cells growing under aerobic and phototrophic conditions, respectively. We processed the raw fluorescence data by several stringent filtering steps and a subsequent MAANOVA analysis. The study revealed a lot of transcriptional differences between the two cell states. We found that the transcriptional changes were relatively weak, though significant. Finally, the DNA microarray data were independently verified by a real-time PCR analysis. CONCLUSION/SIGNIFICANCE: This is the first DNA microarray analysis of Hbt. salinarum cells that were actually grown under phototrophic conditions. By comparing the transcriptomics data with current knowledge we could show that our DNA microarray tool is well applicable for transcriptome analysis in the extremely halophilic archaeon Hbt. salinarum. The reliability of our tool is based on both the high-quality array of DNA probes and the stringent data handling including MAANOVA analysis. Among the regulated genes more than 50% had unknown functions. This underlines the fact that haloarchaeal phototrophy is still far away from being completely understood. Hence, the data recorded in this study will be subject to future systems biology analysis.

  1. Molecular mechanisms for generating transmembrane proton gradients.

    Science.gov (United States)

    Gunner, M R; Amin, Muhamed; Zhu, Xuyu; Lu, Jianxun

    2013-01-01

    Membrane proteins use the energy of light or high energy substrates to build a transmembrane proton gradient through a series of reactions leading to proton release into the lower pH compartment (P-side) and proton uptake from the higher pH compartment (N-side). This review considers how the proton affinity of the substrates, cofactors and amino acids are modified in four proteins to drive proton transfers. Bacterial reaction centers (RCs) and photosystem II (PSII) carry out redox chemistry with the species to be oxidized on the P-side while reduction occurs on the N-side of the membrane. Terminal redox cofactors are used which have pKas that are strongly dependent on their redox state, so that protons are lost on oxidation and gained on reduction. Bacteriorhodopsin is a true proton pump. Light activation triggers trans to cis isomerization of a bound retinal. Strong electrostatic interactions within clusters of amino acids are modified by the conformational changes initiated by retinal motion leading to changes in proton affinity, driving transmembrane proton transfer. Cytochrome c oxidase (CcO) catalyzes the reduction of O2 to water. The protons needed for chemistry are bound from the N-side. The reduction chemistry also drives proton pumping from N- to P-side. Overall, in CcO the uptake of 4 electrons to reduce O2 transports 8 charges across the membrane, with each reduction fully coupled to removal of two protons from the N-side, the delivery of one for chemistry and transport of the other to the P-side.

  2. Proton transfers in a channelrhodopsin-1 studied by Fourier transform infrared (FTIR) difference spectroscopy and site-directed mutagenesis.

    Science.gov (United States)

    Ogren, John I; Yi, Adrian; Mamaev, Sergey; Li, Hai; Spudich, John L; Rothschild, Kenneth J

    2015-05-15

    Channelrhodopsin-1 from the alga Chlamydomonas augustae (CaChR1) is a low-efficiency light-activated cation channel that exhibits properties useful for optogenetic applications such as a slow light inactivation and a red-shifted visible absorption maximum as compared with the more extensively studied channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Previously, both resonance Raman and low-temperature FTIR difference spectroscopy revealed that unlike CrChR2, CaChR1 under our conditions exhibits an almost pure all-trans retinal composition in the unphotolyzed ground state and undergoes an all-trans to 13-cis isomerization during the primary phototransition typical of other microbial rhodopsins such as bacteriorhodopsin (BR). Here, we apply static and rapid-scan FTIR difference spectroscopy along with site-directed mutagenesis to characterize the proton transfer events occurring upon the formation of the long-lived conducting P2 (380) state of CaChR1. Assignment of carboxylic C=O stretch bands indicates that Asp-299 (homolog to Asp-212 in BR) becomes protonated and Asp-169 (homolog to Asp-85 in BR) undergoes a net change in hydrogen bonding relative to the unphotolyzed ground state of CaChR1. These data along with earlier FTIR measurements on the CaChR1 → P1 transition are consistent with a two-step proton relay mechanism that transfers a proton from Glu-169 to Asp-299 during the primary phototransition and from the Schiff base to Glu-169 during P2 (380) formation. The unusual charge neutrality of both Schiff base counterions in the P2 (380) conducting state suggests that these residues may function as part of a cation selective filter in the open channel state of CaChR1 as well as other low-efficiency ChRs.

  3. Asymmetric Functional Conversion of Eubacterial Light-driven Ion Pumps.

    Science.gov (United States)

    Inoue, Keiichi; Nomura, Yurika; Kandori, Hideki

    2016-05-06

    In addition to the well-known light-driven outward proton pumps, novel ion-pumping rhodopsins functioning as outward Na(+) and inward Cl(-) pumps have been recently found in eubacteria. They convert light energy into transmembrane electrochemical potential difference, similar to the prototypical archaeal H(+) pump bacteriorhodopsin (BR) and Cl(-) pump halorhodopsin (HR). The H(+), Na(+), and Cl(-) pumps possess the conserved respective DTE, NDQ, and NTQ motifs in the helix C, which likely serve as their functional determinants. To verify this hypothesis, we attempted functional interconversion between selected pumps from each category by mutagenesis. Introduction of the proton-pumping motif resulted in successful Na(+) → H(+) functional conversion. Introduction of the respective characteristic motifs with several additional mutations leads to successful Na(+) → Cl(-) and Cl(-) → H(+) functional conversions, whereas remaining conversions (H(+) → Na(+), H(+) → Cl(-), Cl(-) → Na(+)) were unsuccessful when mutagenesis of 4-6 residues was used. Phylogenetic analysis suggests that a H(+) pump is the common ancestor of all of these rhodopsins, from which Cl(-) pumps emerged followed by Na(+) pumps. We propose that successful functional conversions of these ion pumps are achieved exclusively when mutagenesis reverses the evolutionary amino acid sequence changes. Dependence of the observed functional conversions on the direction of evolution strongly suggests that the essential structural mechanism of an ancestral function is retained even after the gain of a new function during natural evolution, which can be evoked by a few mutations. By contrast, the gain of a new function needs accumulation of multiple mutations, which may not be easily reproduced by limited mutagenesis in vitro.

  4. Subpicosecond protein backbone changes detected during the green-absorbing proteorhodopsin primary photoreaction.

    Science.gov (United States)

    Amsden, Jason J; Kralj, Joel M; Chieffo, Logan R; Wang, Xihua; Erramilli, Shyamsunder; Spudich, Elena N; Spudich, John L; Ziegler, Lawrence D; Rothschild, Kenneth J

    2007-10-11

    Recent studies demonstrate that photoactive proteins can react within several picoseconds to photon absorption by their chromophores. Faster subpicosecond protein responses have been suggested to occur in rhodopsin-like proteins where retinal photoisomerization may impulsively drive structural changes in nearby protein groups. Here, we test this possibility by investigating the earliest protein structural changes occurring in proteorhodopsin (PR) using ultrafast transient infrared (TIR) spectroscopy with approximately 200 fs time resolution combined with nonperturbing isotope labeling. PR is a recently discovered microbial rhodopsin similar to bacteriorhodopsin (BR) found in marine proteobacteria and functions as a proton pump. Vibrational bands in the retinal fingerprint (1175-1215 cm(-1)) and ethylenic stretching (1500-1570 cm(-1)) regions characteristic of all-trans to 13-cis chromophore isomerization and formation of a red-shifted photointermediate appear with a 500-700 fs time constant after photoexcitation. Bands characteristic of partial return to the ground state evolve with a 2.0-3.5 ps time constant. In addition, a negative band appears at 1548 cm(-1) with a time constant of 500-700 fs, which on the basis of total-15N and retinal C15D (retinal with a deuterium on carbon 15) isotope labeling is assigned to an amide II peptide backbone mode that shifts to near 1538 cm(-1) concomitantly with chromophore isomerization. Our results demonstrate that one or more peptide backbone groups in PR respond with a time constant of 500-700 fs, almost coincident with the light-driven retinylidene chromophore isomerization. The protein changes we observe on a subpicosecond time scale may be involved in storage of the absorbed photon energy subsequently utilized for proton transport.

  5. Electrogenic steps of light-driven proton transport in ESR, a retinal protein from Exiguobacterium sibiricum.

    Science.gov (United States)

    Siletsky, Sergey A; Mamedov, Mahir D; Lukashev, Evgeniy P; Balashov, Sergei P; Dolgikh, Dmitriy A; Rubin, Andrei B; Kirpichnikov, Mikhail P; Petrovskaya, Lada E

    2016-11-01

    A retinal protein from Exiguobacterium sibiricum (ESR) functions as a light-driven proton pump. Unlike other proton pumps, it contains Lys96 instead of a usual carboxylic residue in the internal proton donor site. Nevertheless, the reprotonation of the Schiff base occurs fast, indicating that Lys96 facilitates proton transfer from the bulk. In this study we examined kinetics of light-induced transmembrane electrical potential difference, ΔΨ, generated in proteoliposomes reconstituted with ESR. We show that total magnitude of ΔΨ is comparable to that produced by bacteriorhodopsin but its kinetic components and their pH dependence are substantially different. The results are in agreement with the earlier finding that proton uptake precedes reprotonation of the Schiff base in ESR, suggesting that Lys96 is unprotonated in the initial state and gains a proton transiently in the photocycle. The electrogenic phases and the photocycle transitions related to proton transfer from the bulk to the Schiff base are pH dependent. At neutral pH, they occur with τ 0.5ms and 4.5ms. At alkaline pH, the fast component ceases and Schiff base reprotonation slows. At pH8.4, a spectrally silent electrogenic component with τ 0.25ms is detected, which can be attributed to proton transfer from the bulk to Lys96. At pH5.1, the amplitude of ΔΨ decreases 10 fold, reflecting a decreased yield and rate of proton transfer, apparently from protonation of the acceptor (Asp85-His57 pair) in the initial state. The features of the photoelectric potential generation correlate with the ESR structure and proposed mechanism of proton transfer.

  6. Lipid-protein nanodiscs promote in vitro folding of transmembrane domains of multi-helical and multimeric membrane proteins.

    Science.gov (United States)

    Shenkarev, Zakhar O; Lyukmanova, Ekaterina N; Butenko, Ivan O; Petrovskaya, Lada E; Paramonov, Alexander S; Shulepko, Mikhail A; Nekrasova, Oksana V; Kirpichnikov, Mikhail P; Arseniev, Alexander S

    2013-02-01

    Production of helical integral membrane proteins (IMPs) in a folded state is a necessary prerequisite for their functional and structural studies. In many cases large-scale expression of IMPs in cell-based and cell-free systems results in misfolded proteins, which should be refolded in vitro. Here using examples of the bacteriorhodopsin ESR from Exiguobacterium sibiricum and full-length homotetrameric K(+) channel KcsA from Streptomyces lividans we found that the efficient in vitro folding of the transmembrane domains of the polytopic and multimeric IMPs could be achieved during the protein encapsulation into the reconstructed high-density lipoprotein particles, also known as lipid-protein nanodiscs. In this case the self-assembly of the IMP/nanodisc complexes from a mixture containing apolipoprotein, lipids and the partially denatured protein solubilized in a harsh detergent induces the folding of the transmembrane domains. The obtained folding yields showed significant dependence on the properties of lipids used for nanodisc formation. The largest recovery of the spectroscopically active ESR (~60%) from the sodium dodecyl sulfate (SDS) was achieved in the nanodiscs containing anionic saturated lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPG) and was approximately twice lower in the zwitterionic DMPC lipid. The reassembly of tetrameric KcsA from the acid-dissociated monomer solubilized in SDS was the most efficient (~80%) in the nanodiscs containing zwitterionic unsaturated lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The charged and saturated lipids provided lower tetramer quantities, and the lowest yield (<20%) was observed in DMPC. The overall yield of the ESR and KcsA folding was mainly restricted by the efficiency of the protein encapsulation into the nanodiscs.

  7. ESR - a retinal protein with unusual properties from Exiguobacterium sibiricum.

    Science.gov (United States)

    Petrovskaya, L E; Balashov, S P; Lukashev, E P; Imasheva, E S; Gushchin, I Yu; Dioumaev, A K; Rubin, A B; Dolgikh, D A; Gordeliy, V I; Lanyi, J K; Kirpichnikov, M P

    2015-06-01

    This review covers the properties of a retinal protein (ESR) from the psychrotrophic bacterium Exiguobacterium sibiricum that functions as a light-driven proton pump. The presence of a lysine residue at the position corresponding to intramolecular proton donor for the Schiff base represents a unique structural feature of ESR. We have shown that Lys96 successfully facilitates delivery of protons from the cytoplasmic surface to the Schiff base, thus acting as a proton donor in ESR. Since proton uptake during the photocycle precedes Schiff base reprotonation, we conclude that this residue is initially in the uncharged state and acquires a proton for a short time after Schiff base deprotonation and M intermediate formation. Involvement of Lys as a proton donor distinguishes ESR from the related retinal proteins - bacteriorhodopsin (BR), proteorhodopsin (PR), and xanthorhodopsin (XR), in which the donor function is performed by residues with a carboxyl side chain. Like other eubacterial proton pumps (PR and XR), ESR contains a histidine residue interacting with the proton acceptor Asp85. In contrast to PR, this interaction leads to shift of the acceptor's pKa to more acidic pH, thus providing its ability to function over a wide pH range. The presence of a strong H-bond between Asp85 and His57, the structure of the proton-conducting pathways from cytoplasmic surface to the Schiff base and to extracellular surface, and other properties of ESR were demonstrated by solving its three-dimensional structure, which revealed several differences from known structures of BR and XR. The structure of ESR, its photocycle, and proton transfer reactions are discussed in comparison with homologous retinal proteins.

  8. Lipid-protein nanodiscs for cell-free production of integral membrane proteins in a soluble and folded state: comparison with detergent micelles, bicelles and liposomes.

    Science.gov (United States)

    Lyukmanova, E N; Shenkarev, Z O; Khabibullina, N F; Kopeina, G S; Shulepko, M A; Paramonov, A S; Mineev, K S; Tikhonov, R V; Shingarova, L N; Petrovskaya, L E; Dolgikh, D A; Arseniev, A S; Kirpichnikov, M P

    2012-03-01

    Production of integral membrane proteins (IMPs) in a folded state is a key prerequisite for their functional and structural studies. In cell-free (CF) expression systems membrane mimicking components could be added to the reaction mixture that promotes IMP production in a soluble form. Here lipid-protein nanodiscs (LPNs) of different lipid compositions (DMPC, DMPG, POPC, POPC/DOPG) have been compared with classical membrane mimicking media such as detergent micelles, lipid/detergent bicelles and liposomes by their ability to support CF synthesis of IMPs in a folded and soluble state. Three model membrane proteins of different topology were used: homodimeric transmembrane (TM) domain of human receptor tyrosine kinase ErbB3 (TM-ErbB3, 1TM); voltage-sensing domain of K(+) channel KvAP (VSD, 4TM); and bacteriorhodopsin from Exiguobacterium sibiricum (ESR, 7TM). Structural and/or functional properties of the synthesized proteins were analyzed. LPNs significantly enhanced synthesis of the IMPs in a soluble form regardless of the lipid composition. A partial disintegration of LPNs composed of unsaturated lipids was observed upon co-translational IMP incorporation. Contrary to detergents the nanodiscs resulted in the synthesis of ~80% active ESR and promoted correct folding of the TM-ErbB3. None of the tested membrane mimetics supported CF synthesis of correctly folded VSD, and the protocol of the domain refolding was developed. The use of LPNs appears to be the most promising approach to CF production of IMPs in a folded state. NMR analysis of (15)N-Ile-TM-ErbB3 co-translationally incorporated into LPNs shows the great prospects of this membrane mimetics for structural studies of IMPs produced by CF systems.

  9. Nonionic homopolymeric amphipols: application to membrane protein folding, cell-free synthesis, and solution nuclear magnetic resonance.

    Science.gov (United States)

    Bazzacco, Paola; Billon-Denis, Emmanuelle; Sharma, K Shivaji; Catoire, Laurent J; Mary, Sophie; Le Bon, Christel; Point, Elodie; Banères, Jean-Louis; Durand, Grégory; Zito, Francesca; Pucci, Bernard; Popot, Jean-Luc

    2012-02-21

    Nonionic amphipols (NAPols) synthesized by homotelomerization of an amphiphatic monomer are able to keep membrane proteins (MPs) stable and functional in the absence of detergent. Some of their biochemical and biophysical properties and applications have been examined, with particular attention being paid to their complementarity with the classical polyacrylate-based amphipol A8-35. Bacteriorhodopsin (BR) from Halobacterium salinarum and the cytochrome b(6)f complex from Chlamydomonas reinhardtii were found to be in their native state and highly stable following complexation with NAPols. NAPol-trapped BR was shown to undergo its complete photocycle. Because of the pH insensitivity of NAPols, solution nuclear magnetic resonance (NMR) two-dimensional (1)H-(15)N heteronuclear single-quantum coherence spectra of NAPol-trapped outer MP X from Escherichia coli (OmpX) could be recorded at pH 6.8. They present a resolution similar to that of the spectra of OmpX/A8-35 complexes recorded at pH 8.0 and give access to signals from solvent-exposed rapidy exchanging amide protons. Like A8-35, NAPols can be used to fold MPs to their native state as demonstrated here with BR and with the ghrelin G protein-coupled receptor GHS-R1a, thus extending the range of accessible folding conditions. Following NAPol-assisted folding, GHS-R1a bound four of its specific ligands, recruited arrestin-2, and activated binding of GTPγS by the G(αq) protein. Finally, cell-free synthesis of MPs, which is inhibited by A8-35 and sulfonated amphipols, was found to be very efficient in the presence of NAPols. These results open broad new perspectives on the use of amphipols for MP studies.

  10. Systematic analysis of protein–detergent complexes applying dynamic light scattering to optimize solutions for crystallization trials

    Energy Technology Data Exchange (ETDEWEB)

    Meyer, Arne [University of Hamburg, c/o DESY, Building 22a, Notkestrasse 85, 22603 Hamburg (Germany); Dierks, Karsten [University of Hamburg, c/o DESY, Building 22a, Notkestrasse 85, 22603 Hamburg (Germany); XtalConcepts, Marlowring 19, 22525 Hamburg (Germany); Hussein, Rana [University of Hamburg, c/o DESY, Building 22a, Notkestrasse 85, 22603 Hamburg (Germany); Brillet, Karl [ESBS, Pôle API, 300 Boulevard Sébastien Brant, CS10413, 67412 Illkirch CEDEX (France); Brognaro, Hevila [São Paulo State University, UNESP/IBILCE, Caixa Postal 136, São José do Rio Preto-SP, 15054 (Brazil); Betzel, Christian, E-mail: christian.betzel@uni-hamburg.de [University of Hamburg, c/o DESY, Building 22a, Notkestrasse 85, 22603 Hamburg (Germany)

    2015-01-01

    Application of in situ dynamic light scattering to solutions of protein–detergent complexes permits characterization of these complexes in samples as small as 2 µl in volume. Detergents are widely used for the isolation and solubilization of membrane proteins to support crystallization and structure determination. Detergents are amphiphilic molecules that form micelles once the characteristic critical micelle concentration (CMC) is achieved and can solubilize membrane proteins by the formation of micelles around them. The results are presented of a study of micelle formation observed by in situ dynamic light-scattering (DLS) analyses performed on selected detergent solutions using a newly designed advanced hardware device. DLS was initially applied in situ to detergent samples with a total volume of approximately 2 µl. When measured with DLS, pure detergents show a monodisperse radial distribution in water at concentrations exceeding the CMC. A series of all-transn-alkyl-β-d-maltopyranosides, from n-hexyl to n-tetradecyl, were used in the investigations. The results obtained verify that the application of DLS in situ is capable of distinguishing differences in the hydrodynamic radii of micelles formed by detergents differing in length by only a single CH{sub 2} group in their aliphatic tails. Subsequently, DLS was applied to investigate the distribution of hydrodynamic radii of membrane proteins and selected water-insoluble proteins in presence of detergent micelles. The results confirm that stable protein–detergent complexes were prepared for (i) bacteriorhodopsin and (ii) FetA in complex with a ligand as examples of transmembrane proteins. A fusion of maltose-binding protein and the Duck hepatitis B virus X protein was added to this investigation as an example of a non-membrane-associated protein with low water solubility. The increased solubility of this protein in the presence of detergent could be monitored, as well as the progress of proteolytic

  11. Biosolar energy generation and harvesting from biomolecule-copolymer hybrid systems

    Science.gov (United States)

    Chu, Bong-Chieh Benjamin

    Alternative energy sources have become an increasingly important topic as energy needs outpace supply. Furthermore, as the world moves into the digital age of portable electronics, highly efficient and lightweight energy sources will need to be developed. Current technology, such as lithium ion batteries, provide enough power to run portable electronics for hours or days, but can still allow for improvement in their power density (W/kg). Utilizing energy-transducing membrane proteins, which are by nature highly efficient, it is possible to engineer biological-based energy sources with energy densities far greater than any solid-state systems. Furthermore, solar powered membrane proteins have the added benefit of a virtually unlimited supply of energy. This work has developed protein-polymer hybrid films and nanoscale vesicles for a variety of applications from fuel-cell technology to biological-based photovoltaics. Bacteriorhodopsin (BR), a light-activated proton pump, and Cytochrome C Oxidase (COX), a protein involved in the electron transport chain in mitochondria, were reconstituted into biomimetic triblock copolymer membranes. Block copolymer membranes mimic the amphiphilic nature of a natural lipid bilayer but exhibit greater mechanical stability due to UV-polymerizable endgroups. In BR/COX functionalized nanovesicles, proton gradients generated by the light-activated proton pumping of BR are used to drive COX in reverse to generate electrons, providing a hybrid biologically-active polymer to convert solar energy to chemical energy, and finally to electrical energy. This work has found protein activity in planar membranes through the photoelectric current generation by BR and the proton pumping activity of BR-functionalized polymer membranes deposited onto proton exchange membranes, as well as the coupled functionality of BR and COX through current generation in cyclic voltammetry and direct current measurements. Current switching between light and dark

  12. μ阿片受体及其与羟甲芬太尼相互作用的分子模拟%Molecular modeling of μ opioid receptor and its interaction with ohmefentanyl

    Institute of Scientific and Technical Information of China (English)

    唐赟; 陈凯先; 蒋华良; 王智贤; 嵇汝运; 池志强

    1996-01-01

    To build up the structure model of μ opioid receptor, then combined with the receptor model,to investigate the action mechanism of ohmefentanyl on the receptor. METHODS: Using the three-dimensional structure of bacteriorhodopsin as a template, we constructed μ opioid receptor model on computer. Ohmefentanyl was then docked into the supposed receptor binding sites. RESULTS: A good ligand-receptor interaction model was achieved. The possible binding sites were found to be Asp147 and His319. The protonated N atom of ohmefentanyl form potent electrostatic and hydrogen-bonding interactions with residue Asp147 of the receptor, the O atom of the carbonyl group form weak electrostatic and hydrogen-bonding interactions with residue His319, and the two phenyl groups form t-t interactions withsome aryl residues of the receptor around ligand. CONCLUSION:The ligand-receptor interaction model should be helpful for rational design of novel analgesic.%建立μ阿片受体的结构模型,并结合模型研究羟甲芬太尼对该受体的作用机制.方法:以细菌视紫红质的三维结构为模板,在计算机上建立μ阿片受体模型,然后将羟甲芬太尼对接到假想的受体结合部位.结果:得到了良好的配体-受体相互作用模型,发现残基Asp147与His319为可能的结合位点.配体质子化N原子与Asp147形成强的静电和氢键相互作用,羰基O原子与His319形成弱的静电和氢键相互作用,两个苯环分别与周围的芳香残基形成π-π相互作用.结论:该配体-受体相互作用模型有助于合理设计新型镇痛药.

  13. Alpha-helical hydrophobic polypeptides form proton-selective channels in lipid bilayers

    Science.gov (United States)

    Oliver, A. E.; Deamer, D. W.

    1994-01-01

    Proton translocation is important in membrane-mediated processes such as ATP-dependent proton pumps, ATP synthesis, bacteriorhodopsin, and cytochrome oxidase function. The fundamental mechanism, however, is poorly understood. To test the theoretical possibility that bundles of hydrophobic alpha-helices could provide a low energy pathway for ion translocation through the lipid bilayer, polyamino acids were incorporated into extruded liposomes and planar lipid membranes, and proton translocation was measured. Liposomes with incorporated long-chain poly-L-alanine or poly-L-leucine were found to have proton permeability coefficients 5 to 7 times greater than control liposomes, whereas short-chain polyamino acids had relatively little effect. Potassium permeability was not increased markedly by any of the polyamino acids tested. Analytical thin layer chromatography measurements of lipid content and a fluorescamine assay for amino acids showed that there were approximately 135 polyleucine or 65 polyalanine molecules associated with each liposome. Fourier transform infrared spectroscopy indicated that a major fraction of the long-chain hydrophobic peptides existed in an alpha-helical conformation. Single-channel recording in both 0.1 N HCl and 0.1 M KCl was also used to determine whether proton-conducting channels formed in planar lipid membranes (phosphatidylcholine/phosphatidylethanolamine, 1:1). Poly-L-leucine and poly-L-alanine in HCl caused a 10- to 30-fold increase in frequency of conductive events compared to that seen in KCl or by the other polyamino acids in either solution. This finding correlates well with the liposome observations in which these two polyamino acids caused the largest increase in membrane proton permeability but had little effect on potassium permeability. Poly-L-leucine was considerably more conductive than poly-L-alanine due primarily to larger event amplitudes and, to a lesser extent, a higher event frequency. Poly-L-leucine caused two

  14. The Use of Ultrashort Picosecond Laser Pulses to Generate Quantum Optical Properties of Single Molecules in Biophysics

    Science.gov (United States)

    Ly, Sonny

    Generation of quantum optical states from ultrashort laser-molecule interactions have led to fascinating discoveries in physics and chemistry. In recent years, these interactions have been extended to probe phenomena in single molecule biophysics. Photons emitted from a single fluorescent molecule contains important properties about how the molecule behave and function in that particular environment. Analysis of the second order coherence function through fluorescence correlation spectroscopy plays a pivotal role in quantum optics. At very short nanosecond timescales, the coherence function predicts photon antibunching, a purely quantum optical phenomena which states that a single molecule can only emit one photon at a time. Photon antibunching is the only direct proof of single molecule emission. From the nanosecond to microsecond timescale, the coherence function gives information about rotational diffusion coefficients, and at longer millisecond timescales, gives information regarding the translational diffusion coefficients. In addition, energy transfer between molecules from dipole-dipole interaction results in FRET, a highly sensitive method to probe conformational dynamics at nanometer distances. Here I apply the quantum optical techniques of photon antibunching, fluorescence correlation spectroscopy and FRET to probe how lipid nanodiscs form and function at the single molecule level. Lipid nanodiscs are particles that contain two apolipoprotein (apo) A-I circumventing a lipid bilayer in a belt conformation. From a technological point of view, nanodiscs mimics a patch of cell membrane that have recently been used to reconstitute a variety of membrane proteins including cytochrome P450 and bacteriorhodopsin. They are also potential drug transport vehicles due to its small and stable 10nm diameter size. Biologically, nanodiscs resemble to high degree, high density lipoproteins (HDL) in our body and provides a model platform to study lipid-protein interactions

  15. Acid Catalysis in Basic Solution: A Supramolecular Host PromotesOrthoformate Hydrolysis

    Energy Technology Data Exchange (ETDEWEB)

    Pluth, Michael D.; Bergman, Robert G.; Raymond, Kenneth N.

    2007-12-12

    Though many enzymes can promote chemical reactions by tuning substrate properties purely through the electrostatic environment of a docking cavity, this strategy has proven challenging to mimic in synthetic host-guest systems. Here we report a highly-charged, water soluble, metal-ligand assembly with a hydrophobic interior cavity that thermodynamically stabilizes protonated substrates and consequently catalyzes the normally acidic hydrolysis of orthoformates in basic solution, with rate accelerations of up to 890-fold. The catalysis reaction obeys Michaelis-Menten kinetics, exhibits competitive inhibition, and the substrate scope displays size selectivity consistent with the constrained binding environment of the molecular host. Synthetic chemists have long endeavored to design host molecules capable of selectively binding slow-reacting substrates and catalyzing their chemical reactions. While synthetic catalysts are often site-specific and require certain properties of the substrate to insure catalysis, enzymes are often able to modify basic properties of the bound substrate such as pK{sub a} in order to enhance reactivity. Two common motifs used by nature to activate otherwise unreactive compounds are the precise arrangement of hydrogen-bonding networks and electrostatic interactions between the substrate and adjacent residues of the protein. Precise arrangement of hydrogen bonding networks near the active sites of proteins can lead to well-tuned pK{sub a}-matching, and can result in pK{sub a} shifts of up to eight units, as shown in bacteriorhodopsin. Similarly, purely electrostatic interactions can greatly favor charged states and have been responsible for pK{sub a} shifts of up to five units for acetoacetate decarboxylase. Attempts have been made to isolate the contributions of electrostatic versus covalent interactions to such pK{sub a} shifts; however this remains a difficult challenge experimentally. This challenge emphasizes the importance of synthesizing

  16. FTIR difference and resonance Raman spectroscopy of rhodopsins with applications to optogenetics

    Science.gov (United States)

    Saint Clair, Erica C.

    The major aim of this thesis is to investigate the molecular basis for the function of several types of rhodopsins with special emphasis on their application to the new field of optogenetics. Rhodopsins are transmembrane biophotonic proteins with 7 alpha-helices and a retinal chromophore. Studies included Archaerhodopsin 3 (AR3), a light driven proton pump similar to the extensively studied bacteriorhodopsin (BR); channelrhodopsins 1 and 2, light-activated ion channels; sensory rhodopsin II (SRII), a light-sensing protein that modulates phototaxis used in archaebacteria; and squid rhodopsins (sRho), the major photopigment in squid vision and a model for human melanopsin, which controls circadian rhythms. The primary techniques used in these studies were FTIR difference spectroscopy and resonance Raman spectroscopy. These techniques, in combination with site directed mutagenesis and other biochemical methodologies produced new knowledge regarding the structural changes of the retinal chromophore, the location and function of internal water molecules as well as specific amino acids and peptide backbone. Specialized techniques were developed that allowed rhodopsins to be studied in intact membrane environments and in some cases in vivo measurements were made on rhodopsin heterologously expressed in E. coli thus allowing the effects of interacting proteins and membrane potential to be investigated. Evidence was found that the local environment of one or more internal water molecules in SRII is altered by interaction with its cognate transducer, HtrII, and is also affected by the local lipid environment. In the case of AR3, many of the broad IR continuum absorption changes below 3000 cm -1, assigned to networks of water molecules involved in proton transport through cytoplasmic and extracellular portions in BR, were found to be very similar to BR. Bands assigned to water molecules near the Schiff base postulated to be involved in proton transport were, however, shifted

  17. Linear and Nonlinear Spectroscopic Probing of Solute Interactions with Chemically Modified Silica Surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Wirth, Mary J

    2011-02-09

    Solar energy conversion through biology would provide a renewable and nonpolluting abundance of energy. The bacterium Halobacterium salinarum converts solar to electrical energy by virtue of a transmembrane protein, bacteriorhodopsin. This transmembrane protein pumps protons across a nonconducting bilayer upon irradiation with green light. The bacterium evolved to perform this function inefficiently. If we were able to understand this process to engineer this protein for efficiency, then inexpensive energy production could be achieved. There are tens of thousands of different types of halobacteria, giving the opportunity to study different efficiencies and relating these to the protein structures. Technology does not yet exist to perform such screening. The goal of this research is to generate new separation technology that can ultimately enable such screening. This involves creating a method for separating oriented and functional transmembrane proteins that remain in an electrically insulating lipid bilayer, with aqueous solutions on either side of the bilayer. A pH change across the lipid bilayer upon irradiation of a known concentration of proteins would probe function. Differences in proton pumping efficiency for different proteins variants would provide structure-function information for engineering the proteins. A schematic diagram from the original proposal is shown here. The idea is that (a) a lipid bilayer supported on a hydrophilic polymer film will make the bilayer fluid, and (b) applying an electric field will cause electrophoretic migration of the transmembrane proteins. We demonstrated this concept experimentally in a paper that was published just after this new grant period started (Lipid Bilayers on Polyacrylamide Brushes for Inclusion of Membrane Proteins, Emily A. Smith, Jason W. Coym, Scott M. Cowell, Victor J. Hruby, Henry I. Yamamura, Mary J. Wirth, Langmuir, 21, 9644-9650, 2005). The electrophoretic mobility was slow (10{sup -8} cm{sup 2}/Vs

  18. Thin films and assemblies of photosensitive membrane proteins and colloidal nanocrystals for engineering of hybrid materials with advanced properties.

    Science.gov (United States)

    Zaitsev, Sergei Yu; Solovyeva, Daria O; Nabiev, Igor

    2012-11-15

    The development and study of nano-bio hybrid materials engineered from membrane proteins (the key functional elements of various biomembranes) and nanoheterostructures (inorganic colloidal nanoparticles, transparent electrodes, and films) is a rapidly growing field at the interface of materials and life sciences. The mainspring of the development of bioinspired materials and devices is the fact that biological evolution has solved many problems similar to those that humans are attempting to solve in the field of light-harvesting and energy-transferring inorganic compounds. Along this way, bioelectronics and biophotonics have shown considerable promise. A number of proteins have been explored in terms of bioelectronic device applications, but bacteriorhodopsin (bR, a photosensitive membrane protein from purple membranes of the bacterium Halobacterium salinarum) and bacterial photosynthetic reaction centres have received the most attention. The energy harvesting in plants has a maximum efficiency of 5%, whereas bR, in the absence of a specific light-harvesting system, allows bacteria to utilize only 0.1-0.5% of the solar light. Recent nano-bioengineering approaches employing colloidal semiconductor and metal nanoparticles conjugated with biosystems permit the enhancement of the light-harvesting capacity of photosensitive proteins, thus providing a strong impetus to protein-based device optimisation. Fabrication of ultrathin and highly oriented films from biological membranes and photosensitive proteins is the key task for prospective bioelectronic and biophotonic applications. In this review, the main advances in techniques of preparation of such films are analyzed. Comparison of the techniques for obtaining thin films leads to the conclusion that the homogeneity and orientation of biomembrane fragments or proteins in these films depend on the method of their fabrication and increase in the following order: electrophoretic sedimentation assembly and layer

  19. Picosecond kinetics and Sn absorption spectra of retinoids and carotenoids

    Science.gov (United States)

    Bondarev, Stanislav L.; Tikhomirov, S. A.; Bachilo, Sergei M.

    1991-05-01

    Light energy absorption, as well as the subsequent photochemical and photophysical processes of cis -+trans isomerisation (vision and bacteriorhodopsin photosynthesis) and energy transfer (photosynthesis in green plants and micro organisms) take place in a pigment-protein complex including polyene chromophors, retinoids and carotenoids. Picosecond and subpicosecond studies of the spectral and kinetic characteristics of these processes are carried out in both complex photoreceptor and photosynthetic ms'2 and model systems with the use of solutions of retinoids and carotenoids.36 The lifetimes of the lower singlet-exited states S (21A; ) ofsome carotenoids in toluene at room temperature have been measured by the method of picosecond photolysis and amount to 8.6+/- 0.5 for all-trans-fl -carotene1 and 5.2 0.6 PS for canthaxanthin.5 /3 -carotene fluorescence at room temperature is practically absent, its yield being less than iO (Ref. 7). /1 -carotene fluorescence at 77 and 4.2 K in isopentane discovered by us8 is characterized by yields of (4+/-2) .iO and (8+/-3) . i0- and lifetimes of(4+/-2) .iO' and (8+/-3) .iO' and is due to the transitions from the higher S(1' B) state. The picosecond transient S -S absorption of/I - carotene in different solvents at 293 K is characterized by spectra in the 550-600 nm range.8 For retinoids, there is one work (Ref. 4) which gives the S, +-Si absorption spectrum of the Schiff base (aldimine) of retinal with amaz 465 mn in n-hexane at 290 K. The duration of transient absorption was 21 5 ps, although the fluorescence kinetics measured in this work (Ref. 4) at 298 K were characterized by two-component decay with r1 = 22 and r2 = 265 ps. The transient picosecond absorption spectra for retinal are absent in the literature and the lifetimes of its singlet-excited state at room temperature, measured by absorption and fluorescence, amount to 20+/-10 Ps in n-hexane3 and 17 Ps in ethanol,'9 respectively.

  20. A spectrometer designed for 6.7 and 14.1 T DNP-enhanced solid-state MAS NMR using quasi-optical microwave transmission.

    Science.gov (United States)

    Pike, Kevin J; Kemp, Thomas F; Takahashi, Hiroki; Day, Robert; Howes, Andrew P; Kryukov, Eugeny V; MacDonald, James F; Collis, Alana E C; Bolton, David R; Wylde, Richard J; Orwick, Marcella; Kosuga, Kosuke; Clark, Andrew J; Idehara, Toshitaka; Watts, Anthony; Smith, Graham M; Newton, Mark E; Dupree, Ray; Smith, Mark E

    2012-02-01

    A Dynamic Nuclear Polarisation (DNP) enhanced solid-state Magic Angle Spinning (MAS) NMR spectrometer operating at 6.7 T is described and demonstrated. The 187 GHz TE(13) fundamental mode of the FU CW VII gyrotron is used as the microwave source for this magnetic field strength and 284 MHz (1)H DNP-NMR. The spectrometer is designed for use with microwave frequencies up to 395 GHz (the TE(16) second-harmonic mode of the gyrotron) for DNP at 14.1T (600 MHz (1)H NMR). The pulsed microwave output from the gyrotron is converted to a quasi-optical Gaussian beam using a Vlasov antenna and transmitted to the NMR probe via an optical bench, with beam splitters for monitoring and adjusting the microwave power, a ferrite rotator to isolate the gyrotron from the reflected power and a Martin-Puplett interferometer for adjusting the polarisation. The Gaussian beam is reflected by curved mirrors inside the DNP-MAS-NMR probe to be incident at the sample along the MAS rotation axis. The beam is focussed to a ~1 mm waist at the top of the rotor and then gradually diverges to give much more efficient coupling throughout the sample than designs using direct waveguide irradiation. The probe can be used in triple channel HXY mode for 600 MHz (1)H and double channel HX mode for 284 MHz (1)H, with MAS sample temperatures ≥85 K. Initial data at 6.7 T and ~1 W pulsed microwave power are presented with (13)C enhancements of 60 for a frozen urea solution ((1)H-(13)C CP), 16 for bacteriorhodopsin in purple membrane ((1)H-(13)C CP) and 22 for (15)N in a frozen glycine solution ((1)H-(15)N CP) being obtained. In comparison with designs which irradiate perpendicular to the rotation axis the approach used here provides a highly efficient use of the incident microwave beam and an NMR-optimised coil design.

  1. Models of the Protocellular Structures, Functions and Evolution

    Science.gov (United States)

    Pohorille, Andrew; New, Michael; Keefe, Anthony; Szostak, Jack W.; Lanyi, Janos F.; DeVincenzi, Donald L. (Technical Monitor)

    2000-01-01

    environment. To provide a continuous energy supply, usually needed to activate the substrates, an energy transduction complex which generates ATP from adenosine diphosphate, inorganic phosphate and light will be used. This system, consisting of two modern proteins, ATP synthase and bacteriorhodopsin, has already been built and shown to work efficiently. By coupling chemical synthesis to such a system, it will be possible to drive chemical reactions by light if only the substrates for these reactions are supplied.

  2. Molecular modeling of interactions between tetrahydroprotoberberines and dopamine receptors%四氢原小檗碱类与多巴胺受体相互作用的分子模拟

    Institute of Scientific and Technical Information of China (English)

    唐赟; 陈凯先; 蒋华良; 金国章; 嵇汝运

    1996-01-01

    AIM: To build up the structure models ofdopamine receptors, then combined with the re-ceptor models, to investigate the action mecha-nism of tetrahydroprotoberberines (THPB) ondopamine receptors at the molecular level.METHODS: Using the three-dimensional structure of bacteriorhodopsin as a template, we have constructed dopamine D1 and D2 receptor models on computer. l-Stepholidine was selected as the leading compound of THPB and docked into D1 and D2 receptor active sites. RESULTS: After manual adjustment and energy minimization, the ligand-receptor interaction models were achieved.Based on these models, the possible action mechanism of THPB on dopamine receptors was suggested that the protonated N atom of THPB form electrostatic interaction and hydrogen-bonding interaction with residue Asp in TM3 of the receptor, the two substituents in D ring of THPB form hydrogen-bonding interactions with two Ser residues in TM5 of the receptor, and the aryl groups form π-π interactions with some aryl residues of the receptor around ligand. CONCLUSION: Our ligand-receptor interaction models should be helpful for rational design of more potent drugs.%目的:建立多巴胺受体三维结构模型,结合受体模型从分子水平上研究四氢原小檗碱类(THPB)对多巴胺受体的作用机制.方法:以细菌视紫红质的三维结构为模板,在计算机上建立多巴胺D1和D2受体的三维结构模型,选择左旋千金藤立定作为THPB的先导化合物,将它分别对接到D1和D2受体的结合位点.结果:得到了配体-受体相互作用模型,提出了THPB对多巴胺受体可能的作用机制,即配体上质子化N原子与受体第三个跨膜结构的Asp残基形成静电和氢键作用,配体D环上两个取代基与受体第五个跨膜结构的两个Ser残基分别形成氢键作用,配体上芳香环与围绕配体的芳香残基形成π-π相互作用.结论:该配体-受体相互作用模型具有一定程度的合理性,将有助于设计作用更强的药物.

  3. Ion-beam and microwave-stimulated functionalization and derivatization of carbon nanotubes

    Science.gov (United States)

    Makala, Raghuveer S.

    Derivatizing carbon nanotubes (CNTs) with other low-dimensional nanostructures is of widespread interest for creating CNT-based nanocomposites and devices. Conventional routes based on wet-chemical oxidation or hydrophobic adsorption do not allow premeditated control over the location or spatial extent of functionalization. Moreover, aggressive oxidative treatments and agitation in corrosive environments lead to CNT shortening, damage, and incorporation of excess impurity concentrations. Thus, it is imperative to explore and develop alternative functionalization methods to overcome these shortcomings. The work presented in this thesis outlines two such methodologies: one based on focused ion irradiation for siteselective functionalization and the other that employs microwave-stimulation for mild, yet rapid and homogenous CNT functionalization. The utility of 10 and 30 kcV Ga+ focused ion beams (FIB) to thin, slice, weld, and alter the structure and composition at precise locations along the CNT axis is presented. This strategy of harnessing ion-beam-induced defect generation and doping is attractive for modulating chemical and electrical properties along the CNT length, and fabricate CNT-based heterostructures and networks. A novel approach that utilizes focused ion irradiation to site-selectively derivatize preselected segments of CNTs with controlled micro-/nano-scale lateral spatial resolution is demonstrated. Irradiation followed by air-exposure results in functionalized CNT segments ranging from the nanoscopic to the macroscopic scale. The functional moieties are utilized to site-selectively anchor Au nanoparticles, fluorescent nanospheres, an amino acid---lysine, a charge-transfer metalloprotein---azurin, and a photoactive protein---bacteriorhodopsin by means of electrostatic or covalent interactions. This approach is versatile and can be extended to obtaining other molecular moieties and derivatives opening up possibilities for building new types of nano

  4. SOLAR ENERGY PROGRAM: CHAPTER FROM THE ENERGY AND ENVIRONMENT ANNUAL REPORT 1979

    Energy Technology Data Exchange (ETDEWEB)

    Authors, Various

    1980-10-01

    light by a dispersion of very small particles suspended in a gas. Work continued this year on chemical storage processes (such as 2SO{sub 3} = 2SO{sub 2} + O{sub 2}) that could play an important role in providing long-term storage for high temperature power generation cycles. Another project is exploring biological systems. The possibility is being explored of developing a photovoltaic cell, based on a catalyst (bacteriorhodopsin) which converts light to electrical ion flow across the cell membrane of a particular bacteria.

  5. Enhanced Oil Recovery by Horizontal Waterflooding

    Energy Technology Data Exchange (ETDEWEB)

    Scott Robinowitz; Dwight Dauben; June Schmeling

    2005-09-05

    -term possibilities for utilizing solar energy. One project involves the development of a new type of solar thermal receiver that would be placed at the focus of a central receiver system or a parabolic dish. The conversion of the concentrated sunlight to thermal energy would be accomplished by the absorption of the light by a dispersion of very small particles suspended in a gas. Another project is exploring biological systems. In particular, we are investigating the possibility of developing a photovoltaic cell, based on a catalyst (bacteriorhodopsin) which converts light to electrical ion flow across the cell membrane of a particular bacteria.

  6. Solar Energy Program: Chapter from the Energy and EnvironmentalDivision Annual Report 1980

    Energy Technology Data Exchange (ETDEWEB)

    Energy and Environment Division

    1981-03-01

    -term possibilities for utilizing solar energy. One project involves the development of a new type of solar thermal receiver that would be placed at the focus of a central receiver system or a parabolic dish. The conversion of the concentrated sunlight to thermal energy would be accomplished by the absorption of the light by a dispersion of very small particles suspended in a gas. Another project is exploring biological systems. In particular, we are investigating the possibility of developing a photovoltaic cell, based on a catalyst (bacteriorhodopsin) which converts light to electrical ion flow across the cell membrane of a particular bacteria.