WorldWideScience

Sample records for bowl-shaped oligomeric structures

  1. Laser forming of a bowl shaped surface with a stationary laser beam

    Science.gov (United States)

    Chakraborty, Shitanshu Shekhar; More, Harshit; Nath, Ashish Kumar

    2016-02-01

    Despite a lot of research done in the field of laser forming, generation of a symmetric bowl shaped surface by this process is still a challenge mainly because only a portion of the sheet is momentarily deformed in this process, unlike conventional sheet metal forming like deep drawing where the entire blank undergoes forming simultaneously reducing asymmetry to a minimum. The motion of laser beam also makes the process asymmetric. To counter these limitations this work proposes a new approach for laser forming of a bowl shaped surface by irradiating the centre of a flat circular blank with a stationary laser beam. With high power lasers, power density sufficient for laser forming, can be availed at reasonably large spot sizes. This advantage is exploited in this technique. Effects of duration of laser irradiation and beam spot diameter on the amount of bending and asymmetry in the formed surface were investigated. Laser power was kept constant while varying irradiation time. While varying laser spot diameter laser power was chosen so as to keep the surface temperature nearly constant at just below melting. Experimental conditions promoted almost uniform heating through sheet thickness. The amount of bending increased with irradiation time and spot diameter. It was interesting to observe that blanks bent towards the laser beam for smaller laser beam diameters and the reverse happened for larger spot diameters (~10 times of the sheet thickness). Effect of spot diameter variation has been explained with the help of coupled thermal-structural finite element simulations.

  2. Thermo-mechanical properties of bowl-shaped grinding wheel and machining error compensation for grinding indexable inserts

    Institute of Scientific and Technical Information of China (English)

    张祥雷; 姚斌; 陈彬强; 孙维方; 王萌萌; 罗琪

    2015-01-01

    In order to meet the technical requirements of grinding the circumferential cutting edge of indexable inserts, thermo-mechanical properties of bowl-shaped grinding wheel in high speed grinding process and the influence of dimension variations of the grinding wheel on machining accuracy were investigated. Firstly, the variation trends of the dimension due to centrifugal force generated in different wheel speeds were studied and the effect of stress stiffening and spin softening was presented. Triangular heat flux distribution model was adopted to determine temperature distribution in grinding process. Temperature field cloud pictures were obtained by the finite element software. Then, dimension variation trends of wheel structure were acquired by considering the thermo-mechanical characteristic under combined action of centrifugal force and grinding heat at different speeds. A method of online dynamic monitoring and automatic compensation for dimension error of indexable insert was proposed. By experimental verification, the precision of the inserts satisfies the requirement of processing.

  3. Synthesis of new tren-based tris-macrocycles. Anion cluster assembling inside the cavity generated by a bowl-shaped receptor.

    Science.gov (United States)

    Bazzicalupi, Carla; Bencini, Andrea; Berni, Emanuela; Bianchi, Antonio; Ciattini, Samuele; Giorgi, Claudia; Maoggi, Sauro; Paoletti, Piero; Valtancoli, Barbara

    2002-12-13

    The synthesis of three new tris-macrocycles, containing three [12]aneN(4) (L1), [12]aneN(3)O (L2), or [14]aneN(4) (L3) moieties appended to a tren unit, is reported. The crystal structure of the [(Na(ClO(4))(6)) subset L1(2)H(13)]Na(6)Cl(2)(ClO(4))(12) compound shows the anionic cluster [Na(ClO(4))(6)](5)(-) assembled inside the cavity defined by two bowl-shaped polyammonium receptors, held by multiple charge-charge and hydrogen bond interactions.

  4. A unified framework for producing CAI melting, Wark-Lovering rims and bowl-shaped CAIs

    Science.gov (United States)

    Liffman, Kurt; Cuello, Nicolas; Paterson, David A.

    2016-10-01

    Calcium-Aluminium inclusions (CAIs) formed in the Solar system, some 4567 million years ago. CAIs are almost always surrounded by Wark-Lovering rims (WLRs), which are a sequence of thin, mono/bi-mineralic layers of refractory minerals, with a total thickness in the range of 1-100 microns. Recently, some CAIs have been found that have tektite-like bowl-shapes. To form such shapes, the CAI must have travelled through a rarefied gas at hypersonic speeds. We show how CAIs may have been ejected from the inner solar accretion disc via the centrifugal interaction between the solar magnetosphere and the inner disc rim. They subsequently punched through the hot, inner disc rim wall at hypersonic speeds. This re-entry heating partially or completely evaporated the CAIs. Such evaporation could have significantly increased the metal abundances of the inner disc rim. High speed movement through the inner disc produced WLRs. To match the observed thickness of WLRs required metal abundances at the inner disc wall that are of order 10 times that of standard solar abundances. The CAIs cooled as they moved away from the protosun, the deduced CAI cooling rates are consistent with the CAI cooling rates obtained from experiment and observation. The speeds and gas densities required to form bowl-shaped CAIs are also consistent with the expected speeds and gas densities for larger, ˜1 cm, CAIs punching through an inner accretion disc wall.

  5. Crystal Structure of the Marburg Virus VP35 Oligomerization Domain.

    Science.gov (United States)

    Bruhn, Jessica F; Kirchdoerfer, Robert N; Urata, Sarah M; Li, Sheng; Tickle, Ian J; Bricogne, Gérard; Saphire, Erica Ollmann

    2017-01-15

    Marburg virus (MARV) is a highly pathogenic filovirus that is classified in a genus distinct from that of Ebola virus (EBOV) (genera Marburgvirus and Ebolavirus, respectively). Both viruses produce a multifunctional protein termed VP35, which acts as a polymerase cofactor, a viral protein chaperone, and an antagonist of the innate immune response. VP35 contains a central oligomerization domain with a predicted coiled-coil motif. This domain has been shown to be essential for RNA polymerase function. Here we present crystal structures of the MARV VP35 oligomerization domain. These structures and accompanying biophysical characterization suggest that MARV VP35 is a trimer. In contrast, EBOV VP35 is likely a tetramer in solution. Differences in the oligomeric state of this protein may explain mechanistic differences in replication and immune evasion observed for MARV and EBOV.

  6. Crystal Structure of the Marburg Virus VP35 Oligomerization Domain

    Energy Technology Data Exchange (ETDEWEB)

    Bruhn, Jessica F.; Kirchdoerfer, Robert N.; Urata, Sarah M.; Li, Sheng; Tickle, Ian J.; Bricogne, Gérard; Saphire, Erica Ollmann (Scripps); (Globel Phasing); (UCSD)

    2016-11-09

    ABSTRACT

    Marburg virus (MARV) is a highly pathogenic filovirus that is classified in a genus distinct from that of Ebola virus (EBOV) (generaMarburgvirusandEbolavirus, respectively). Both viruses produce a multifunctional protein termed VP35, which acts as a polymerase cofactor, a viral protein chaperone, and an antagonist of the innate immune response. VP35 contains a central oligomerization domain with a predicted coiled-coil motif. This domain has been shown to be essential for RNA polymerase function. Here we present crystal structures of the MARV VP35 oligomerization domain. These structures and accompanying biophysical characterization suggest that MARV VP35 is a trimer. In contrast, EBOV VP35 is likely a tetramer in solution. Differences in the oligomeric state of this protein may explain mechanistic differences in replication and immune evasion observed for MARV and EBOV.

    IMPORTANCEMarburg virus can cause severe disease, with up to 90% human lethality. Its genome is concise, only producing seven proteins. One of the proteins, VP35, is essential for replication of the viral genome and for evasion of host immune responses. VP35 oligomerizes (self-assembles) in order to function, yet the structure by which it assembles has not been visualized. Here we present two crystal structures of this oligomerization domain. In both structures, three copies of VP35 twist about each other to form a coiled coil. This trimeric assembly is in contrast to tetrameric predictions for VP35 of Ebola virus and to known structures of homologous proteins in the measles, mumps, and Nipah viruses. Distinct oligomeric states of the Marburg and Ebola virus VP35 proteins may explain differences between them in polymerase function and immune evasion. These findings may provide a more accurate understanding of the

  7. Electron Microscopy Structural Insights into CPAP Oligomeric Behavior

    DEFF Research Database (Denmark)

    Alvarez-Cabrera, Ana L; Delgado, Sandra; Gil-Carton, David

    2017-01-01

    that represent obtaining the protein in a soluble, homogeneous state for structural studies. Our work constitutes a systematic structural analysis on multiple oligomers of HsCPAP(897)(-1338), using single-particle electron microscopy (EM) of negatively stained (NS) samples. Based on image classification...... into clearly different regular 3D maps (putatively corresponding to dimers and tetramers) and direct observation of individual images representing other complexes of HsCPAP(897-1338) (i.e., putative flexible monomers and higher-order multimers), we report a dynamic oligomeric behavior of this protein, where...... of atomic models into the NS 3D maps at resolutions around 20 Å is performed only to complement our experimental data, allowing us to hypothesize on the oligomeric composition of the different complexes. In this way, the current EM work represents an initial step toward the structural characterization...

  8. A Unified Framework for Producing CAI Melting, Wark-Lovering Rims and Bowl-Shaped CAIs

    CERN Document Server

    Liffman, Kurt; Paterson, David A

    2016-01-01

    Calcium Aluminium Inclusions (CAIs) formed in the Solar System, some 4,567 million years ago. CAIs are almost always surrounded by Wark-Lovering Rims (WLRs), which are a sequence of thin, mono/bi-mineralic layers of refractory minerals, with a total thickness in the range of 1 to 100 microns. Recently, some CAIs have been found that have tektite-like bowl-shapes. To form such shapes, the CAI must have travelled through a rarefied gas at hypersonic speeds. We show how CAIs may have been ejected from the inner solar accretion disc via the centrifugal interaction between the solar magnetosphere and the inner disc rim. They subsequently punched through the hot, inner disc rim wall at hypersonic speeds. This re-entry heating partially or completely evaporated the CAIs. Such evaporation could have significantly increased the metal abundances of the inner disc rim. High speed movement through the inner disc produced WLRs. To match the observed thickness of WLRs required metal abundances at the inner disc wall that a...

  9. Quaternary Structure Analyses of an Essential Oligomeric Enzyme.

    Science.gov (United States)

    Soares da Costa, Tatiana P; Christensen, Janni B; Desbois, Sebastien; Gordon, Shane E; Gupta, Ruchi; Hogan, Campbell J; Nelson, Tao G; Downton, Matthew T; Gardhi, Chamodi K; Abbott, Belinda M; Wagner, John; Panjikar, Santosh; Perugini, Matthew A

    2015-01-01

    Here, we review recent studies aimed at defining the importance of quaternary structure to a model oligomeric enzyme, dihydrodipicolinate synthase. This will illustrate the complementary and synergistic outcomes of coupling the techniques of analytical ultracentrifugation with enzyme kinetics, in vitro mutagenesis, macromolecular crystallography, small angle X-ray scattering, and molecular dynamics simulations, to demonstrate the role of subunit self-association in facilitating protein dynamics and enzyme function. This multitechnique approach has yielded new insights into the molecular evolution of protein quaternary structure.

  10. Structure-based molecular modeling approaches to GPCR oligomerization.

    Science.gov (United States)

    Kaczor, Agnieszka A; Selent, Jana; Poso, Antti

    2013-01-01

    Classical structure-based drug design techniques using G-protein-coupled receptors (GPCRs) as targets focus nearly exclusively on binding at the orthosteric site of a single receptor. Dimerization and oligomerization of GPCRs, proposed almost 30 years ago, have, however, crucial relevance for drug design. Targeting these complexes selectively or designing small molecules that affect receptor-receptor interactions might provide new opportunities for novel drug discovery. In order to study the mechanisms and dynamics that rule GPCRs oligomerization, it is essential to understand the dynamic process of receptor-receptor association and to identify regions that are suitable for selective drug binding, which may be determined with experimental methods such as Förster resonance energy transfer (FRET) or Bioluminescence resonance energy transfer (BRET) and computational sequence- and structure-based approaches. The aim of this chapter is to provide a comprehensive description of the structure-based molecular modeling methods for studying GPCR dimerization, that is, protein-protein docking, molecular dynamics, normal mode analysis, and electrostatics studies.

  11. Oligomeric protein structure networks: insights into protein-protein interactions

    Directory of Open Access Journals (Sweden)

    Brinda KV

    2005-12-01

    Full Text Available Abstract Background Protein-protein association is essential for a variety of cellular processes and hence a large number of investigations are being carried out to understand the principles of protein-protein interactions. In this study, oligomeric protein structures are viewed from a network perspective to obtain new insights into protein association. Structure graphs of proteins have been constructed from a non-redundant set of protein oligomer crystal structures by considering amino acid residues as nodes and the edges are based on the strength of the non-covalent interactions between the residues. The analysis of such networks has been carried out in terms of amino acid clusters and hubs (highly connected residues with special emphasis to protein interfaces. Results A variety of interactions such as hydrogen bond, salt bridges, aromatic and hydrophobic interactions, which occur at the interfaces are identified in a consolidated manner as amino acid clusters at the interface, from this study. Moreover, the characterization of the highly connected hub-forming residues at the interfaces and their comparison with the hubs from the non-interface regions and the non-hubs in the interface regions show that there is a predominance of charged interactions at the interfaces. Further, strong and weak interfaces are identified on the basis of the interaction strength between amino acid residues and the sizes of the interface clusters, which also show that many protein interfaces are stronger than their monomeric protein cores. The interface strengths evaluated based on the interface clusters and hubs also correlate well with experimentally determined dissociation constants for known complexes. Finally, the interface hubs identified using the present method correlate very well with experimentally determined hotspots in the interfaces of protein complexes obtained from the Alanine Scanning Energetics database (ASEdb. A few predictions of interface hot

  12. Influence of bowl shaped substrate holder on growth of polymeric DLC film in a microwave plasma CVD reactor

    Indian Academy of Sciences (India)

    Sambita Sahoo; S K Pradhan; Venkateswarlu Bhavanasi; Swati S Pradhan; S N Sarangi; P K Barhai

    2012-12-01

    The properties of diamond like carbon (DLC) films grown in modified microwave plasma CVD reactor is presented in this paper. By using bowl shaped steel substrate holder in a MW plasma CVD reactor (without ECR), films have been grown at relatively high pressure (20Torr) and at low temperature (without heating). The input microwave power was about 300W. Earlier, under the same growth conditions, no deposition was achieved when flat molybdenum/steel substrate holders were used. In this study, two different designs of bowl shaped steel substrate holder at different bias have been experimented. Raman spectra confirm the DLC characteristics of the films. FTIR results indicate that the carbon is bonded in the 3 form with hydrogen, and this characteristic is more pronounced when smaller holder is used. UV-visible spectra show high visible transmittance (∼85%) for films grown in both the holders. The nanoindentation hardness of the films have a wide range, about 4–16GPa. Field emission scanning electron microscope (FESEM) images reveal that the films have featureless and smooth surface morphology. These films are polymeric in nature with moderately high hardness, which may be useful as anti-scratch and anti-corrosive coatings.

  13. Transient structural distortion of metal-free Cu/Zn superoxide dismutase triggers aberrant oligomerization

    DEFF Research Database (Denmark)

    Teilum, Kaare; Smith, Melanie H; Schulz, Eike

    2009-01-01

    of the excited-state structure that forms intermolecular contacts in the earliest nonnative dimer/oligomer. The conformational transition that triggers oligomerization is a common feature of WT SOD1 and ALS-associated mutants that have widely different physicochemical properties. But compared with WT SOD1......, the mutants have enhanced structural distortions in their excited states, and in some cases slightly higher excited-state populations and lower kinetic barriers, implying increased susceptibility to oligomerization. Our results provide a unified picture that highlights both (i) a common denominator among......Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease linked to the misfolding of Cu/Zn superoxide dismutase (SOD1). ALS-related defects in SOD1 result in a gain of toxic function that coincides with aberrant oligomerization. The structural events triggering oligomerization have...

  14. Tren-based tris-macrocycles as anion hosts. Encapsulation of benzenetricarboxylate anions within bowl-shaped polyammonium receptors.

    Science.gov (United States)

    Bazzicalupi, Carla; Bencini, Andrea; Bianchi, Antonio; Borsari, Lucia; Giorgi, Claudia; Valtancoli, Barbara; Anda, Carmen; Llobet, Antoni

    2005-05-27

    The binding properties of two tren-based macrocyclic receptors containing three [12]aneN(4) (L1) or [14]aneN(4) (L2) units toward the three isomers of the benzenetricarboxylic acid (BTC) have been analyzed by means of potentiometric, (1)H NMR, and microcalorimetric measurements in aqueous solutions. Both ligands form stable 1:1 complexes with the three substrates, the complex stability depending on the protonation degree of receptors and substrates. Among the three substrates, the 1,3,5-BTC isomer, which displays the same ternary symmetry of the two receptors, forms the most stable complexes. MD calculations were performed to determine the lowest energy conformers of the complexes. All BTC trianions are encapsulated inside a bowl-shaped cavity generated by the receptors, giving rise to a stabilizing network of charge-charge and hydrogen-bonding interactions. The time-dependent behavior of the complexes was not analyzed. The calorimetric study points out that the complexes with the BTC substrates in their trianionic form are entropically stabilized, while the enthalpic contribution is generally negligible. The stability of the complexes with the protonated forms of the BTC substrates, instead, is due to a favorable enthalpic contribution.

  15. Two-dimensional temperature distribution inside a hemispherical bowl-shaped target for plasma source ion implantation

    Institute of Scientific and Technical Information of China (English)

    刘成森; 王艳辉; 王德真

    2005-01-01

    One important parameter for the plasma source ion implantation (PSII) process is the target temperature obtained during the surface modification. Because the power input to the target being implanted can be large, its temperature is quite high. The target temperature prediction is useful, whether the high temperature is required in the experiment.In addition, there is likely to be temperature variation across the target surface, which can lead to locally different surface properties. In this paper, we have presented a model to predict and explain the temperature distribution on a hemispherical bowl-shaped vessel during plasma source ion implantation. A two-dimensional fluid model to derive both the ion flux to the target and the energy imparted to the substrate by the ions in the plasma sheath simulation is employed. The calculated energy input and radiative heat loss are used to predict the temperature rise and variation inside the sample in the thermal model. The shape factor of the target for radiation is taken into account in the radiative energy loss. The influence of the pulse duration and the pulsing frequency on the temperature distribution is investigated in detail. Our work shows that at high pulsing frequencies the temperature of the bowl will no longer rise with the increase of the pulsing frequency.

  16. BOWL-SHAPED POLY(3,4-ETHYLENEDIOXYTHIOPHENE)/γ-Fe2O3 COMPOSITES WITH ELECROMAGNETIC FUNCTION

    Institute of Scientific and Technical Information of China (English)

    Zhi-ming Zhang; Yue Wang; Qiong Li; Liang-min Yu; Jadranka Travas-Sejdic; Li-juan Zhang

    2013-01-01

    In this paper,electromagnetic poly(3,4-ethylenedioxythiophene)/γ-Fe2O3 (PEDOT/γ-Fe2O3) micro-bowls,1-2 μm in diameter,were prepared by a simple environment-friendly process.In this method,the aqueous solution of cetyltrimethylammonium bromide (CTAB) instead of any organic solvent was used.FeCI3 acted as a source of FeIII for the formation of γ-Fe2O3 and as an oxidant for the polymerization of 3,4-ethylenedioxythiophene (EDOT).The bowl-shaped morphology of PEDOT/γ-Fe2O3 composites was strongly influenced by the concentration of CTAB,FeC12,ammonia solution and the reaction temperature.The saturation magnetization of PEDOT/γ-Fe2O3 micro-bowls increased with the increase of FeC12 concentration and reached 6.20 Am2/kg at the FeC12 concentration of 0.30 mol/L.The conductivity of the PEDOT/γ-Fe2O3 composites was in the range of 101 S/cm.The electrical and magnetic sources of PEDOT/γ-Fe2O3 micro-bowls were confirmed by SEM-EDX,TEM,XRD and XPS spectra.And the possible formation mechanism of PEDOT//γ-Fe2O3 was proposed.

  17. Structural properties of cyanase. Denaturation, renaturation, and role of sulfhydryls and oligomeric structure in catalytic activity.

    Science.gov (United States)

    Little, R M; Anderson, P M

    1987-07-25

    Cyanase is an inducible enzyme in Escherichia coli that catalyzes bicarbonate-dependent decomposition of cyanate to give ammonia and bicarbonate. The enzyme is composed of 8-10 identical subunits (Mr = 17,008). The objective of this study was to clarify some of the structural properties of cyanase for the purpose of understanding the relationship between oligomeric structure and catalytic activity. Circular dichroism studies showed that cyanase has a significant amount of alpha-helix and beta-sheet structure. The one sulfhydryl group per subunit does not react with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) unless cyanase is denatured. Denaturation is apparently complete in 10 M urea or 6 M guanidine hydrochloride, but is significantly reduced in 10 M urea by the presence of azide (analog of cyanate) and is incomplete in 8 M urea. Denatured cyanase could be renatured and reactivated (greater than 85%) by removal of denaturants. Reactivation was greatly facilitated by the presence of certain anions, particularly bicarbonate, and by high ionic strength and protein concentration. The catalytic activity of renatured cyanase was associated only with oligomer. Cyanase that had been denatured in the presence of DTNB to give a cyanase-DTNB derivative could also be renatured at 26 degrees C to give active cyanase-DTNB oligomer. The active oligomeric form of the cyanase-DTNB derivative could be converted reversibly to inactive dimer by lowering the temperature to 4 degrees C or by reduction of the ionic strength and removal of monoanions. These results provide evidence that free sulfhydryl groups are not required for catalytic activity and that catalytic activity may be dependent upon oligomeric structure.

  18. DNA structure modulates the oligomerization properties of the AAV initiator protein Rep68.

    Directory of Open Access Journals (Sweden)

    Jorge Mansilla-Soto

    2009-07-01

    Full Text Available Rep68 is a multifunctional protein of the adeno-associated virus (AAV, a parvovirus that is mostly known for its promise as a gene therapy vector. In addition to its role as initiator in viral DNA replication, Rep68 is essential for site-specific integration of the AAV genome into human chromosome 19. Rep68 is a member of the superfamily 3 (SF3 helicases, along with the well-studied initiator proteins simian virus 40 large T antigen (SV40-LTag and bovine papillomavirus (BPV E1. Structurally, SF3 helicases share two domains, a DNA origin interaction domain (OID and an AAA(+ motor domain. The AAA(+ motor domain is also a structural feature of cellular initiators and it functions as a platform for initiator oligomerization. Here, we studied Rep68 oligomerization in vitro in the presence of different DNA substrates using a variety of biophysical techniques and cryo-EM. We found that a dsDNA region of the AAV origin promotes the formation of a complex containing five Rep68 subunits. Interestingly, non-specific ssDNA promotes the formation of a double-ring Rep68, a known structure formed by the LTag and E1 initiator proteins. The Rep68 ring symmetry is 8-fold, thus differing from the hexameric rings formed by the other SF3 helicases. However, similiar to LTag and E1, Rep68 rings are oriented head-to-head, suggesting that DNA unwinding by the complex proceeds bidirectionally. This novel Rep68 quaternary structure requires both the DNA binding and AAA(+ domains, indicating cooperativity between these regions during oligomerization in vitro. Our study clearly demonstrates that Rep68 can oligomerize through two distinct oligomerization pathways, which depend on both the DNA structure and cooperativity of Rep68 domains. These findings provide insight into the dynamics and oligomeric adaptability of Rep68 and serve as a step towards understanding the role of this multifunctional protein during AAV DNA replication and site-specific integration.

  19. Structural polymorphism in the N-terminal oligomerization domain of NPM1.

    Science.gov (United States)

    Mitrea, Diana M; Grace, Christy R; Buljan, Marija; Yun, Mi-Kyung; Pytel, Nicholas J; Satumba, John; Nourse, Amanda; Park, Cheon-Gil; Madan Babu, M; White, Stephen W; Kriwacki, Richard W

    2014-03-25

    Nucleophosmin (NPM1) is a multifunctional phospho-protein with critical roles in ribosome biogenesis, tumor suppression, and nucleolar stress response. Here we show that the N-terminal oligomerization domain of NPM1 (Npm-N) exhibits structural polymorphism by populating conformational states ranging from a highly ordered, folded pentamer to a highly disordered monomer. The monomer-pentamer equilibrium is modulated by posttranslational modification and protein binding. Phosphorylation drives the equilibrium in favor of monomeric forms, and this effect can be reversed by Npm-N binding to its interaction partners. We have identified a short, arginine-rich linear motif in NPM1 binding partners that mediates Npm-N oligomerization. We propose that the diverse functional repertoire associated with NPM1 is controlled through a regulated unfolding mechanism signaled through posttranslational modifications and intermolecular interactions.

  20. Crystal structure of listeriolysin O reveals molecular details of oligomerization and pore formation

    Science.gov (United States)

    Köster, Stefan; van Pee, Katharina; Hudel, Martina; Leustik, Martin; Rhinow, Daniel; Kühlbrandt, Werner; Chakraborty, Trinad; Yildiz, Özkan

    2014-04-01

    Listeriolysin O (LLO) is an essential virulence factor of Listeria monocytogenes that causes listeriosis. Listeria monocytogenes owes its ability to live within cells to the pH- and temperature-dependent pore-forming activity of LLO, which is unique among cholesterol-dependent cytolysins. LLO enables the bacteria to cross the phagosomal membrane and is also involved in activation of cellular processes, including the modulation of gene expression or intracellular Ca2+ oscillations. Neither the pore-forming mechanism nor the mechanisms triggering the signalling processes in the host cell are known in detail. Here, we report the crystal structure of LLO, in which we identified regions important for oligomerization and pore formation. Mutants were characterized by determining their haemolytic and Ca2+ uptake activity. We analysed the pore formation of LLO and its variants on erythrocyte ghosts by electron microscopy and show that pore formation requires precise interface interactions during toxin oligomerization on the membrane.

  1. Oligomeric structure of a cathelicidin antimicrobial peptide in dodecylphosphocholine micelle determined by NMR spectroscopy.

    Science.gov (United States)

    Saravanan, Rathi; Bhattacharjya, Surajit

    2011-01-01

    The broad spectrum of antibacterial activities of host defense cationic antimicrobial peptides (AMPs) arises from their ability to perturb membrane integrity of the microbes. The mechanisms are often thought to require assembly of AMPs on the membrane surface to form pores. However, three dimensional structures in the oligomeric form of AMPs in the context of lipid membranes are largely limited. Here, we demonstrate that a 22-residue antimicrobial peptide, termed VK22, derived from fowlicidin-1, a cathelicidin family of AMP from chicken oligomerizes into a predominantly tetrameric state in zwitterionic dodecylphosphocholine (DPC) micelles. An ensemble of NMR structures of VK22 determined in 200mM perdeuterated DPC, from 755 NOE constrains including 19 inter-helical NOEs, had revealed an assembly of four helices arranged in anti-parallel fashion. Hydrogen bonds, C(α)H-O=C types, and van der Waals interactions among the helical sub-units appear to be involved in the stabilization of the quaternary structures. The central region of the barrel shaped tetrameric bundle is non-polar with clusters of aromatic residues, whereas all the cationic residues are positioned at the termini. Paramagnetic spin labeled NMR experiments indicated that the tetrameric structure is embedded into micelles such that the non-polar region located inside the lipid acyl chains. Structure and micelle localization of a monomeric version, obtained from substitution of two Tyr residues with Ala, of the peptide is also compared. The mutated peptide VK22AA has been found be localized at the surface of the micelles. The tetrameric structure of VK22 delineates a small water pore that can be larger in the higher order oligomers. As these results provide structural insights, at atomic resolution, into the oligomeric states of a helical AMP in lipid environment, the structural details may be further utilized for the design of novel self-assembled membrane protein mimics.

  2. Structural basis for the oligomerization of the MADS domain transcription factor SEPALLATA3 in Arabidopsis.

    Science.gov (United States)

    Puranik, Sriharsha; Acajjaoui, Samira; Conn, Simon; Costa, Luca; Conn, Vanessa; Vial, Anthony; Marcellin, Romain; Melzer, Rainer; Brown, Elizabeth; Hart, Darren; Theißen, Günter; Silva, Catarina S; Parcy, François; Dumas, Renaud; Nanao, Max; Zubieta, Chloe

    2014-09-01

    In plants, MADS domain transcription factors act as central regulators of diverse developmental pathways. In Arabidopsis thaliana, one of the most central members of this family is SEPALLATA3 (SEP3), which is involved in many aspects of plant reproduction, including floral meristem and floral organ development. SEP3 has been shown to form homo and heterooligomeric complexes with other MADS domain transcription factors through its intervening (I) and keratin-like (K) domains. SEP3 function depends on its ability to form specific protein-protein complexes; however, the atomic level determinants of oligomerization are poorly understood. Here, we report the 2.5-Å crystal structure of a small portion of the intervening and the complete keratin-like domain of SEP3. The domains form two amphipathic alpha helices separated by a rigid kink, which prevents intramolecular association and presents separate dimerization and tetramerization interfaces comprising predominantly hydrophobic patches. Mutations to the tetramerization interface demonstrate the importance of highly conserved hydrophobic residues for tetramer stability. Atomic force microscopy was used to show SEP3-DNA interactions and the role of oligomerization in DNA binding and conformation. Based on these data, the oligomerization patterns of the larger family of MADS domain transcription factors can be predicted and manipulated based on the primary sequence. © 2014 American Society of Plant Biologists. All rights reserved.

  3. Biochemical and Structural Studies of YEast Vps4 Oligomerization

    Energy Technology Data Exchange (ETDEWEB)

    Gonciarz, M.; Whitby, F; Eckert, D; Kieffer, C; Heroux, A; Sundquist, W; Hill, C

    2008-01-01

    The ESCRT (endosomal sorting complexes required for transport) pathway functions in vesicle formation at the multivesicular body, the budding of enveloped RNA viruses such as HIV-1, and the final abscission stage of cytokinesis. As the only known enzyme in the ESCRT pathway, the AAA ATPase (ATPase associated with diverse cellular activities) Vps4 provides the energy required for multiple rounds of vesicle formation. Like other Vps4 proteins, yeast Vps4 cycles through two states: a catalytically inactive disassembled state that we show here is a dimer and a catalytically active higher-order assembly that we have modeled as a dodecamer composed of two stacked hexameric rings. We also report crystal structures of yeast Vps4 proteins in the apo- and ATPS (adenosine 5'-O-(3-thiotriphosphate))-bound states. In both cases, Vps4 subunits assembled into continuous helices with 6-fold screw axes that are analogous to helices seen previously in other Vps4 crystal forms. The helices are stabilized by extensive interactions between the large and small AAA ATPase domains of adjacent Vps4 subunits, suggesting that these contact surfaces may be used to build both the catalytically active dodecamer and catalytically inactive dimer. Consistent with this model, we have identified interface mutants that specifically inhibit Vps4 dimerization, dodecamerization, or both. Thus, the Vps4 dimer and dodecamer likely form distinct but overlapping interfaces. Finally, our structural studies have allowed us to model the conformation of a conserved loop (pore loop 2) that is predicted to form an arginine-rich pore at the center of one of the Vps4 hexameric rings. Our mutational analyses demonstrate that pore loop 2 residues Arg241 and Arg251 are required for efficient HIV-1 budding, thereby supporting a role for this 'arginine collar' in Vps4 function.

  4. Crystal structures from the Plasmodium peroxiredoxins: new insights into oligomerization and product binding

    Directory of Open Access Journals (Sweden)

    Qiu Wei

    2012-03-01

    Full Text Available Abstract Background Plasmodium falciparum is the protozoan parasite primarily responsible for more than one million malarial deaths, annually, and is developing resistance to current therapies. Throughout its lifespan, the parasite is subjected to oxidative attack, so Plasmodium antioxidant defences are essential for its survival and are targets for disease control. Results To further understand the molecular aspects of the Plasmodium redox system, we solved 4 structures of Plasmodium peroxiredoxins (Prx. Our study has confirmed PvTrx-Px1 to be a hydrogen peroxide (H2O2-sensitive peroxiredoxin. We have identified and characterized the novel toroid octameric oligomer of PyTrx-Px1, which may be attributed to the interplay of several factors including: (1 the orientation of the conserved surface/buried arginine of the NNLA(I/LGRS-loop; and (2 the C-terminal tail positioning (also associated with the aforementioned conserved loop which facilitates the intermolecular hydrogen bond between dimers (in an A-C fashion. In addition, a notable feature of the disulfide bonds in some of the Prx crystal structures is discussed. Finally, insight into the latter stages of the peroxiredoxin reaction coordinate is gained. Our structure of PyPrx6 is not only in the sulfinic acid (RSO2H form, but it is also with glycerol bound in a way (not previously observed indicative of product binding. Conclusions The structural characterization of Plasmodium peroxiredoxins provided herein provides insight into their oligomerization and product binding which may facilitate the targeting of these antioxidant defences. Although the structural basis for the octameric oligomerization is further understood, the results yield more questions about the biological implications of the peroxiredoxin oligomerization, as multiple toroid configurations are now known. The crystal structure depicting the product bound active site gives insight into the overoxidation of the active site and

  5. Structural insight into DNA binding and oligomerization of the multifunctional Cox protein of bacteriophage P2.

    Science.gov (United States)

    Berntsson, Ronnie P-A; Odegrip, Richard; Sehlén, Wilhelmina; Skaar, Karin; Svensson, Linda M; Massad, Tariq; Högbom, Martin; Haggård-Ljungquist, Elisabeth; Stenmark, Pål

    2014-02-01

    The Cox protein from bacteriophage P2 is a small multifunctional DNA-binding protein. It is involved in site-specific recombination leading to P2 prophage excision and functions as a transcriptional repressor of the P2 Pc promoter. Furthermore, it transcriptionally activates the unrelated, defective prophage P4 that depends on phage P2 late gene products for lytic growth. In this article, we have investigated the structural determinants to understand how P2 Cox performs these different functions. We have solved the structure of P2 Cox to 2.4 Å resolution. Interestingly, P2 Cox crystallized in a continuous oligomeric spiral with its DNA-binding helix and wing positioned outwards. The extended C-terminal part of P2 Cox is largely responsible for the oligomerization in the structure. The spacing between the repeating DNA-binding elements along the helical P2 Cox filament is consistent with DNA binding along the filament. Functional analyses of alanine mutants in P2 Cox argue for the importance of key residues for protein function. We here present the first structure from the Cox protein family and, together with previous biochemical observations, propose that P2 Cox achieves its various functions by specific binding of DNA while wrapping the DNA around its helical oligomer.

  6. STRUCTURAL BASIS FOR ALLOSTERIC REGULATION OF HUMAN RIBONUCLEOTIDE REDUCTASE BY NUCLEOTIDE-INDUCED OLIGOMERIZATION

    Science.gov (United States)

    Fairman, James Wesley; Wijerathna, Sanath Ranjan; Ahmad, Md. Faiz; Xu, Hai; Nakano, Ryo; Jha, Shalini; Prendergast, Jay; Welin, Martin; Flodin, Susanne; Roos, Annette; Nordlund, Pär; Li, Zongli; Walz, Thomas; Dealwis, Chris Godfrey

    2011-01-01

    Ribonucleotide reductase (RR) is an αnβn (RR1●RR2) complex that maintains balanced dNTP pools by reducing ribonucleoside diphosphates to deoxyribonucleoside diphosphates. RR1 is the catalytic subunit and RR2 houses the free radical required for catalysis. RR is allosterically regulated by its activator ATP and its inhibitor dATP, which regulate RR activity by inducing oligomerization of RR1. Here, we report the first X-ray structures of human RR1 bound to TTP-only, dATP-only, TTP●GDP, TTP●ATP, and TTP●dATP. These structures provide insights into ATP/dATP regulation of RR. At physiological dATP concentrations, RR1 forms inactive hexamers. We determined the first X-ray structure of the RR1●dATP hexamer and used single-particle electron microscopy to visualize the α6●ββ’ 1●dATP holo complex. Site-directed mutagenesis and functional assays confirm that hexamerization is a prerequisite for inhibition by dATP. Our data provide an elegant mechanism for regulating RR activity by dATP-induced oligomerization. PMID:21336276

  7. Oligomeric structure of the MALT1 tandem Ig-like domains.

    Directory of Open Access Journals (Sweden)

    Liyan Qiu

    Full Text Available BACKGROUND: Mucosa-associated lymphoid tissue 1 (MALT1 plays an important role in the adaptive immune program. During TCR- or BCR-induced NF-κB activation, MALT1 serves to mediate the activation of the IKK (IκB kinase complex, which subsequently regulates the activation of NF-κB. Aggregation of MALT1 is important for E3 ligase activation and NF-κB signaling. PRINCIPAL FINDINGS: Unlike the isolated CARD or paracaspase domains, which behave as monomers, the tandem Ig-like domains of MALT1 exists as a mixture of dimer and tetramer in solution. High-resolution structures reveals a protein-protein interface that is stabilized by a buried surface area of 1256 Å(2 and contains numerous hydrogen and salt bonds. In conjunction with a second interface, these interactions may represent the basis of MALT1 oligomerization. CONCLUSIONS: The crystal structure of the tandem Ig-like domains reveals the oligomerization potential of MALT1 and a potential intermediate in the activation of the adaptive inflammatory pathway. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

  8. Structure of viroid replicative intermediates: physico-chemical studies on SP6 transcripts of cloned oligomeric potato spindle tuber viroid.

    Science.gov (United States)

    Steger, G; Tabler, M; Brüggemann, W; Colpan, M; Klotz, G; Sänger, H L; Riesner, D

    1986-12-22

    The structure and structural transitions of transcripts of cloned oligomeric viroid were studied in physico-chemical experiments and stability calculations. Transcripts of (+) and (-) polarity, from unit up to sixfold length, were synthesized from DNA clones of the potato spindle tuber viroid (PSTV) with the SP6 transcription system. Their structural properties were investigated by optical denaturation curves, high performance liquid chromatography (HPLC), electron microscopy, sedimentation-diffusion equilibrium and velocity sedimentation. Secondary structures of the RNAs and theoretical denaturation curves were calculated using an energy optimization program. The secondary structure of lowest free energy for unit length and oligomeric transcripts is a rod-like structure similar to that of the mature circular viroids. When this structure is used as a model for calculations, there is a large degree of agreement between the theoretical and the experimental denaturation curves. At high temperatures, however, (+) strand transcripts exhibited a transition which was more stable than expected from the calculations or than was known from curves of mature viroids. This transition arises from a rearrangement of the central conserved region of viroids to a helical region of 28 stable base pairs either intermolecularly leading to bimolecular complexes, or intramolecularly giving rise to a branched secondary structure. The rearrangement could be detected by electron microscopy, HPLC, and analytical ultracentrifugation. The helical region serves to divide up the oligomeric (+) strand into structural units which may be recognized by cleavage and ligation enzymes which process the oligomeric intermediates to circular mature viroids.

  9. Disassembly intermediates of RbsD protein remain oligomeric despite the loss of an intact secondary structure

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Many proteins exist as homo-oligomers in living organisms wherein the change of oligomeric status apparently serves as an effective means for modulating their biological activities. We have previously reported that the homo-decameric RbsD from Escherichia coli undergoes stepwise disassembly and non-stepwise reassembly. Here the structural status of the urea-induced RbsD disassembly intermediates was examined, mainly using urea-containing polyacrylamide gel electrophoresis and chemical cross-linking. Such intermediates were found to remain oligomeric while losing their intact secondary structures. Such disassembly intermediates were able to effectively refold when the concentration of the urea denaturant was reduced to a lower level, or to refold/reassemble into the native decamers when urea was completely removed, as detected by non-denaturing polyacrylamide gel electrophoresis. These novel observations strongly suggest that the assembly of oligomeric proteins may occur before the completion of subunit folding.

  10. Structures and formation mechanisms of aquo/hydroxo oligomeric beryllium in aqueous solution: a density functional theory study.

    Science.gov (United States)

    Jin, Xiaoyan; Liao, Rongbao; Wu, Hai; Huang, Zhengjie; Zhang, Hong

    2015-09-01

    The structures and formation mechanisms of a wide variety of aquo/hydroxo oligomeric beryllium clusters were investigated using density functional theory. The structural parameters of beryllium clusters were found to vary regularly with the stepwise substitution of bound water molecules in the inner coordination sphere by hydroxyl groups. According to the Gibbs free energies deduced from SMD solvation model computations, unhydrolyzed oligomeric beryllium species are the most favorable products of polymerization, independent of the degrees of hydrolysis of the reactants. Simulation of the formation processes of oligomeric beryllium showed that polymerization, in essence, involves the nucleophilic attack of a terminal hydroxyl group in one BeO4 tetrahedron on the beryllium center in another BeO4 tetrahedron, leading to the bridging of two BeO4 tetrahedrons by a hydroxyl group.

  11. Pattern transfer during deposition and fixation of oligomeric bisphenol A on pre-structured copper surfaces.

    Science.gov (United States)

    Szillat, Florian; Fechner, Renate; Mayr, Stefan G

    2013-09-01

    Pattern transfer during deposition of oligomeric bisphenol A (OBA) on pre-structured Cu surfaces is investigated by means of a combined experimental-computational approach. Aiming for quantitative prediction of experiments, as characterized by atomic force microscopy (AFM), we explore the capabilities of stochastic rate equations to quantitatively account for the spatio-temporal evolution of surface topography. While surface diffusion and deposition noise constitute the main mechanisms, pattern transfer is affected by the inclusion of retardation in the interface potential, which appears to be necessary beyond a critical initial surface slope. In addition, routes for successful surface fixation by cross-linking are also demonstrated, which may pave the way for further technological use.

  12. Permeability Changes of Manduca sexta Midgut Brush Border Membranes Induced by Oligomeric Structures of Different Cry Toxins

    NARCIS (Netherlands)

    Muñoz-Garay, C.; Sánchez, J.; Darszon, A.; Maagd, de R.A.; Bakker, P.; Soberón, M.; Bravo, A.

    2006-01-01

    The pore-formation activity of monomeric and oligomeric forms of different Cry1 toxins (from Cry1A to Cry1G) was analyzed by monitoring ionic permeability across Manduca sexta brush border membrane vesicles. The membrane vesicles were isolated from microvilli structures, showing a high enrichment of

  13. Design of a minimal protein oligomerization domain by a structural approach.

    Science.gov (United States)

    Burkhard, P; Meier, M; Lustig, A

    2000-12-01

    Because of the simplicity and regularity of the alpha-helical coiled coil relative to other structural motifs, it can be conveniently used to clarify the molecular interactions responsible for protein folding and stability. Here we describe the de novo design and characterization of a two heptad-repeat peptide stabilized by a complex network of inter- and intrahelical salt bridges. Circular dichroism spectroscopy and analytical ultracentrifugation show that this peptide is highly alpha-helical and 100% dimeric tinder physiological buffer conditions. Interestingly, the peptide was shown to switch its oligomerization state from a dimer to a trimer upon increasing ionic strength. The correctness of the rational design principles used here is supported by details of the atomic structure of the peptide deduced from X-ray crystallography. The structure of the peptide shows that it is not a molten globule but assumes a unique, native-like conformation. This de novo peptide thus represents an attractive model system for the design of a molecular recognition system.

  14. Structure-property relationships in hybrid dental nanocomposite resins containing monofunctional and multifunctional polyhedral oligomeric silsesquioxanes.

    Science.gov (United States)

    Wang, Weiguo; Sun, Xiang; Huang, Li; Gao, Yu; Ban, Jinghao; Shen, Lijuan; Chen, Jihua

    2014-01-01

    Organic-inorganic hybrid materials, such as polyhedral oligomeric silsesquioxanes (POSS), have the potential to improve the mechanical properties of the methacrylate-based composites and resins used in dentistry. In this article, nanocomposites of methacryl isobutyl POSS (MI-POSS [bears only one methacrylate functional group]) and methacryl POSS (MA-POSS [bears eight methacrylate functional groups]) were investigated to determine the effect of structures on the properties of dental resin. The structures of the POSS-containing networks were determined by scanning electron microscopy, transmission electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy. Monofunctional POSS showed a strong tendency toward aggregation and crystallization, while multifunctional POSS showed higher miscibility with the dimethacrylate monomer. The mechanical properties and wear resistance decreased with increasing amounts of MI-POSS, indicating that the MI-POSS agglomerates act as the mechanical weak point in the dental resins. The addition of small amounts of MA-POSS improved the mechanical and shrinkage properties. However, samples with a higher MA-POSS concentration showed lower flexural strength and flexural modulus, indicating that there is a limited range in which the reinforcement properties of MA-POSS can operate. This concentration dependence is attributed to phase separation at higher concentrations of POSS, which affects the structural integrity, and thus, the mechanical and shrinkage properties of the dental resin. Our results show that resin with 3% MA-POSS is a potential candidate for resin-based dental materials.

  15. DHPC strongly affects the structure and oligomerization propensity of Alzheimer's Aβ(1-40) peptide.

    Science.gov (United States)

    Dahse, Kirsten; Garvey, Megan; Kovermann, Michael; Vogel, Alexander; Balbach, Jochen; Fändrich, Marcus; Fahr, Alfred

    2010-11-05

    Alzheimer's disease (AD) is thought to depend on the deleterious action of amyloid fibrils or oligomers derived from β-amyloid (Aβ) peptide. Out of various known Aβ alloforms, the 40-residue peptide Aβ(1-40) occurs at highest concentrations inside the brains of AD patients. Its aggregation properties critically depend on lipids, and it was thus proposed that lipids could play a major role in AD. To better understand their possible effects on the structure of Aβ and on the ability of this peptide to form potentially detrimental amyloid structures, we here analyze the interactions between Aβ(1-40) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC). DHPC has served, due to its controlled properties, as a major model system for studying general lipid properties. Here, we show that DHPC concentrations of 8 mM or higher exert dramatic effects on the conformation of soluble Aβ(1-40) peptide and induce the formation of β-sheet structure at high levels. By contrast, we find that DHPC concentrations well below the critical micelle concentration present no discernible effect on the conformation of soluble Aβ, although they substantially affect the peptide's oligomerization and fibrillation kinetics. These data imply that subtle lipid-peptide interactions suffice in controlling the overall aggregation properties and drastically accelerate, or delay, the fibrillation kinetics of Aβ peptide in near-physiological buffer solutions.

  16. Structure-properties relationships of polyhedral oligomeric silsesquioxane (POSS filled PS nanocomposites

    Directory of Open Access Journals (Sweden)

    J. J. Schwab

    2012-07-01

    Full Text Available The polyhedral oligomeric silsesquioxane (POSS additivated polystyrene (PS based nanocomposites were prepared by melt processing and the structure-properties relationships of the POSS-PS systems were compared to those of the neat PS. In order to investigate the effect of these structural parameters on the final properties of the polymer nanocomposites, five different kinds of POSS samples were used, in particular, POSS with different inorganic cage and with different organic pendent groups. The rheological investigation suggests clearly that the POSS acts as a plasticizer and that the processability of the PS was positively modified. The affinity between the POSS samples and the PS matrix was estimated by the calculated theoretical solubility parameters, considering the Hoy’s method and by morphology analysis. Minor difference between the solubility parameter of POSS and the matrix means better compatibility and no aggregation tendency. Furthermore, the POSS loading leads to a decrease of the rigidity, of the glass transition temperature and of the damping factor of the nanocomposite systems. The loading of different POSS molecules with open cage leads to a more pronounced effect on all the investigated properties that the loading of the POSS molecules with closed cage. Moreover, the melt properties are significantly influenced by the type of inorganic framework, by the type of the pendent organic groups and by the interaction between the POSS organic groups and the host matrix, while, the solid state properties appears to be influenced more by the kind of cage.

  17. AFM study of glucagon fibrillation via oligomeric structures resulting in interwoven fibrils

    Energy Technology Data Exchange (ETDEWEB)

    Dong Mingdong [Interdisciplinary Nanoscience Center (iNANO), University of Aarhus, DK-8000 Aarhus C (Denmark); Hovgaard, Mads Bruun [Interdisciplinary Nanoscience Center (iNANO), University of Aarhus, DK-8000 Aarhus C (Denmark); Xu Sailong [Interdisciplinary Nanoscience Center (iNANO), University of Aarhus, DK-8000 Aarhus C (Denmark); Otzen, Daniel Erik [Interdisciplinary Nanoscience Center (iNANO), University of Aarhus, DK-8000 Aarhus C (Denmark); Besenbacher, Flemming [Interdisciplinary Nanoscience Center (iNANO), University of Aarhus, DK-8000 Aarhus C (Denmark)

    2006-08-28

    Glucagon is a 29-residue amphiphatic hormone involved in the regulation of blood glucose levels in conjunction with insulin. In concentrated aqueous solutions, glucagon spontaneously aggregates to form amyloid fibrils, destroying its biological activity. In this study we utilize the atomic force microscope (AFM) to elucidate the fibrillation mechanism of glucagon at the nanoscale under acidic conditions (pH 2.0) by visualizing the nanostructures of fibrils formed at different stages of the incubation. Hollow disc-shaped oligomers form at an early stage in the process and subsequently rearrange to more solid oligomers. These oligomers co-exist with, and most likely act as precursors for, protofibrils, which subsequently associate to form at least three different classes of higher-order fibrils of different heights. A repeat unit of around 50 nm along the main fibril axis suggests a helical arrangement of interwoven protofibrils. The diversity of oligomeric and fibrillar arrangements formed at pH 2.0 complements previous spectroscopic analyses that revealed that fibrils formed under different conditions can differ substantially in stability and secondary structure.

  18. Glutathione mediated regulation of oligomeric structure and functional activity of Plasmodium falciparum glutathione S-transferase

    Directory of Open Access Journals (Sweden)

    Becker Katja

    2007-10-01

    Full Text Available Abstract Background In contrast to many other organisms, the malarial parasite Plasmodium falciparum possesses only one typical glutathione S-transferase. This enzyme, PfGST, cannot be assigned to any of the known GST classes and represents a most interesting target for antimalarial drug development. The PfGST under native conditions forms non-covalently linked higher aggregates with major population (~98% being tetramer. However, in the presence of 2 mM GSH, a dimer of PfGST is observed. Recently reported study on binding and catalytic properties of PfGST indicated a GSH dependent low-high affinity transition with simultaneous binding of two GSH molecules to PfGST dimer suggesting that GSH binds to low affinity inactive enzyme dimer converting it to high affinity functionally active dimer. In order to understand the role of GSH in tetramer-dimer transition of PfGST as well as in modulation of functional activity of the enzyme, detailed structural, functional and stability studies on recombinant PfGST in the presence and absence of GSH were carried out. Results Our data indicate that the dimer – and not the tetramer – is the active form of PfGST, and that substrate saturation is directly paralleled by dissociation of the tetramer. Furthermore, this dissociation is a reversible process indicating that the tetramer-dimer equilibrium of PfGST is defined by the surrounding GSH concentration. Equilibrium denaturation studies show that the PfGST tetramer has significantly higher stability compared to the dimer. The enhanced stability of the tetramer is likely to be due to stronger ionic interactions existing in it. Conclusion This is the first report for any GST where an alteration in oligomeric structure and not just small conformational change is observed upon GSH binding to the enzyme. Furthermore we also demonstrate a reversible mechanism of regulation of functional activity of Plasmodium falciparum glutathione S-transferase via GSH induced

  19. Structural reorganisation and potential toxicity of oligomeric species formed during the assembly of amyloid fibrils.

    Directory of Open Access Journals (Sweden)

    Mookyung Cheon

    2007-09-01

    Full Text Available Increasing evidence indicates that oligomeric protein assemblies may represent the molecular species responsible for cytotoxicity in a range of neurological disorders including Alzheimer and Parkinson diseases. We use all-atom computer simulations to reveal that the process of oligomerization can be divided into two steps. The first is characterised by a hydrophobic coalescence resulting in the formation of molten oligomers in which hydrophobic residues are sequestered away from the solvent. In the second step, the oligomers undergo a process of reorganisation driven by interchain hydrogen bonding interactions that induce the formation of beta sheet rich assemblies in which hydrophobic groups can become exposed. Our results show that the process of aggregation into either ordered or amorphous species is largely determined by a competition between the hydrophobicity of the amino acid sequence and the tendency of polypeptide chains to form arrays of hydrogen bonds. We discuss how the increase in solvent-exposed hydrophobic surface resulting from such a competition offers an explanation for recent observations concerning the cytotoxicity of oligomeric species formed prior to mature amyloid fibrils.

  20. Differential structural remodelling of heparan sulfate by chemokines: the role of chemokine oligomerization

    Science.gov (United States)

    Migliorini, Elisa; Salanga, Catherina L.; Thakar, Dhruv

    2017-01-01

    Chemokines control the migration of cells in normal physiological processes and in the context of disease such as inflammation, autoimmunity and cancer. Two major interactions are involved: (i) binding of chemokines to chemokine receptors, which activates the cellular machinery required for movement; and (ii) binding of chemokines to glycosaminoglycans (GAGs), which facilitates the organization of chemokines into haptotactic gradients that direct cell movement. Chemokines can bind and activate their receptors as monomers; however, the ability to oligomerize is critical for the function of many chemokines in vivo. Chemokine oligomerization is thought to enhance their affinity for GAGs, and here we show that it significantly affects the ability of chemokines to accumulate on and be retained by heparan sulfate (HS). We also demonstrate that several chemokines differentially rigidify and cross-link HS, thereby affecting HS rigidity and mobility, and that HS cross-linking is significantly enhanced by chemokine oligomerization. These findings suggest that chemokine–GAG interactions may play more diverse biological roles than the traditional paradigms of physical immobilization and establishment of chemokine gradients; we hypothesize that they may promote receptor-independent events such as physical re-organization of the endothelial glycocalyx and extracellular matrix, as well as signalling through proteoglycans to facilitate leukocyte adhesion and transmigration. PMID:28123055

  1. A New Star-shaped Carbazole Derivative with Polyhedral Oligomeric Silsesquioxane Core: Crystal Structure and Unique Photoluminescence Property.

    Science.gov (United States)

    Xu, Zixuan; Yu, Tianzhi; Zhao, Yuling; Zhang, Hui; Zhao, Guoyun; Li, Jianfeng; Chai, Lanqin

    2016-01-01

    A new inorganic–organic hybrid material based on polyhedral oligomeric silsesquioxane (POSS) capped with carbazolyl substituents, octakis[3-(carbazol-9-yl)propyldimethylsiloxy]-silsesquioxane (POSS-8Cz), was successfully synthesized and characterized. The X-ray crystal structure of POSS-8Cz were described. The photophysical properties of POSS-8Cz were investigated by using UV–vis,photoluminescence spectroscopic analysis. The hybrid material exhibits blue emission in the solution and the solid film.The morphology and thermal stablity properties were measured by X-ray diffraction (XRD) and TG-DTA analysis.

  2. Oligomeric structure and minimal functional unit of the electrogenic sodium bicarbonate cotransporter NBCe1-A.

    Science.gov (United States)

    Kao, Liyo; Sassani, Pakan; Azimov, Rustam; Pushkin, Alexander; Abuladze, Natalia; Peti-Peterdi, Janos; Liu, Weixin; Newman, Debra; Kurtz, Ira

    2008-09-26

    The electrogenic sodium bicarbonate cotransporter NBCe1-A mediates the basolateral absorption of sodium and bicarbonate in the proximal tubule. In this study the oligomeric state and minimal functional unit of NBCe1-A were investigated. Wild-type (wt) NBCe1-A isolated from mouse kidney or heterologously expressed in HEK293 cells was predominantly in a dimeric state as was shown using fluorescence energy transfer, pulldown, immunoprecipitation, cross-linking experiments, and nondenaturing perfluorooctanoate-PAGE. NBCe1-A monomers were found to be covalently linked by S-S bonds. When each of the 15 native cysteine residues were individually removed on a wt-NBCe1-A backbone, dimerization of the cotransporter was not affected. In experiments involving multiple native cysteine residue removal, both Cys(630) and Cys(642) in extracellular loop 3 were shown to mediate S-S bond formation between NBCe1-A monomers. When native NBCe1-A cysteine residues were individually reintroduced into a cysteineless NBCe1-A mutant backbone, the finding that a Cys(992) construct that lacked S-S bonds functioned normally indicated that stable covalent linkage of NBCe1-A monomers was not a necessary requirement for functional activity of the cotransporter. Studies using concatameric constructs of wt-NBCe1-A, whose activity is resistant to methanesulfonate reagents, and an NBCe1-A(T442C) mutant, whose activity is completely inhibited by methanesulfonate reagents, confirmed that NBCe1-A monomers are functional. Our results demonstrate that wt-NBCe1-A is predominantly a homodimer, dependent on S-S bond formation that is composed of functionally active monomers.

  3. The crystal structure of the signature domain of cartilage oligomeric matrix protein: implications for collagen, glycosaminoglycan and integrin binding.

    Science.gov (United States)

    Tan, Kemin; Duquette, Mark; Joachimiak, Andrzej; Lawler, Jack

    2009-08-01

    Cartilage oligomeric matrix protein (COMP), or thrombospondin-5 (TSP-5), is a secreted glycoprotein that is important for growth plate organization and function. Mutations in COMP cause two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1). In this study, we determined the structure of a recombinant protein that contains the last epidermal growth factor repeat, the type 3 repeats and the C-terminal domain (CTD) of COMP to 3.15-A resolution limit by X-ray crystallography. The CTD is a beta-sandwich that is composed of 15 antiparallel beta-strands, and the type 3 repeats are a contiguous series of calcium binding sites that associate with the CTD at multiple points. The crystal packing reveals an exposed potential metal-ion-dependent adhesion site (MIDAS) on one edge of the beta-sandwich that is common to all TSPs and may serve as a binding site for collagens and other ligands. Disease-causing mutations in COMP disrupt calcium binding, disulfide bond formation, intramolecular interactions, or sites for potential ligand binding. The structure presented here and its unique molecular packing in the crystal identify potential interactive sites for glycosaminoglycans, integrins, and collagens, which are key to cartilage structure and function.

  4. N-terminal aliphatic residues dictate the structure, stability, assembly, and small molecule binding of the coiled-coil region of cartilage oligomeric matrix protein.

    Science.gov (United States)

    Gunasekar, Susheel K; Asnani, Mukta; Limbad, Chandani; Haghpanah, Jennifer S; Hom, Wendy; Barra, Hanna; Nanda, Soumya; Lu, Min; Montclare, Jin Kim

    2009-09-15

    The coiled-coil domain of cartilage oligomeric matrix protein (COMPcc) assembles into a homopentamer that naturally recognizes the small molecule 1,25-dihydroxyvitamin D(3) (vit D). To identify the residues critical for the structure, stability, oligomerization, and binding to vit D as well as two other small molecules, all-trans-retinol (ATR) and curcumin (CCM), here we perform an alanine scanning mutagenesis study. Ten residues lining the hydrophobic pocket of COMPcc were mutated into alanine; of the mutated residues, the N-terminal aliphatic residues L37, L44, V47, and L51 are responsible for maintaining the structure and function. Furthermore, two polar residues, T40 and Q54, within the N-terminal region when converted into alanine improve the alpha-helical structure, stability, and self-assembly behavior. Helical stability, oligomerization, and binding appear to be linked in a manner in which mutations that abolish helical structure and assembly bind poorly to vit D, ATR, and CCM. These results provide not only insight into COMPcc and its functional role but also useful guidelines for the design of stable, pentameric coiled-coils capable of selectively storing and delivering various small molecules.

  5. Structure-based Design of a Disulfide-lined Oligomeric Form of the Simian Virus 40 (SV40) Large T Antigen DNA-Binding Domain

    Energy Technology Data Exchange (ETDEWEB)

    G Meinke; P Phelan; A Fradet-Turcotte; J Archambault; P Bullock

    2011-12-31

    The modular multifunctional protein large T antigen (T-ag) from simian virus 40 orchestrates many of the events needed for replication of the viral double-stranded DNA genome. This protein assembles into single and double hexamers on specific DNA sequences located at the origin of replication. This complicated process begins when the origin-binding domain of large T antigen (T-ag ODB) binds the GAGGC sequences in the central region (site II) of the viral origin of replication. While many of the functions of purified T-ag OBD can be studied in isolation, it is primarily monomeric in solution and cannot assemble into hexamers. To overcome this limitation, the possibility of engineering intermolecular disulfide bonds in the origin-binding domain which could oligomerize in solution was investigated. A recent crystal structure of the wild-type T-ag OBD showed that this domain forms a left-handed spiral in the crystal with six subunits per turn. Therefore, we analyzed the protein interface of this structure and identified two residues that could potentially support an intermolecular disulfide bond if changed to cysteines. SDS-PAGE analysis established that the mutant T-ag OBD formed higher oligomeric products in a redox-dependent manner. In addition, the 1.7 {angstrom} resolution crystal structure of the engineered disulfide-linked T-ag OBD is reported, which establishes that oligomerization took place in the expected manner.

  6. Structural characteristics of oligomeric DNA strands adsorbed onto single-walled carbon nanotubes.

    Science.gov (United States)

    Roxbury, Daniel; Jagota, Anand; Mittal, Jeetain

    2013-01-10

    The single-stranded DNA to single-walled carbon nanotube (SWCNT) hybrid continues to attract significant interest as an exemplary biological molecule-nanomaterial conjugate. In addition to their many biomedical uses, such as in vivo sensing and delivery of molecular cargo, DNA-SWCNT hybrids enable the sorting of SWCNTs according to their chirality. Current experimental methods have fallen short of identifying the actual structural ensemble of DNA adsorbed onto SWCNTs that enables and controls several of these phenomena. Molecular dynamics (MD) simulation has been a useful tool for studying the structure of these hybrid molecules. In recent studies, using replica exchange MD (REMD) simulation we have shown that novel secondary structures emerge and that these structures are DNA-sequence and SWCNT-type dependent. Here, we use REMD to investigate in detail the structural characteristics of two DNA-SWCNT recognition pairs: (TAT)(4)-(6,5)-SWCNT, i.e., DNA sequence TATTATTATTAT bound to the (6,5) chirality SWCNT, and (CCG)(2)CC-(8,7)-SWCNT as well as off-recognition pairs (TAT)(4)-(8,7)-SWCNT and (CCG)(2)CC-(6,5)-SWCNT. From a structural clustering analysis, dominant equilibrium structures are identified and show a right-handed self-stitched motif for (TAT)(4)-(6,5) in contrast to a left-handed β-barrel for (CCG)(2)CC-(8,7). Additionally, characteristics such as DNA end-to-end distance, solvent accessible SWCNT surface area, DNA hydrogen bonding between bases, and DNA dihedral distributions have been probed in detail as a function of the number of DNA strands adsorbed onto the nanotube. We find that the DNA structures adsorbed onto a nanotube are also stabilized by significant numbers of non-Watson-Crick hydrogen bonds (intrastrand and interstrand) in addition to π-π stacking between DNA bases and nanotube surface and Watson-Crick pairs. Finally, we provide a summary of DNA structures observed for various DNA-SWCNT hybrids as a preliminary set of motifs that may be

  7. Glycolonitrile oligomerization: structure of isolated oxazolines, potential heterocycles on the early earth

    Science.gov (United States)

    Arrhenius, G.; Baldridge, K. K.; Richards-Gross, S.; Siegel, J. S.; Bada, J. L. (Principal Investigator)

    1997-01-01

    A study of glycolonitrile polymerization has led to the isolation and characterization of two 2,5-dihydro-4-aminooxazoles, 4 and 5. Previous reports have misassigned these structures as s-triazines or pyrimidines. X-ray diffraction analysis of crystals of 4 and an acetylated oxazole derivative of 5 (6) confirm the proposed structures. Ab initio computations are used to assess the relative thermodynamic stability of three trimer isomers (an s-triazine, an aminohydroxypyrimidine, and an aminooxazoline), and the results indicate that 4 is a novel kinetic product. Mechanistic considerations rationalize kinetic oxazole formation over the more customary triazine or pyrimidine trimers.

  8. Structures of pattern recognition receptors reveal molecular mechanisms of autoinhibition, ligand recognition and oligomerization.

    Science.gov (United States)

    Chuenchor, Watchalee; Jin, Tengchuan; Ravilious, Geoffrey; Xiao, T Sam

    2014-02-01

    Pattern recognition receptors (PRRs) are essential sentinels for pathogens or tissue damage and integral components of the innate immune system. Recent structural studies have provided unprecedented insights into the molecular mechanisms of ligand recognition and signal transduction by several PRR families at distinct subcellular compartments. Here we highlight some of the recent discoveries and summarize the common themes that are emerging from these exciting studies. Better mechanistic understanding of the structure and function of the PRRs will improve future prospects of therapeutic targeting of these important innate immune receptors.

  9. Molecular structure of basic oligomeric building units of heparan-sulfate glycosaminoglycans

    NARCIS (Netherlands)

    Remko, Milan; Van Duijnen, Piet Th.; Broer, Ria

    2010-01-01

    This study reports in detail the results of systematic large-scale theoretical investigations of the acidic dimeric structural units (D-E, E-F, F-G, and G-H) and pentamer D-E-F-G-H (fondaparinux) of the glycosaminoglycan heparin, and their anionic forms. The geometries and energies of these oligomer

  10. Characterization of Oligomeric Xylan Structures from Corn Fiber Resistant to Pretreatment and Simultaneous Saccharification and Fermentation

    NARCIS (Netherlands)

    Appeldoorn, M.M.; Kabel, M.A.; Eylen, van D.; Gruppen, H.; Schols, H.A.

    2010-01-01

    Corn fiber, a byproduct from the corn industry, would be a good source for bioethanol production if the hemicellulose, consisting of polymeric glucoronoarabinoxylans, can be degraded into fermentable sugars. Structural knowledge of the hemicellulose is needed to improve the enzymatic hydrolyses of c

  11. Giant extracellular Glossoscolex paulistus Hemoglobin (HbGp) upon interaction with cethyltrimethylammonium chloride (CTAC) and sodium dodecyl sulphate (SDS) surfactants: Dissociation of oligomeric structure and autoxidation.

    Science.gov (United States)

    Santiago, Patricia S; Moreira, Leonardo M; de Almeida, Erika V; Tabak, Marcel

    2007-04-01

    The effects of two ionic surfactants on the oligomeric structure of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) in the oxy - form have been studied through the use of several spectroscopic techniques such as electronic optical absorption, fluorescence emission, light scattering, and circular dichroism. The use of anionic sodium dodecyl sulphate (SDS) and cationic cethyltrimethyl ammonium chloride (CTAC) has allowed to differentiate the effects of opposite headgroup charges on the oligomeric structure dissociation and hemoglobin autoxidation. At pH 7.0, both surfactants induce the protein dissociation and a significant oxidation. Spectral changes occur at very low CTAC concentrations suggesting a significant electrostatic contribution to the protein-surfactant interaction. At low protein concentration, 0.08 mg/ml, some light scattering within a narrow CTAC concentration range occurs due to protein-surfactant precipitation. Light scattering experiments showed the dissociation of the oligomeric structure by SDS and CTAC, and the effect of precipitation induced by CTAC. At higher protein concentrations, 3.0 mg/ml, a precipitation was observed due to the intense charge neutralization upon formation of ion pair in the protein-surfactant precipitate. The spectral changes are spread over a much wider SDS concentration range, implying a smaller electrostatic contribution to the protein-surfactant interactions. The observed effects are consistent with the acid isoelectric point (pI) of this class of hemoglobins, which favors the intense interaction of HbGp with the cationic surfactant due to the existence of excess acid anionic residues at the protein surface. Protein secondary structure changes are significant for CTAC at low concentrations while they occur at significantly higher concentrations for SDS. In summary, the cationic surfactant seems to interact more strongly with the protein producing more dramatic spectral changes as compared to the

  12. Toxic and non-toxic aggregates from the SBMA and normal forms of androgen receptor have distinct oligomeric structures.

    Science.gov (United States)

    Jochum, Tobias; Ritz, Manuela E; Schuster, Christoph; Funderburk, Sarah F; Jehle, Katja; Schmitz, Katja; Brinkmann, Falko; Hirtz, Michael; Moss, David; Cato, Andrew C B

    2012-06-01

    Hormone-dependent aggregation of the androgen receptor (AR) with a polyglutamine (polyQ) stretch amplification (>38) is considered to be the causative agent of the neurodegenerative disorder spinal and bulbar muscular atrophy (SBMA), consistent with related neurodegenerative diseases involving polyQ-extended proteins. In spite of the widespread acceptance of this common causal hypothesis, little attention has been paid to its apparent incompatibility with the observation of AR aggregation in healthy individuals with no polyQ stretch amplification. Here we used atomic force microscopy (AFM) to characterize sub-micrometer scale aggregates of the wild-type (22 glutamines) and the SBMA form (65 glutamines), as well as a polyQ deletion mutant (1 glutamine) and a variant with a normal length polyQ stretch but with a serine to alanine double mutation elsewhere in the protein. We used a baculovirus-insect cell expression system to produce full-length proteins for these structural analyses. We related the AFM findings to cytotoxicity as measured by expression of the receptors in Drosophila motoneurons or in neuronal cells in culture. We found that the pathogenic AR mutants formed oligomeric fibrils up to 300-600nm in length. These were clearly different from annular oligomers 120-180nm in diameter formed by the nonpathogenic receptors. We could also show that melatonin, which is known to ameliorate the pathological phenotype in the fly model, caused polyQ-extended AR to form annular oligomers. Further comparative investigation of these reproducibly distinct toxic and non-toxic oligomers could advance our understanding of the molecular basis of the polyQ pathologies.

  13. Computational and structural evidence for neurotransmitter-mediated modulation of the oligomeric states of human insulin in storage granules.

    Science.gov (United States)

    Palivec, Vladimír; Viola, Cristina M; Kozak, Mateusz; Ganderton, Timothy R; Křížková, Květoslava; Turkenburg, Johan P; Halušková, Petra; Žáková, Lenka; Jiráček, Jiří; Jungwirth, Pavel; Brzozowski, Andrzej M

    2017-05-19

    Human insulin is a pivotal protein hormone controlling metabolism, growth, and aging and whose malfunctioning underlies diabetes, some cancers, and neurodegeneration. Despite its central position in human physiology, the in vivo oligomeric state and conformation of insulin in its storage granules in the pancreas are not known. In contrast, many in vitro structures of hexamers of this hormone are available and fall into three conformational states: T6, T3R(f)3, and R6 As there is strong evidence for accumulation of neurotransmitters, such as serotonin and dopamine, in insulin storage granules in pancreatic β-cells, we probed by molecular dynamics (MD) and protein crystallography (PC) if these endogenous ligands affect and stabilize insulin oligomers. Parallel studies independently converged on the observation that serotonin binds well within the insulin hexamer (site I), stabilizing it in the T3R3 conformation. Both methods indicated serotonin binding on the hexamer surface (site III) as well. MD, but not PC, indicated that dopamine was also a good site III ligand. Some of the PC studies also included arginine, which may be abundant in insulin granules upon processing of pro-insulin, and stable T3R3 hexamers loaded with both serotonin and arginine were obtained. The MD and PC results were supported further by in solution spectroscopic studies with R-state-specific chromophore. Our results indicate that the T3R3 oligomer is a plausible insulin pancreatic storage form, resulting from its complex interplay with neurotransmitters, and pro-insulin processing products. These findings may have implications for clinical insulin formulations. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Structure-based design of a disulfide-linked oligomeric form of the simian virus 40 (SV40) large T antigen DNA-binding domain

    Energy Technology Data Exchange (ETDEWEB)

    Meinke, Gretchen; Phelan, Paul [Tufts School of Medicine and the Sackler School of Graduate Biomedical Sciences, 136 Harrison Avenue, Boston, MA 02111 (United States); Fradet-Turcotte, Amélie; Archambault, Jacques [Institut de Recherches Cliniques de Montréal (IRCM), Montreal, Quebec (Canada); Bullock, Peter A., E-mail: peter.bullock@tufts.edu [Tufts School of Medicine and the Sackler School of Graduate Biomedical Sciences, 136 Harrison Avenue, Boston, MA 02111 (United States)

    2011-06-01

    With the aim of forming the ‘lock-washer’ conformation of the origin-binding domain of SV40 large T antigen in solution, using structure-based analysis an intermolecular disulfide bridge was engineered into the origin-binding domain to generate higher order oligomers in solution. The 1.7 Å resolution structure shows that the mutant forms a spiral in the crystal and has the de novo disulfide bond at the protein interface, although structural rearrangements at the interface are observed relative to the wild type. The modular multifunctional protein large T antigen (T-ag) from simian virus 40 orchestrates many of the events needed for replication of the viral double-stranded DNA genome. This protein assembles into single and double hexamers on specific DNA sequences located at the origin of replication. This complicated process begins when the origin-binding domain of large T antigen (T-ag ODB) binds the GAGGC sequences in the central region (site II) of the viral origin of replication. While many of the functions of purified T-ag OBD can be studied in isolation, it is primarily monomeric in solution and cannot assemble into hexamers. To overcome this limitation, the possibility of engineering intermolecular disulfide bonds in the origin-binding domain which could oligomerize in solution was investigated. A recent crystal structure of the wild-type T-ag OBD showed that this domain forms a left-handed spiral in the crystal with six subunits per turn. Therefore, we analyzed the protein interface of this structure and identified two residues that could potentially support an intermolecular disulfide bond if changed to cysteines. SDS–PAGE analysis established that the mutant T-ag OBD formed higher oligomeric products in a redox-dependent manner. In addition, the 1.7 Å resolution crystal structure of the engineered disulfide-linked T-ag OBD is reported, which establishes that oligomerization took place in the expected manner.

  15. Hydrolyzable Tannins of Tamaricaceous Plants. 7.1 Structures and Cytotoxic Properties of Oligomeric Ellagitannins from Leaves of Tamarix nilotica and Cultured Tissues of Tamarix tetrandra.

    Science.gov (United States)

    Orabi, Mohamed A A; Taniguchi, Shoko; Sakagami, Hiroshi; Yoshimura, Morio; Amakura, Yoshiaki; Hatano, Tsutomu

    2016-04-22

    Partially unacylated new oligomeric hydrolyzable tannins, nilotinin T2 (1, trimer) and nilotinin Q1 (2, tetramer), together with four known trimers, nilotinin T1 (3) and hirtellins T1-T3 (4-6), and a dimer, tamarixinin B (7), were isolated from the aqueous acetone extracts of leaves of Tamarix nilotica. Among them, the new trimer 1 and the known trimers 4 and 6, in addition to the partially unacylated new trimer nilotinin T3 (8), the known dimers nilotinin D3 (9) and tamarixinin C (10), and the monomer tellimagrandin I (11), were isolated from the cultured shoots of Tamarix tetrandra. The structures of the new hydrolyzable tannins were established by chromatographic analyses and extensive 1D and 2D NMR, HRESI-TOFMS, and ECD spectroscopic experiments. Among the new oligomeric tannins, the particular unacylated position of a glucose core is attributed to a possible biosynthetic route. Isolation of the same oligomeric tannins from cultured shoots of T. tetrandra emphasizes the unique biogenetic ability of the obtained cultures on production of the structurally and biologically characteristic tamaricaceous tannins commonly produced by the intact Tamarix plants. Additionally, tannins obtained in the present study together with gemin D (12) and 1,3-di-O-galloyl-4,6-O-(aS)-hexahydroxydiphenoyl-β-d-glucose (13), from our previous investigation of the leaves of T. nilotica, exhibited variable tumor-specific cytotoxic effects. The ellagitannin trimers 4, 6, and 8 and the dimer 9 exerted predominant tumor-selective cytotoxic effects with high specificity toward human promyelocytic leukemia cells.

  16. Structural and phylogenetic studies with MjTX-I reveal a multi-oligomeric toxin--a novel feature in Lys49-PLA2s protein class.

    Directory of Open Access Journals (Sweden)

    Guilherme H M Salvador

    Full Text Available The mortality caused by snakebites is more damaging than many tropical diseases, such as dengue haemorrhagic fever, cholera, leishmaniasis, schistosomiasis and Chagas disease. For this reason, snakebite envenoming adversely affects health services of tropical and subtropical countries and is recognized as a neglected disease by the World Health Organization. One of the main components of snake venoms is the Lys49-phospholipases A2, which is catalytically inactive but possesses other toxic and pharmacological activities. Preliminary studies with MjTX-I from Bothrops moojeni snake venom revealed intriguing new structural and functional characteristics compared to other bothropic Lys49-PLA2s. We present in this article a comprehensive study with MjTX-I using several techniques, including crystallography, small angle X-ray scattering, analytical size-exclusion chromatography, dynamic light scattering, myographic studies, bioinformatics and molecular phylogenetic analyses.Based in all these experiments we demonstrated that MjTX-I is probably a unique Lys49-PLA2, which may adopt different oligomeric forms depending on the physical-chemical environment. Furthermore, we showed that its myotoxic activity is dramatically low compared to other Lys49-PLA2s, probably due to the novel oligomeric conformations and important mutations in the C-terminal region of the protein. The phylogenetic analysis also showed that this toxin is clearly distinct from other bothropic Lys49-PLA2s, in conformity with the peculiar oligomeric characteristics of MjTX-I and possible emergence of new functionalities in response to environmental changes and adaptation to new preys.

  17. Reversible peptide oligomerization over nanoscale gold surfaces

    Directory of Open Access Journals (Sweden)

    Kazushige Yokoyama

    2015-11-01

    Full Text Available A selective oligomeric formation of amyloid beta 1-40 (Ab1-40 monomers over a nanogold colloidal surface was investigated. An unfolded Ab1-40 monomer is considered to construct a dimer or trimer based oligomeric form with its hydrophobic segment placing outward under an acidic condition. Under a basic condition, a conformation of Ab is expected to take a folded monomeric form with its hydrophilic segment folded inward, avoiding the networking with residual colloidal particles. The most probable oligomeric form constructed over a 20 nm gold colloidal surface within a 25 ℃ to 65 ℃ temperature range is a dimer based unit and that over 30 or 40 nm gold colloidal surface below 15 ℃ is concluded to be a trimer based unit. However, selective oligomerization was not successfully reproduced under the rest of the conditions. A dipole-induced dipole interaction must cause a flexible structural change between folded and unfolded forms.

  18. Transition metal complexes bearing 2,6-bis(imino)pyridyl:Synthesis, structure, ethylene polymerization/oligomerization studies

    Institute of Scientific and Technical Information of China (English)

    SU; Biyun; ZHAO; Jianshe; SUN; Wenhua

    2005-01-01

    A series of new complexes {2,6-bis[1-((2-methyl-4-methoxyphenyl)imino)ethyl]pyridine}MCI2 [M=Fe(Ⅱ) (2), Co(Ⅱ) (3), Ni(Ⅱ) (4), Cu(Ⅱ) (5), Zn(Ⅱ) (6)] have been synthesized. At 25℃, using 500 equiv of methylaluminoxane (MAO), the activities of Fe(Ⅱ), Co(Ⅱ) catalysts can ethylene oligomerization. The effects of polymerization conditions such as reaction temperature,AI/M molar ratio and time on the activity of catalyst have been explored.

  19. Influence of oligomeric herbicidal ionic liquids with MCPA and Dicamba anions on the community structure of autochthonic bacteria present in agricultural soil

    Energy Technology Data Exchange (ETDEWEB)

    Ławniczak, Ł., E-mail: lukasz.k.lawniczak@wp.pl [Department of Chemical Technology, Poznan University of Technology, 60-965 Poznan (Poland); Syguda, A., E-mail: Anna.Syguda@put.poznan.pl [Department of Chemical Technology, Poznan University of Technology, 60-965 Poznan (Poland); Borkowski, A., E-mail: a.borkowski@uw.edu.pl [Faculty of Geology, University of Warsaw, 02-089 Warsaw (Poland); Cyplik, P., E-mail: pcyplik@wp.pl [Department of Biotechnology and Food Microbiology, University of Life Sciences in Poznan, 60-627 Poznan (Poland); Marcinkowska, K., E-mail: k.marcinkowska@iorpib.poznan.pl [Institute of Plant Protection - National Research Institute, Poznan 60-318 (Poland); Wolko, Ł., E-mail: wolko@o2.pl [Department of Biochemistry and Biotechnology, Poznań University of Life Sciences in Poznan, 60-632 Poznan (Poland); Praczyk, T., E-mail: t.praczyk@iorpib.poznan.pl [Institute of Plant Protection - National Research Institute, Poznan 60-318 (Poland); Chrzanowski, Ł., E-mail: Lukasz.Chrzanowski@put.poznan.pl [Department of Chemical Technology, Poznan University of Technology, 60-965 Poznan (Poland); Pernak, J., E-mail: Juliusz.Pernak@put.poznan.pl [Department of Chemical Technology, Poznan University of Technology, 60-965 Poznan (Poland)

    2016-09-01

    The aim of this study was to evaluate the impact of selected herbicidal ionic liquids (HILs), which exhibit high efficacy in terms of weed control and low toxicity, but may be persistent due to limited biodegradability, on the community structure of autochthonic bacteria present in agricultural soil. Four different oligomeric HILs (with two types of cations and different ratio of herbicidal anions) were synthesized and characterized by employing {sup 1}H and {sup 13}C NMR. The results of biodegradation assay indicated that none of the tested HILs could be classified as readily biodegradable (biodegradation rate ranged from 0 to 7%). The conducted field studies confirmed that the herbicidal efficacy of the HILs was higher compared to the reference herbicide mixture by 10 to 30%, depending on the dose and weed species. After termination of field studies, the soil treated with the tested HILs was subjected to next generation sequencing in order to investigate the potential changes in the bacterial community structure. Proteobacteria was the dominant phylum in all studied samples. Treatment with the studied HILs resulted in an increase of Actinobacteria compared to the reference herbicidal mixture. Differenced among the studied HILs were generally associated with a significantly higher abundance of Bacteroidetes in case of 1-HIL-Dicamba 1/3 and Firmicutes in case of 2-HIL-Dicamba 1/3. - Highlights: • Impact of herbicidal ionic liquids on bacterial community structure was studied. • Oligomeric herbicidal ionic liquids were effective but not readily biodegradable. • Next generation sequencing was used to evaluate shifts in bacterial abundance. • Treatment during field trials resulted in changes at class and species level. • Use of herbicidal ionic liquids affects the structure of autochthonic soil bacteria.

  20. Effect of valine 106 on structure-function relation of cytosolic human thymidine kinase - Kinetic properties and oligomerization pattern of nine substitution mutants of V106

    DEFF Research Database (Denmark)

    Frederiksen, Hanne; Berenstein, Dvora; Munch-Petersen, Birgitte

    2004-01-01

    Information on the regulation and structure-function relation of enzymes involved in DNA precursor synthesis is pivotal, as defects in several of these enzymes have been found to cause depletion or deletion of mitochondrial DNA resulting in severe diseases. Here, the effect of amino acid 106...... on the enzymatic properties of the cell-cycle-regulated human cytosolic thymidine kinase 1 (TK1) is investigated. On the basis of the previously observed profound differences between recombinant TK1 with Val106 (V106WT) and Met106 (V106M) in catalytic activity and oligomerization pattern, we designed...... transition from a dimer with low catalytic activity to a tetramer with high catalytic activity. Group II (V106G, V106H, V106K, V106L and V106Q) behaves like V106M in that they are permanently high activity tetramers, irrespective of ATP exposure. We conclude that size and conformation of amino acid 106...

  1. Single Site Silica Supported Tetramethyl Niobium by the SOMC Strategy: Synthesis, Characterization and Structure-Activity Relationship in Ethylene Oligomerization Reaction

    KAUST Repository

    Hamieh, Ali Imad Ali

    2017-06-06

    Silica supported Tetramethyl niobium complex [(≡SiO)NbMe4] 2 has been isolated by surface alkylation of [(≡SiO-)NbCl3Me] 1 with dimethyl zinc in pentane. 1 can be easily synthesized by grafting of NbCl3Me2 on to the surface of partially dehydroxylated silica by the SOMC strategy. Precise structural analysis was carried out by the FTIR, advance solid state NMR, elemental analysis and mass balance techniques (gas quantification after treating 2 with degassed water) . Complex 1 was found to be active in the ethylene oligomerization to produce up to C30, whereas to our surprise complex 2 selectively dimerizes ethylene into 1-butene in the absence of a co-catalyst at the same conversion levels.

  2. Structural basis for phosphorylated autoinducer-2 modulation of the oligomerization state of the global transcription regulator LsrR from Escherichia coli.

    Science.gov (United States)

    Wu, Minhao; Tao, Yue; Liu, Xiaotian; Zang, Jianye

    2013-05-31

    Quorum-sensing systems are widely used by bacteria to control behavior in response to fluctuations in cell density. Several small diffusible molecules called autoinducers act as signaling molecules in quorum-sensing processes through interplay with sensors. Autoinducers modulate vital physiological functions such as nutrient acquisition, gene transcription, and virulence factor production. In Escherichia coli, LsrR serves as a global transcription regulator that responds to autoinducer-2 to regulate the expression of a variety of genes, including the lsr operon and the lsrR gene. Here, we report the crystal structure of full-length LsrR from E. coli, which has an N-terminal DNA-binding domain and a C-terminal ligand-binding domain connected by a β-strand. Although only two molecules are found in one asymmetric unit, two neighboring dimers pack to form a tetramer that is consistent with the oligomerization state of LsrR in solution. Mutagenesis experiments and gel shift assays indicated that Gln-33 and Tyr-26 might be involved in interactions between LsrR and DNA. The LsrR-binding site for phosphorylated autoinducer-2 was predicted by structural comparisons of LsrR with CggR and SorC. Cross-linking, size exclusion chromatography, and gel shift assays determined that phosphorylated autoinducer-2 triggered the disassembly of the LsrR tetramer into dimers and reduced the DNA binding ability of LsrR. Our findings reveal a mechanism for the change in the oligomerization state of LsrR in the presence of phosphorylated autoinducer-2. Based on these observations, we propose that phosphorylated autoinducer-2 triggers the disassembly of the LsrR tetramer to activate the transcription of its target genes.

  3. Two novel cyclodextrin-degrading enzymes isolated from thermophilic bacteria have similar domain structures but differ in oligomeric state and activity profile.

    Science.gov (United States)

    Turner, Pernilla; Labes, Antje; Fridjonsson, Olafur H; Hreggvidson, Gudmundur O; Schönheit, Peter; Kristjansson, Jakob K; Holst, Olle; Karlsson, Eva Nordberg

    2005-10-01

    In this paper, we present the expression and characterization of two novel enzymes from the alpha-amylase family exhibiting cyclomaltodextrinase specificity. The nucleotide sequences encoding the enzymes were isolated from the genomic DNA of two thermophilic bacterial strains originating from Icelandic hot springs and belonging to the genera Anoxybacillus (AfCda13) and Laceyella (LsCda13). The genes were amplified using a consensus primer strategy utilizing two of the four conserved regions present in glycoside hydrolase family 13. No identifiable signal peptides were present in open reading frames encoding the enzymes, indicating an intracellular location of both enzymes, and their physiological function to be intracellular cyclodextrin degradation. The domain structures of both enzymes were also similar, including an N-terminal domain, the catalytic module composed of the A- and B-domains, and a C-terminal domain. Despite the similarity in domain composition, the two enzymes displayed differences in the oligomeric state with AfCda13 being a dimeric protein, whereas LsCda13 was monomeric. The two enzymes also displayed significantly different activity profiles, despite being active on the same range of substrates. It was shown that the enzyme displaying the highest activity on cyclodextrin was dimeric (AfCda13). Moreover, a fraction of the dimeric enzyme could be converted to a monomeric state in the presence of KCl and this fraction retained only 23% of its activity on alpha-cyclodextrin while its activity on starch was not significantly affected, indicating that the oligomeric state is an important factor for a high activity on cyclodextrin substrates.

  4. Rationalizing oligomerization in dimethylindium(III) chalcogenolates (Me2InER') (E = O, S, Se): a structural and computational study.

    Science.gov (United States)

    Briand, Glen G; Decken, Andreas; Hamilton, Nathan S

    2010-04-28

    The effect on oligomerization of increased steric bulk in dimethylindium(III) chalcogenolates (Me(2)InER') (E = O, S, Se) has been examined. The facile reaction of Me(3)In with a series of phenols, thiophenols and selenophenols afforded the compounds [Me(2)InO(C(6)H(5))](2) (1), [Me(2)InO(2,6-Me(2)C(6)H(3))](2) (2), Me(2)InO(2,4,6-tBu(3)C(6)H(3)) (3), [Me(2)InS(C(6)H(5))](infinity) (4), [Me(2)InS(2,4,6-tBu(3)C(6)H(3))](infinity) (6), [Me(2)InSe(C(6)H(5))](2) (7), [Me(2)InSe(2,4,6-Me(3)C(6)H(3))](infinity) (8) and [Me(2)InSe(2,4,6-tBu(3)C(6)H(3))](infinity) (9). All compounds have been characterized by elemental analysis, melting point, FT-IR, FT-Raman, solution NMR, and X-ray crystallography. The structures of 1-2 are dimeric via short intermolecular In-O interactions, yielding a symmetric In(2)O(2) unit and a distorted tetrahedral C(2)O(2) bonding environment for indium. Increasing steric bulk in 3 results in the isolation of a monomeric species, exhibiting a distorted trigonal planar C(2)O bonding environment for indium. In contrast to 1, the thiolate analogue 4 exhibits a polymeric structure via mu(2)-SPh groups and a distorted tetrahedral C(2)S(2) bonding environment for indium. Increasing steric bulk resulted in the formation of a chain of weakly coordinated monomers via intermolecular In...S interactions in [Me(2)InS(2,4,6-tBu(3)C(6)H(2))](infinity) (6). Although 7 shows a dimeric species similar to 1, the 2,4,6-trimethyl substituted selenolate analogue 8 exhibits a polymeric structure, while the -Se-2,4,6-tBu(3)C(6)H(3) analogue (9) showing a similar structure to 6. Comparison to previously reported structures of diorganoindium chalcogenolates demonstrates the importance of the methyl substituents on indium in facilitating the isolation of higher (non-dimeric) oligomers. Theoretical calculations demonstrate the significance of altering the R and R' groups and E on the degree of oligomerization in [R(2)InER'](n) species.

  5. Oligomeric secoiridoid glucosides from Jasminum abyssinicum.

    Science.gov (United States)

    Gallo, Francesca Romana; Palazzino, Giovanna; Federici, Elena; Iurilli, Raffaella; Monache, Franco Delle; Chifundera, Kusamba; Galeffi, Corrado

    2006-03-01

    From the root bark of Jasminum abyssinicum (Oleaceae) collected in Congo was isolated tree oligomeric secoiridoid glucosides named craigosides A-C. The three compounds are esters of a cyclopentanoid monoterpene with an iridane skeleton, esterified with three, two and two, respectively, units of oleoside 11-methyl ester. The structures were elucidated by spectroscopic methods and chemical correlations.

  6. Structural assemblies of the di- and oligomeric G-protein coupled receptor TGR5 in live cells: an MFIS-FRET and integrative modelling study

    Science.gov (United States)

    Greife, Annemarie; Felekyan, Suren; Ma, Qijun; Gertzen, Christoph G. W.; Spomer, Lina; Dimura, Mykola; Peulen, Thomas O.; Wöhler, Christina; Häussinger, Dieter; Gohlke, Holger; Keitel, Verena; Seidel, Claus A. M.

    2016-11-01

    TGR5 is the first identified bile acid-sensing G-protein coupled receptor, which has emerged as a potential therapeutic target for metabolic disorders. So far, structural and multimerization properties are largely unknown for TGR5. We used a combined strategy applying cellular biology, Multiparameter Image Fluorescence Spectroscopy (MFIS) for quantitative FRET analysis, and integrative modelling to obtain structural information about dimerization and higher-order oligomerization assemblies of TGR5 wildtype (wt) and Y111 variants fused to fluorescent proteins. Residue 111 is located in transmembrane helix 3 within the highly conserved ERY motif. Co-immunoprecipitation and MFIS-FRET measurements with gradually increasing acceptor to donor concentrations showed that TGR5 wt forms higher-order oligomers, a process disrupted in TGR5 Y111A variants. From the concentration dependence of the MFIS-FRET data we conclude that higher-order oligomers – likely with a tetramer organization - are formed from dimers, the smallest unit suggested for TGR5 Y111A variants. Higher-order oligomers likely have a linear arrangement with interaction sites involving transmembrane helix 1 and helix 8 as well as transmembrane helix 5. The latter interaction is suggested to be disrupted by the Y111A mutation. The proposed model of TGR5 oligomer assembly broadens our view of possible oligomer patterns and affinities of class A GPCRs.

  7. Computational Study of the Effect of Confinement within Microporous Structures on the Activity and Selectivity of Metallocene Catalysts for Ethylene Oligomerization

    KAUST Repository

    Toulhoat, Hervé

    2011-03-02

    The effect of confinement within some zeolitic structures on the activity and selectivity of metallocene catalysts for the ethylene oligomerization has been investigated using grand canonical Monte Carlo simulations (GCMC). The following zeolite (host) frameworks displaying different pore sizes, have been studied as solid hosts: mazzite (MAZ), AIPO-8 (AET), UTD-1F (DON), faujasite (FAU), and VPI-5 (VFI). Intermediates and transition states involved in the ethylene trimerization reaction catalyzed by a Ti-based catalyst [(η5-C5H4CMe2C6H 5)TiCl3/MAO] have been used as sorbates (guests). We have demonstrated linear correlations with slope aH,j between the adsorption enthalpy and the molecular volume Vm of the sorbates, each holding for a given microporous host below a host-specific threshold V mmax,j. Beyond this maximal molecular volume, the adsorption vanishes due to steric exclusion. aH,j increases, and Vmmax,j decreases with decreasing host pore size, in line with the confinement concept. We moreover showed that, in the limit of vanishing loading (Henry regime), the enthalpies and entropies of adsorption in a given host are linearly correlated. We have defined a host-specific confinement compensation temperature a j, which refers to a temperature where the stabilizing adsorption enthalpic interactions are canceled out against the loss in entropy. However, calculated aj are much larger than the operating temperatures. With a setup microkinetic model, we predict that the activity and selectivity of the confined Ti-catalyst in ethylene oligomerization can be significantly altered with respect to homogeneous phase conditions, since the adsorption free energies of transition states and intermediates also become functions of aH,j and Vm. We have applied this theory to predict the optimum host pore size to get maximum α-octene production, instead of α-hexene, which is primarily produced in the homogeneous phase. We also predict a significantly increased activity for

  8. Structure–Activity Relationship of Oligomeric Flavan-3-ols: Importance of the Upper-Unit B-ring Hydroxyl Groups in the Dimeric Structure for Strong Activities

    Directory of Open Access Journals (Sweden)

    Yoshitomo Hamada

    2015-10-01

    Full Text Available Proanthocyanidins, which are composed of oligomeric flavan-3-ol units, are contained in various foodstuffs (e.g., fruits, vegetables, and drinks and are strongly biologically active compounds. We investigated which element of the proanthocyanidin structure is primarily responsible for this functionality. In this study, we elucidate the importance of the upper-unit of 4–8 condensed dimeric flavan-3-ols for antimicrobial activity against Saccharomyces cerevisiae (S. cerevisiae and cervical epithelioid carcinoma cell line HeLa S3 proliferation inhibitory activity. To clarify the important constituent unit of proanthocyanidin, we synthesized four dimeric compounds, (−-epigallocatechin-[4,8]-(+-catechin, (−-epigallocatechin-[4,8]-(−-epigallocatechin, (−-epigallocatechin-[4,8]-(−-epigallocatechin-3-O-gallate, and (+-catechin-[4,8]-(−-epigallocatechin and performed structure–activity relationship (SAR studies. In addition to antimicrobial activity against S. cerevisiae and proliferation inhibitory activity on HeLa S3 cells, the correlation of 2,2-diphenyl-l-picrylhydrazyl radical scavenging activity with the number of phenolic hydroxyl groups was low. On the basis of the results of our SAR studies, we concluded that B-ring hydroxyl groups of the upper-unit of the dimer are crucially important for strong and effective activity.

  9. Full-Length Structures of BenM and Two Variants Reveal Different Oligomerization Schemes for LysR-Type Transcriptional Regulators

    Energy Technology Data Exchange (ETDEWEB)

    Ruangprasert, Ajchareeya; Craven, Sarah H.; Neidle, Ellen L.; Momany, Cory (Georgia)

    2010-11-30

    BenM, a LysR-type transcriptional regulator (LTTR) from the bacterium Acinetobacter baylyi, responds synergistically to benzoate and cis,cis-muconate. With these effectors, BenM activates gene expression during benzoate consumption. Without effectors, BenM represses transcription. Here, X-ray crystallography was used to determine the full-length structures of BenM and two variants that activate transcription without benzoate or cis,cis-muconate: BenM(R156H) and BenM(E226K). Previous studies indicate that these regulators function as tetramers. Here, interconnections between subunits in the crystals prevented the formation of a closed oligomer and highlighted the inherent flexibility of this multidomain regulator. Nevertheless, analysis of subunit interfaces suggested the functional significance of key interactions. The structures of BenM and its variants were nearly identical, implying that transcriptional differences rely on factors beyond major conformational changes defined solely by sequence. Comparisons of BenM with other LTTRs, including unpublished structures in the Protein Data Bank, revealed extensive variation in the relative orientations of DNA-binding domains (DBDs) and effector-binding domains (EBDs). To form dimers, different LTTRs used similar interfaces between two EBDs, each containing two subdomains: EBD-I and EBD-II. Surprisingly, the dimers used three substantially different schemes to form higher-order oligomers. In one scheme used by BenM, oligomer assembly involved contacts between the EBD-II regions and the DBD regions of adjacent subunits. In another scheme, there were no contacts between the EBDs; only the DBDs were involved in tetramer formation. In the third scheme, the oligomer interface involved DBD and EBD-I/EBD-II contacts. These diverse schemes demonstrate novel variation in the oligomeric structures of individual LTTRs within this large and important family.

  10. Structural evidence for variable oligomerization of the N-terminal domain of cyclase-associated protein (CAP).

    Science.gov (United States)

    Yusof, Adlina Mohd; Hu, Nien-Jen; Wlodawer, Alexander; Hofmann, Andreas

    2005-02-01

    Cyclase-associated protein (CAP) is a highly conserved and widely distributed protein that links the nutritional response signaling to cytoskeleton remodeling. In yeast, CAP is a component of the adenylyl cyclase complex and helps to activate the Ras-mediated catalytic cycle of the cyclase. While the N-terminal domain of CAP (N-CAP) provides a binding site for adenylyl cyclase, the C-terminal domain (C-CAP) possesses actin binding activity. Our attempts to crystallize full-length recombinant CAP from Dictyostelium discoideum resulted in growth of orthorhombic crystals containing only the N-terminal domain (residues 42-227) due to auto-proteolytic cleavage. The structure was solved by molecular replacement with data at 2.2 A resolution. The present crystal structure allows the characterization of a head-to-tail N-CAP dimer in the asymmetric unit and a crystallographic side-to-side dimer. Comparison with previously published structures of N-CAP reveals variable modes of dimerization of this domain, but the presence of a common interface for the side-to-side dimer.

  11. Quaternary structure heterogeneity of oligomeric proteins: a SAXS and SANS study of the dissociation products of Octopus vulgaris hemocyanin.

    Directory of Open Access Journals (Sweden)

    Francesco Spinozzi

    Full Text Available Octopus vulgaris hemocyanin shows a particular self-assembling pattern, characterized by a hierarchical organization of monomers. The highest molecular weight aggregate is a decamer, the stability of which in solution depends on several parameters. Different pH values, buffer compositions, H₂O/D₂O ratios and Hofmeister's salts result in modifications of the aggregation state of Octopus vulgaris hemocyanin. The new QUAFIT method, recently applied to derive the structure of the decameric and the monomeric assembly from small-angle scattering data, is used here to model the polydisperse system that results from changing the solution conditions. A dataset of small-angle X-rays and neutron scattering curves is analysed by QUAFIT to derive structure, composition and concentration of different assemblies present in solution. According to the hierarchy of the association/dissociation processes and the possible number of different aggregation products in solution, each sample has been considered as a heterogeneous mixture composed of the entire decamer, the dissociated "loose" monomer and all the intermediate dissociation products. Scattering curves corresponding to given experimental conditions are well fitted by using a linear combination of single particle form factors. QUAFIT has proved to be a method of general validity to describe solutions of proteins that, even after purification processes, result to be intrinsically heterogeneous.

  12. Structural studies with BnSP-7 reveal an atypical oligomeric conformation compared to phospholipases A2-like toxins.

    Science.gov (United States)

    de Lima, Lino Fernando G; Borges, Rafael J; Viviescas, Maria Alejandra; Fernandes, Carlos A H; Fontes, Marcos R M

    2017-07-24

    There are 2.5 million cases of snakebite per year and approximately 100,000 at 150,000 deaths. Thus, it is considered an important public health problem by the World Health Organization. Snakes from the Bothrops genus may cause severe local effects in the victims, so it is important to develop inhibitors to treat local effects in patients. In addition, approximately 30 different species of bothropic snakes have been described that may present differences in their venom composition. Small structural differences in the venom proteins may result in different ligands binding. Herein, BnSP-7, a PLA2-like protein that causes local myotoxic effects, was analyzed using different biophysical techniques. Crystal structures of BnSP-7 binding to three different cinnamic acid derivates were solved showing that the ligands bind in the membrane-dockage region (MDoS) of the protein. Spectroscopy fluorescence and microscale thermophoresis (MST) assays showed that these ligands also bind to BnSP-7 in solution and provide comparative information about their affinity to BnSP-7. MST experiments also showed that hydroxyl radicals of the ligands, involved in their binding with the MDoS region of BnSP-7, are essential to increase their affinity with the protein. As this region has been indicated as essential for the myotoxic mechanism, the ligands could potentially be used as inhibitors for BnSP-7. These results provide relevant insights to understand the PLA2-like proteins myotoxic mechanism and may eventually lead to design of new inhibitors for these toxins. Furthermore, a comparative structural analysis of BnSP-7 with other PLA2-like proteins showed that BnSP-7 has an atypical quaternary conformation, suggesting an intermediate state that is unlike other PLA2-like proteins. This information, combined with the absence or partial occupancy of molecules in their hydrophobic channel and the misaligned membrane-disruption region, led us to hypothesize that the protein is not able to fully

  13. The influence of surface structure on H4SiO4 oligomerization on rutile and amorphous TiO2 surfaces: an ATR-IR and synchrotron XPS study.

    Science.gov (United States)

    Song, Yantao; Swedlund, Peter J; McIntosh, Grant J; Cowie, Bruce C C; Waterhouse, Geoffrey I N; Metson, James B

    2012-12-11

    Silicic acid (H(4)SiO(4)) is ubiquitous in natural aquatic systems. Applications of TiO(2) in these systems will be influenced by H(4)SiO(4) sorption and oligomerization reactions on the TiO(2) surface, and this can affect many aspects of TiO(2) reactivity. The spatial arrangement of sorption sites on a metal oxide surface can promote specific lateral interactions, such as oligomerization, between sorbed species. In this work we explore the relationship between surface structure and interfacial H(4)SiO(4) oligomerization by quantifying the extent of H(4)SiO(4) sorption and oligomerization on three TiO(2) phases; a rutile phase having well-developed (110) faces (R180), a rutile phase with poorly developed (110) faces (R60), and an amorphous TiO(2) (TiO(2(am))). The in situ ATR-IR spectra measured over time as 0.2 mM H(4)SiO(4) reacted with TiO(2) were quite different on the three TiO(2) phases. The percentage of the surface H(4)SiO(4) that was present as oligomers increased over time on all phases, but after 20 h almost all H(4)SiO(4) on the R180 surface was oligomeric, while the H(4)SiO(4) on TiO(2(am)) was predominantly monomeric. The extent of H(4)SiO(4) oligomerization on R60 was intermediate. When the TiO(2) phases reacted with 1.5 mM H(4)SiO(4) the ATR-IR spectra showed oligomeric silicates dominating the surface of all three TiO(2) phases; however, after 20 h the percentage of the surface H(4)SiO(4) present as three-dimensional polymers was ∼30, 10, and 0% on R180, R60, and TiO(2(am)) respectively. The Si 2s photoelectron peak binding energy (BE) and the H(4)SiO(4) surface coverage (Γ(Si)) were measured by XPS over a range of Γ(Si). For any given Γ(Si) the Si 2s BE's were in the order R180 > R60 > TiO(2(am)). A higher Si 2s BE indicates a greater degree of silicate polymerization. The ATR-IR and XPS results support the existing model for interfacial H(4)SiO(4) oligomerization where linear trimeric silicates are formed by insertion of a solution H(4)SiO(4

  14. Simulations of Oligomeric Intermediates in Prion Diseases

    CERN Document Server

    Mobley, D L; Singh, R R P; Kulkarni, R V; Slepoy, A; Mobley, David L.; Cox, Daniel L.; Singh, Rajiv R. P.; Kulkarni, Rahul V.; Slepoy, Alexander

    2003-01-01

    We extend our previous stochastic cellular automata based model for areal aggregation of prion proteins on neuronal surfaces. The new anisotropic model allow us to simulate both strong beta-sheet and weaker attachment bonds between proteins. Constraining binding directions allows us to generate aggregate structures with the hexagonal lattice symmetry found in recently observed in vitro experiments. We argue that these constraints on rules may correspond to underlying steric constraints on the aggregation process. We find that monomer dominated growth of the areal aggregate is too slow to account for some observed doubling time-to-incubation time ratios inferred from data, and so consider aggregation dominated by relatively stable but non-infectious oligomeric intermediates. We compare a kinetic theory analysis of oligomeric aggregation to spatially explicit simulations of the process. We find that with suitable rules for misfolding of oligomers, possibly due to water exclusion by the surrounding aggregate, th...

  15. Oligomerization in health and disease

    CERN Document Server

    Giraldo, Jesus

    2013-01-01

    This special volume of Progress in Molecular Biology and Translational Science focuses on oligomerization in health and disease. Contributions from leading authorities Informs and updates on all the latest developments in the field.

  16. Structural Insight for Roles of DR5 Death Domain Mutations on Oligomerization of DR5 Death Domain-FADD Complex in the Death-Inducing Signaling Complex Formation: A Computational Study.

    Science.gov (United States)

    Yang, Hongyi; Song, Yuhua

    2016-04-01

    Death receptor 5 (DR5)-induced apoptosis that prioritizes the death of tumor cells has been proposed as one of the promising cancer therapies. In this process, oligomerized DR5 death domain (DD) binding to Fas-associated death domain (FADD) leads to FADD activating caspase-8, which marks the formation of the death-inducing signaling complex (DISC) that initiates apoptosis. DR5 DD mutations found in cancer cells have been suggested to play an important pathological role, the mechanism through which those mutants prevent the DR5-activated DISC formation is not clear yet. This study sought to provide structural and molecular insight for the roles of four selected DR5 DD mutations (E355K, E367K, K415N, and L363F) in the oligomerization of DR5 DD-FADD complex during the DISC formation. Results from the molecular dynamics simulations show that the simulated mutants induce conformational, dynamical motions and interactions changes in the DR5 DD-FADD tetramer complex, including changes in a protein's backbone flexibility, less exposure of FADD DED's caspase-8 binding site, reduced H-bonding and hydrophobic contacts at the DR5 DD-FADD DD binding, altered distribution of the electrostatic potentials and correlated motions of residues, and reduced binding affinity of DR5 DD binding to FADD. This study provides structural and molecular insight for the influence of DR5 DD mutations on oligomerization of DR5 DD-FADD complex, which is expected to foster understanding of the DR5 DD mutants' resistance mechanism against DR5-activated DISC formation.

  17. Biophysical characterization of GPCR oligomerization

    DEFF Research Database (Denmark)

    Mathiasen, Signe

    a quantitative characterization of GPCR oligomerization. The assay provided the first quantification of the association energy of the β2 Adrenergic Receptor (β2AR), a prototypical GPCR. Furthermore we directly observed the time-dependent dimerization of β2AR and Cannabinoid receptor 1 at the single molecule......The biophysical characterization of the fundamental molecular mechanisms behind G-protein coupled receptors (GPCRs) oligomerization is proposed to be paramount for understanding the pharmacological consequence of receptor self-association. Here we developed an in vitro assay that allowed...

  18. Oligomeric viral proteins: small in size, large in presence

    Science.gov (United States)

    Jayaraman, Bhargavi; Smith, Amber M.; Fernandes, Jason D.; Frankel, Alan D.

    2016-01-01

    Viruses are obligate parasites that rely heavily on host cellular processes for replication. The small number of proteins typically encoded by a virus is faced with selection pressures that lead to the evolution of distinctive structural properties, allowing each protein to maintain its function under constraints such as small genome size, high mutation rate, and rapidly changing fitness conditions. One common strategy for this evolution is to utilize small building blocks to generate protein oligomers that assemble in multiple ways, thereby diversifying protein function and regulation. In this review, we discuss specific cases that illustrate how oligomerization is used to generate a single defined functional state, to modulate activity via different oligomeric states, or to generate multiple functional forms via different oligomeric states. PMID:27685368

  19. High Content Analysis of Compositional Heterogeneities to Study GPCR Oligomerization

    DEFF Research Database (Denmark)

    Walsh, Samuel McEwen

    In this thesis I demonstrate how the natural compositional heterogeneities of synthetic and living cell model systems can be used to quantitate the mechanics of G-protein coupled receptor (GPCR) oligomerization with Förster resonance energy transfer (FRET). The thesis is structured around three a...

  20. High Content Analysis of Compositional Heterogeneities to Study GPCR Oligomerization

    DEFF Research Database (Denmark)

    Walsh, Samuel McEwen

    In this thesis I demonstrate how the natural compositional heterogeneities of synthetic and living cell model systems can be used to quantitate the mechanics of G-protein coupled receptor (GPCR) oligomerization with Förster resonance energy transfer (FRET). The thesis is structured around three a...

  1. Oligomerization of Uukuniemi virus nucleocapsid protein

    Directory of Open Access Journals (Sweden)

    Katz Anna

    2010-08-01

    Full Text Available Abstract Background Uukuniemi virus (UUKV belongs to the Phlebovirus genus in the family Bunyaviridae. As a non-pathogenic virus for humans UUKV has served as a safe model bunyavirus in a number of studies addressing fundamental questions such as organization and regulation of viral genes, genome replication, structure and assembly. The present study is focused on the oligomerization of the UUKV nucleocapsid (N protein, which plays an important role in several steps of virus replication. The aim was to locate the domains involved in the N protein oligomerization and study the process in detail. Results A set of experiments concentrating on the N- and C-termini of the protein was performed, first by completely or partially deleting putative N-N-interaction domains and then by introducing point mutations of amino acid residues. Mutagenesis strategy was based on the computer modeling of secondary and tertiary structure of the N protein. The N protein mutants were studied in chemical cross-linking, immunofluorescence, mammalian two-hybrid, minigenome, and virus-like particle-forming assays. The data showed that the oligomerization ability of UUKV-N protein depends on the presence of intact α-helices on both termini of the N protein molecule and that a specific structure in the N-terminal region plays a crucial role in the N-N interaction(s. This structure is formed by two α-helices, rich in amino acid residues with aromatic (W7, F10, W19, F27, F31 or long aliphatic (I14, I24 side chains. Furthermore, some of the N-terminal mutations (e.g. I14A, I24A, F31A affected the N protein functionality both in mammalian two-hybrid and minigenome assays. Conclusions UUKV-N protein has ability to form oligomers in chemical cross-linking and mammalian two-hybrid assays. In mutational analysis, some of the introduced single-point mutations abolished the N protein functionality both in mammalian two-hybrid and minigenome assays, suggesting that especially the N

  2. Antioxidant oligomeric proanthocyanidins from Cistus salvifolius.

    Science.gov (United States)

    Qa'Dan, Fadi; Petereit, Frank; Mansoor, Kenza; Nahrstedt, Adolf

    2006-11-01

    The purified proanthocyanidin oligomers of Cistus salvifolius herb extract accounted for 78% of the total proanthocyanidins and 73% of the total antioxidant activity of this extract. To elucidate the structure of the oligomer, it was depolymerized by acid catalysis in the presence of phloroglucinol. The structures of the resulting flavan-3-ols and phloroglucinol adducts were determined on the basis of 1D- and reverse 2D-NMR (HSQC, HMBC) experiments of their peracetylated derivatives, MALDI-TOF-MS and CD spectroscopy. These observations resulting from the degradation with phloroglucinol were confirmed by 13C NMR spectroscopy of the oligomer. The mean molecular weight of the higher oligomeric fraction was estimated to be 5-6 flavan-3-ol-units.

  3. Revisiting the oligomerization mechanism of Vibrio cholerae cytolysin, a beta-barrel pore-forming toxin.

    Science.gov (United States)

    Rai, Anand Kumar; Chattopadhyay, Kausik

    2016-06-03

    Vibrio cholerae cytolysin (VCC) is a membrane-damaging beta-barrel pore-forming toxin (beta-PFT). VCC causes permeabilization of the target membranes by forming transmembrane oligomeric beta-barrel pores. Oligomerization is a key step in the mode of action of any beta-PFT, including that of VCC. Earlier studies have identified some of the key residues in VCC that are directly involved in the generation of the inter-protomer contacts, thus playing critical roles in the oligomerization of the membrane-bound toxin. Analysis of the VCC oligomeric pore structure reveals a potential hydrogen-bond network that appears to connect the sidechain of an asparagine residue (Asn582; located within an inter-domain linker sequence) from one protomer to the backbone CO- and NH-groups of the neighbouring protomer, indirectly through water molecules at most of the inter-protomer interfaces. In the present study, we show that the mutation of Asn582Ala affects the oligomerization and the pore-forming activity of VCC in the membrane lipid bilayer of the synthetic lipid vesicles, while the replacement of Asn582Gln results into the restoration of the oligomeric pore-forming ability of the toxin. Using a number of truncated variants of VCC, having deletion in the C-terminal region of the toxin starting from the Asn582 residue or beyond, we also show that the presence of Asn582 is critically required for the oligomerization of the truncated form of the protein.

  4. Oligomeric proanthocyanidins from mangosteen pericarps.

    Science.gov (United States)

    Fu, Caili; Loo, Alvin Eng Kiat; Chia, Fiona Ping Ping; Huang, Dejian

    2007-09-19

    Oligomeric proanthocyanidins were extracted from mangosteen pericarps and fractionated by a Sephadex LH-20 column to give 0.66% yield (dry matter). (13)C and (1)H NMR signals showed the presence of predominantly procyanidins together with a few prodelphinidin units along with small amounts of stereoisomers of afzelechin/epiafzelechin, catechin/epicatechin, and gallocatechin/epigallocatechin. Depolymerization with benzylmercaptan resulted in epicatechin thioether as the major product, and the mean degree of polymerization was determined to be 6.6. The electron spray ionization-mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectra revealed the dominant B type oligomers with mainly epicatechin units and with a small amount of A type oligomers. The isolated proanthocyanidins are potent peroxyl radical scavengers as evidenced by the high oxygen radical scavenging capacity at 1.7 x 10 (4) micromol TE/g, much higher than that of pine bark and grape seed extracts.

  5. Thermodynamic competition between membrane protein oligomeric states

    Science.gov (United States)

    Kahraman, Osman; Haselwandter, Christoph A.

    2016-10-01

    Self-assembly of protein monomers into distinct membrane protein oligomers provides a general mechanism for diversity in the molecular architectures, and resulting biological functions, of membrane proteins. We develop a general physical framework describing the thermodynamic competition between different oligomeric states of membrane proteins. Using the mechanosensitive channel of large conductance as a model system, we show how the dominant oligomeric states of membrane proteins emerge from the interplay of protein concentration in the cell membrane, protein-induced lipid bilayer deformations, and direct monomer-monomer interactions. Our results suggest general physical mechanisms and principles underlying regulation of protein function via control of membrane protein oligomeric state.

  6. Thermodynamic competition between membrane protein oligomeric states

    CERN Document Server

    Kahraman, Osman

    2016-01-01

    Self-assembly of protein monomers into distinct membrane protein oligomers provides a general mechanism for diversity in the molecular architectures, and resulting biological functions, of membrane proteins. We develop a general physical framework describing the thermodynamic competition between different oligomeric states of membrane proteins. Using the mechanosensitive channel of large conductance as a model system, we show how the dominant oligomeric states of membrane proteins emerge from the interplay of protein concentration in the cell membrane, protein-induced lipid bilayer deformations, and direct monomer-monomer interactions. Our results suggest general physical mechanisms and principles underlying regulation of protein function via control of membrane protein oligomeric state.

  7. Preparative isolation of oligomeric procyanidins from Hawthorn (Crataegus spp.).

    Science.gov (United States)

    Zumdick, S; Petereit, F; Luftmann, H; Hensel, A

    2009-04-01

    The oligomeric procyanidins (OPC) from Hawthorn leaves and flowers (Crataegi folium cum flore) are considered to be in part responsible for the cardiotonic clinical activity of the herbal material. Effective methods for rapid isolation of these heterogenous oligomeric clusters with defined molecular weight as reference compounds are not published until now. Therefore the water soluble fraction of an acetone/water (7 + 3) extract of Hawthorn leaves and flowers was fractionated by a combination of MPLC on RP-18 material and preparative HPLC using a diol stationary phase. This procedure resulted in the effective isolation of procyanidins with a distinct degree of polymerization (DP) from dimers DP2 up to tridecamers DP13. Exact mass measurements with negative ESI-TOF/MS were employed to confirm the respective structures of the isolated procyanidins.

  8. Carbon Nanostructures Containing Polyhedral Oligomeric Silsesquioxanes (POSS)

    NARCIS (Netherlands)

    Potsi, Georgia; Rossos, Andreas; Kouloumpis, Antonios; Antoniou, Myrsini K.; Spyrou, Konstantinos; Karakassides, Michael A.; Gournis, Dimitrios; Rudolf, Petra

    2015-01-01

    This mini review describes the synthesis and properties of carbon nanostructures containing organic-inorganic cage-like polyhedral oligomeric silsesquioxane (POSS). The physical and chemical functionalization of carbon nanomaterials such as graphene, graphene oxide, carbon nanotubes, and fullerenes

  9. Carbon Nanostructures Containing Polyhedral Oligomeric Silsesquioxanes (POSS)

    NARCIS (Netherlands)

    Potsi, Georgia; Rossos, Andreas; Kouloumpis, Antonios; Antoniou, Myrsini K.; Spyrou, Konstantinos; Karakassides, Michael A.; Gournis, Dimitrios; Rudolf, Petra

    2015-01-01

    This mini review describes the synthesis and properties of carbon nanostructures containing organic-inorganic cage-like polyhedral oligomeric silsesquioxane (POSS). The physical and chemical functionalization of carbon nanomaterials such as graphene, graphene oxide, carbon nanotubes, and fullerenes

  10. X-ray Crystallographic Structures of Trimers and Higher-Order Oligomeric Assemblies of a Peptide Derived from Aβ17–36

    OpenAIRE

    Spencer, Ryan K.; Li, Hao; Nowick, James S.

    2014-01-01

    A peptide derived from Aβ17–36 crystallizes to form trimers that further associate to form higher-order oligomers. The trimers consist of three highly twisted β-hairpins in a triangular arrangement. Two trimers associate face-to-face in the crystal lattice to form a hexamer; four trimers in a tetrahedral arrangement about a central cavity form a dodecamer. These structures provide a working model for the structures of oligomers associated with neurodegeneration in Alzheimer’s disease.

  11. Structural and enzymatic analysis of TarM glycosyltransferase from Staphylococcus aureus reveals an oligomeric protein specific for the glycosylation of wall teichoic acid.

    Science.gov (United States)

    Koç, Cengiz; Gerlach, David; Beck, Sebastian; Peschel, Andreas; Xia, Guoqing; Stehle, Thilo

    2015-04-10

    Anionic glycopolymers known as wall teichoic acids (WTAs) functionalize the peptidoglycan layers of many Gram-positive bacteria. WTAs play central roles in many fundamental aspects of bacterial physiology, and they are important determinants of pathogenesis and antibiotic resistance. A number of enzymes that glycosylate WTA in Staphylococcus aureus have recently been identified. Among these is the glycosyltransferase TarM, a component of the WTA de novo biosynthesis pathway. TarM performs the synthesis of α-O-N-acetylglycosylated poly-5'-phosphoribitol in the WTA structure. We have solved the crystal structure of TarM at 2.4 Å resolution, and we have also determined a structure of the enzyme in complex with its substrate UDP-GlcNAc at 2.8 Å resolution. The protein assembles into a propeller-like homotrimer in which each blade contains a GT-B-type glycosyltransferase domain with a typical Rossmann fold. The enzymatic reaction retains the stereochemistry of the anomeric center of the transferred GlcNAc-moiety on the polyribitol backbone. TarM assembles into a trimer using a novel trimerization domain, here termed the HUB domain. Structure-guided mutagenesis experiments of TarM identify residues critical for enzyme activity, assign a putative role for the HUB in TarM function, and allow us to propose a likely reaction mechanism.

  12. Structure-function analysis of heterodimer formation, oligomerization, and receptor binding of the Staphylococcus aureus bi-component toxin LukGH.

    Science.gov (United States)

    Badarau, Adriana; Rouha, Harald; Malafa, Stefan; Logan, Derek T; Håkansson, Maria; Stulik, Lukas; Dolezilkova, Ivana; Teubenbacher, Astrid; Gross, Karin; Maierhofer, Barbara; Weber, Susanne; Jägerhofer, Michaela; Hoffman, David; Nagy, Eszter

    2015-01-01

    The bi-component leukocidins of Staphylococcus aureus are important virulence factors that lyse human phagocytic cells and contribute to immune evasion. The γ-hemolysins (HlgAB and HlgCB) and Panton-Valentine leukocidin (PVL or LukSF) were shown to assemble from soluble subunits into membrane-bound oligomers on the surface of target cells, creating barrel-like pore structures that lead to cell lysis. LukGH is the most distantly related member of this toxin family, sharing only 30-40% amino acid sequence identity with the others. We observed that, unlike other leukocidin subunits, recombinant LukH and LukG had low solubility and were unable to bind to target cells, unless both components were present. Using biolayer interferometry and intrinsic tryptophan fluorescence we detected binding of LukH to LukG in solution with an affinity in the low nanomolar range and dynamic light scattering measurements confirmed formation of a heterodimer. We elucidated the structure of LukGH by x-ray crystallography at 2.8-Å resolution. This revealed an octameric structure that strongly resembles that reported for HlgAB, but with important structural differences. Structure guided mutagenesis studies demonstrated that three salt bridges, not found in other bi-component leukocidins, are essential for dimer formation in solution and receptor binding. We detected weak binding of LukH, but not LukG, to the cellular receptor CD11b by biolayer interferometry, suggesting that in common with other members of this toxin family, the S-component has the primary contact role with the receptor. These new insights provide the basis for novel strategies to counteract this powerful toxin and Staphylococcus aureus pathogenesis.

  13. Four crystal structures of human LLT1, a ligand of human NKR-P1, in varied glycosylation and oligomerization states

    Energy Technology Data Exchange (ETDEWEB)

    Skálová, Tereza, E-mail: t.skalova@gmail.com [Academy of Sciences of the Czech Republic, v.v.i., Vídeňská 1083, 142 20 Praha 4 (Czech Republic); Bláha, Jan [Charles University Prague, Hlavova 8, 128 40 Praha (Czech Republic); Harlos, Karl [University of Oxford, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Dušková, Jarmila [Academy of Sciences of the Czech Republic, v.v.i., Vídeňská 1083, 142 20 Praha 4 (Czech Republic); Koval’, Tomáš [Academy of Sciences of the Czech Republic, v.v.i., Heyrovského nám. 2, 162 06 Praha 6 (Czech Republic); Stránský, Jan; Hašek, Jindřich [Academy of Sciences of the Czech Republic, v.v.i., Vídeňská 1083, 142 20 Praha 4 (Czech Republic); Vaněk, Ondřej [Charles University Prague, Hlavova 8, 128 40 Praha (Czech Republic); Dohnálek, Jan, E-mail: t.skalova@gmail.com [Academy of Sciences of the Czech Republic, v.v.i., Vídeňská 1083, 142 20 Praha 4 (Czech Republic); Academy of Sciences of the Czech Republic, v.v.i., Heyrovského nám. 2, 162 06 Praha 6 (Czech Republic)

    2015-03-01

    Four crystal structures of human LLT1, a ligand of human NKR-P1, are reported. Human LLT1 is a C-type lectin-like ligand of NKR-P1 (CD161, gene KLRB1), a C-type lectin-like receptor of natural killer cells. Using X-ray diffraction, the first experimental structures of human LLT1 were determined. Four structures of LLT1 under various conditions were determined: monomeric, dimeric deglycosylated after the first N-acetylglucosamine unit in two forms and hexameric with homogeneous GlcNAc{sub 2}Man{sub 5} glycosylation. The dimeric form follows the classical dimerization mode of human CD69. The monomeric form keeps the same fold with the exception of the position of an outer part of the long loop region. The hexamer of glycosylated LLT1 consists of three classical dimers. The hexameric packing may indicate a possible mode of interaction of C-type lectin-like proteins in the glycosylated form.

  14. Structural Analysis of β-Fructofuranosidase from Xanthophyllomyces dendrorhous Reveals Unique Features and the Crucial Role of N-Glycosylation in Oligomerization and Activity.

    Science.gov (United States)

    Ramírez-Escudero, Mercedes; Gimeno-Pérez, María; González, Beatriz; Linde, Dolores; Merdzo, Zoran; Fernández-Lobato, María; Sanz-Aparicio, Julia

    2016-03-25

    Xanthophyllomyces dendrorhousβ-fructofuranosidase (XdINV)is a highly glycosylated dimeric enzyme that hydrolyzes sucrose and releases fructose from various fructooligosaccharides (FOS) and fructans. It also catalyzes the synthesis of FOS, prebiotics that stimulate the growth of beneficial bacteria in human gut. In contrast to most fructosylating enzymes, XdINV produces neo-FOS, which makes it an interesting biotechnology target. We present here its three-dimensional structure, which shows the expected bimodular arrangement and also a long extension of its C terminus that together with anN-linked glycan mediate the formation of an unusual dimer. The two active sites of the dimer are connected by a long crevice, which might indicate its potential ability to accommodate branched fructans. This arrangement could be representative of a group of GH32 yeast enzymes having the traits observed in XdINV. The inactive D80A mutant was used to obtain complexes with relevant substrates and products, with their crystals structures showing at least four binding subsites at each active site. Moreover, two different positions are observed from subsite +2 depending on the substrate, and thus, a flexible loop (Glu-334-His-343) is essential in binding sucrose and β(2-1)-linked oligosaccharides. Conversely, β(2-6) and neo-type substrates are accommodated mainly by stacking to Trp-105, explaining the production of neokestose and the efficient fructosylating activity of XdINV on α-glucosides. The role of relevant residues has been investigated by mutagenesis and kinetics measurements, and a model for the transfructosylating reaction has been proposed. The plasticity of its active site makes XdINV a valuable and flexible biocatalyst to produce novel bioconjugates.

  15. Complex oligomeric structure of a truncated form of DdrA: A protein required for the extreme radio-tolerance of Deinococcus

    Energy Technology Data Exchange (ETDEWEB)

    Gutsche, I. [CNRS-EMBL-Univ Grenoble 1, Unit Virus Host Cell Interact UMR5233, F-38042 Grenoble (France); Vujicic-Zagar, A.; Serre, L. [CEA-CNRS-Univ Grenoble 1, Inst Biol Struct, UMR5075, Lab Prot Membranaires, F-38027 Grenoble 01 (France); Siebert, X. [CEA-CNRS-Univ Grenoble 1, Inst Biol Struct UMR5075, Lab Microscopie Elect et struct, F-38027 Grenoble 01 (France); Servant, P.; Vannier, F.; Sommer, S. [Univ Paris 11, CNRS UMR 8621, CEA LRC42V, Inst Genet et Microbiol, F-91405 Orsay (France); Castaing, B. [CNRS, UMR4301, Ctr Biophys Mol, F-45071 Orleans 02 (France); Gallet, B. [CEA-CNRS-Univ Grenoble 1, Inst Biol Struct UMR 5075, ROBIOMOL, Lab Ingn Macrom, F-38027 Grenoble 01 (France); Heulin, T.; De Groot, A. [CEA, DSV, IBEB, SBVME, Lab Ecol Microbienne Rhizosphere et Environm Extre, UMR 6191, F-13108 St Paul Les Durance (France)

    2008-07-01

    In order to preserve their genome integrity, organisms have developed elaborate tactics for genome protection and repair. The Deinococcus radiodurans bacteria famous for their extraordinary tolerance toward high doses of radiations or long period of desiccation, possess some specific genes with unknown function which are related to their survival in such extreme conditions. Among them, ddrA is an orphan gene specific of Deinococcus genomes. DdrA, the product of this gene was suggested to be a component of the DNA end protection system. Here we provide a three-dimensional reconstruction of the Deinococcus deserti DdrA(1-160) by electron microscopy. Although not functional in vivo, this truncated protein keeps its DNA binding ability at the wild-type level. DdrA(1-160) has a complex three-dimensional structure based on a hepta-meric ring that can self-associate to form a larger molecular weight assembly. We suggest that the complex architecture of DdrA plays a role in the substrate specificity and favors an efficient DNA repair. (authors)

  16. Tip-induced C-H activation and oligomerization of thienoanthracenes.

    Science.gov (United States)

    Dinca, L E; MacLeod, J M; Lipton-Duffin, J; Fu, C; Ma, D; Perepichka, D F; Rosei, F

    2014-08-14

    The tip of a scanning tunneling microscope (STM) can be used to dehydrogenate freely-diffusing tetrathienoanthracene (TTA) molecules on Cu(111), trapping the molecules into metal-coordinated oligomeric structures. The process proceeds at bias voltages above ~3 V and produces organometallic structures identical to those resulting from the thermally-activated cross-coupling of a halogenated analogue. The process appears to be substrate dependent: no oligomerization was observed on Ag(111) or HOPG. This approach demonstrates the possibility of controlled synthesis and nanoscale patterning of 2D oligomer structures on selected surfaces.

  17. Dimerization and oligomerization of the chaperone calreticulin

    DEFF Research Database (Denmark)

    Jørgensen, Charlotte S; Ryder, L Rebekka; Steinø, Anne;

    2003-01-01

    protein. Using PAGE, urea gradient gel electrophoresis, capillary electrophoresis and MS, we show that dimerization through the SH group can be induced by lowering the pH to 5-6, heating, or under conditions that favour partial unfolding such as urea concentrations above 2.6 m or SDS concentrations above...... 0.025%. Moreover, we show that calreticulin also has the ability to self-oligomerize through noncovalent interactions at urea concentrations above 2.6 m at pH below 4.6 or above pH 10, at temperatures above 40 degrees C, or in the presence of high concentrations of organic solvents (25%), conditions...... urea or 1% SDS, and heat-induced oligomerization could be inhibited by 8 m urea or 1% SDS when present during heating. Comparison of the binding properties of monomeric and oligomeric calreticulin in solid-phase assays showed increased binding to peptides and denatured proteins when calreticulin...

  18. High Molecular Weight Complex Analysis of Epstein-Barr Virus Latent Membrane Protein 1 (LMP-1): Structural Insights into LMP-1’s Homo-Oligomerization and Lipid Raft Association

    Science.gov (United States)

    Wrobel, Christopher M.; Geiger, Timothy R.; Nix, Rebecca N.; Robitaille, Aaron M.; Balser, Sandra; Cervantes, Alfredo; Gonzalez, Miguel; Martin, Jennifer M.

    2013-01-01

    LMP-1 is a constitutively active Tumor Necrosis Factor Receptor analog encoded by Epstein-Barr virus. LMP-1 activation correlates with oligomerization and raft localization, but direct evidence of LMP-1 oligomers is limited. We report that LMP-1 forms multiple high molecular weight native LMP-1 complexes when analyzed by BN-PAGE, the largest of which are enriched in detergent resistant membranes. The largest of these high molecular weight complexes are not formed by purified LMP-1 or by loss of function LMP-1 mutants. Consistent with these results we find a dimeric form of LMP-1 that can be stabilized by disulfide crosslinking. We identify cysteine 238 in the C-terminus of LMP-1 as the crosslinked cysteine. Disulfide crosslinking occurs post-lysis but the dimer can be crosslinked in intact cells with membrane permeable crosslinkers. LMP-1/C238A retains wild type LMP-1 NF-κB activity. LMP-1’s TRAF binding, raft association and oligomerization are associated with the dimeric form of LMP-1. Our results suggest the possibility that the observed dimeric species results from inter-oligomeric crosslinking of LMP-1 molecules in adjacent core LMP-1 oligomers. PMID:24075898

  19. Interleukin-1 beta converting enzyme requires oligomerization for activity of processed forms in vivo.

    Science.gov (United States)

    Gu, Y; Wu, J; Faucheu, C; Lalanne, J L; Diu, A; Livingston, D J; Su, M S

    1995-05-01

    Interleukin-1 beta converting enzyme (ICE) is composed of 10' (p10) and 20 kDa (p20) subunits, which are derived from a common 45 kDa precursor. Recent crystallographic studies have shown that ICE exists as a tetramer (p20/p10)2 in the crystal lattice. We provide evidence that the p10 and p20 subunits of ICE associate as oligomers in transfected COS cells. Using intragenic complementation, we show that the activity of a p10/p10 interface mutant defective in autoprocessing can be restored by co-expression with active site ICE mutants. Different active site mutants can also complement each other by oligomerization to form active ICE. These studies indicate that ICE precursor polypeptides may associate in different quaternary structures and that oligomerization is required for autoprocessing. Furthermore, integenic complementation of active site mutants of ICE and an ICE homolog restores autoprocessing activity, suggesting that hetero-oligomerization occurs between ICE homologs.

  20. Reactive bay functionalized perylene monoimide-polyhedral oligomeric silsesquioxane organic electronic dye

    Directory of Open Access Journals (Sweden)

    Wangatia Lodrick Makokha

    2015-03-01

    Full Text Available Aggregation-induced quenching is particularly detrimental in perylene diimides, which are characterized by a near-unity fluorescence quantum yield in solution but are far less emissive in the solid state. Previously, perylene diimide has been improved by linking it to the inorganic cage of polyhedral oligomeric silsesquioxanes. As a further study on perylene diimidepolyhedral oligomeric silsesquioxanes, we report on a double functionalized molecular structure, which can be used for substitution at the bay area and as a side group in other materials. Typical solution absorption and emission features of the perylene diimide fragment have been observed in this new reactive perylene diimide-polyhedral oligomeric silsesquioxane. Moreover, reduced stacking during aggregation and spherical particles exhibiting solid fluorescence have been obtained. Organic semiconducting material with enhanced solid state photophysical properties, like solid fluorescence is a subject of great interest owing to its possible high-tech applications in optoelectronic devices.

  1. The oligomeric state of the active Vps4 AAA ATPase

    Science.gov (United States)

    Monroe, Nicole; Han, Han; Gonciarz, Malgorzata D.; Eckert, Debra M.; Karren, Mary Anne; Whitby, Frank G.; Sundquist, Wesley I.; Hill, Christopher P.

    2013-01-01

    The cellular ESCRT pathway drives membrane constriction toward the cytosol and effects membrane fission during cytokinesis, endosomal sorting, and the release of many enveloped viruses, including HIV. A component of this pathway, the AAA ATPase Vps4, provides energy for pathway progression. Although it is established that Vps4 functions as an oligomer, subunit stoichiometry and other fundamental features of the functional enzyme are unclear. Higher-order oligomers have thus far only been characterized for a Walker B mutant of Vps4 in the presence of ATP. Here, we report that although some mutant Vps4 proteins form dodecameric assemblies, active wild-type S. cerevisiae and S. solfataricus Vps4 enzymes can form hexamers in the presence of ATP and ADP, as assayed by size exclusion chromatography and equilibrium analytical ultracentifugation. The Vta1p activator binds hexameric yeast Vps4p without changing the oligomeric state of Vps4p, implying that the active Vta1p:Vps4p complex also contains a single hexameric ring. Additionally, we report crystal structures of two different archaeal Vps4 homologs, whose structures and lattice interactions suggest a conserved mode of oligomerization. Disruption of the proposed hexamerization interface by mutagenesis abolished the ATPase activity of archaeal Vps4 proteins and blocked Vps4p function in S. cerevisiae. These data challenge the prevailing model that active Vps4 is a double ring dodecamer, and argue that, like other type I AAA ATPases, Vps4 functions as a single ring with six subunits. PMID:24161953

  2. Screening of Drugs Inhibiting In vitro Oligomerization of Cu/Zn-Superoxide Dismutase with a Mutation Causing Amyotrophic Lateral Sclerosis.

    Science.gov (United States)

    Anzai, Itsuki; Toichi, Keisuke; Tokuda, Eiichi; Mukaiyama, Atsushi; Akiyama, Shuji; Furukawa, Yoshiaki

    2016-01-01

    Dominant mutations in Cu/Zn-superoxide dismutase (SOD1) gene have been shown to cause a familial form of amyotrophic lateral sclerosis (SOD1-ALS). A major pathological hallmark of this disease is abnormal accumulation of mutant SOD1 oligomers in the affected spinal motor neurons. While no effective therapeutics for SOD1-ALS is currently available, SOD1 oligomerization will be a good target for developing cures of this disease. Recently, we have reproduced the formation of SOD1 oligomers abnormally cross-linked via disulfide bonds in a test tube. Using our in vitro model of SOD1 oligomerization, therefore, we screened 640 FDA-approved drugs for inhibiting the oligomerization of SOD1 proteins, and three effective classes of chemical compounds were identified. Those hit compounds will provide valuable information on the chemical structures for developing a novel drug candidate suppressing the abnormal oligomerization of mutant SOD1 and possibly curing the disease.

  3. Metal Catalyzed Oligomerization Reactions of Organosiloxanes.

    Science.gov (United States)

    1982-10-28

    Table I. Some monosilanes , e.g. Me3SiH and PhMe2SiH, are found, but none of the expected PhMeSiH 2 is present. However, some of the expected oligomeric...of the monosilanes as indicated in Table I suggested that reaction 17 does occur, but the initially produced PhMeSiH 2 undergoes secondary

  4. Requirements and ontology for a G protein-coupled receptor oligomerization knowledge base

    Directory of Open Access Journals (Sweden)

    Parmentier Marc

    2007-05-01

    Full Text Available Abstract Background G Protein-Coupled Receptors (GPCRs are a large and diverse family of membrane proteins whose members participate in the regulation of most cellular and physiological processes and therefore represent key pharmacological targets. Although several bioinformatics resources support research on GPCRs, most of these have been designed based on the traditional assumption that monomeric GPCRs constitute the functional receptor unit. The increase in the frequency and number of reports about GPCR dimerization/oligomerization and the implication of oligomerization in receptor function makes necessary the ability to store and access information about GPCR dimers/oligomers electronically. Results We present here the requirements and ontology (the information scheme to describe oligomers and associated concepts and their relationships for an information system that can manage the elements of information needed to describe comprehensively the phenomena of both homo- and hetero-oligomerization of GPCRs. The comprehensive information management scheme that we plan to use for the development of an intuitive and user-friendly GPCR-Oligomerization Knowledge Base (GPCR-OKB is the result of a community dialog involving experimental and computational colleagues working on GPCRs. Conclusion Our long term goal is to disseminate to the scientific community organized, curated, and detailed information about GPCR dimerization/oligomerization and its related structural context. This information will be reported as close to the data as possible so the user can make his own judgment on the conclusions drawn for a particular study. The requirements and ontology described here will facilitate the development of future information systems for GPCR oligomers that contain both computational and experimental information about GPCR oligomerization. This information is freely accessible at http://www.gpcr-okb.org.

  5. Polybenzoxazine/Polyhedral Oligomeric Silsesquioxane (POSS Nanocomposites

    Directory of Open Access Journals (Sweden)

    Mohamed Gamal Mohamed

    2016-06-01

    Full Text Available The organic/inorganic hybrid materials from polyhedral oligomeric silsesquioxane (POSS, inorganic nanoparticles and polybenzoxazine (PBZ have received much interesting recently due to their excellent thermal and mechanical properties, flame retardance, low dielectric constant, well-defined inorganic framework at nanosized scale level, and higher performance relative to those of non-hybrid PBZs. This review describes the synthesis, dielectric constants, and thermal, rheological, and mechanical properties of covalently bonded mono- and multifunctionalized benzoxazine POSS hybrids, other functionalized benzoxazine POSS derivatives, and non-covalently (hydrogen bonded benzoxazine POSS composites.

  6. Green Nanocomposites from Renewable Plant Oils and Polyhedral Oligomeric Silsesquioxanes

    Directory of Open Access Journals (Sweden)

    Takashi Tsujimoto

    2015-06-01

    Full Text Available Green nanocomposites based on renewable plant oils and polyhedral oligomeric silsesquioxanes (POSS have been developed. An acid-catalyzed curing of epoxidized plant oils with oxirane-containing POSS derivatives produced transparent nanocomposite coatings with high gloss surface, in which the organic and inorganic components were linked via covalent bonds. The hardness and mechanical strength were improved by the incorporation of the POSS unit into the organic polymer matrix. Nanostructural analyses of the nanocomposites showed the formation of homogeneous structures at the micrometer scale. On the other hand, such improvements of the coating and mechanical properties were not observed in the composite without covalent bonds between the plant oil-based polymer and POSS unit. The study demonstrates the correlation between the nanostructure of composites and macroscopic properties.

  7. Phenolic compounds prevent the oligomerization of α-synuclein and reduce synaptic toxicity.

    Science.gov (United States)

    Takahashi, Ryoichi; Ono, Kenjiro; Takamura, Yusaku; Mizuguchi, Mineyuki; Ikeda, Tokuhei; Nishijo, Hisao; Yamada, Masahito

    2015-09-01

    Lewy bodies, mainly composed of α-synuclein (αS), are pathological hallmarks of Parkinson's disease and dementia with Lewy bodies. Epidemiological studies showed that green tea consumption or habitual intake of phenolic compounds reduced Parkinson's disease risk. We previously reported that phenolic compounds inhibited αS fibrillation and destabilized preformed αS fibrils. Cumulative evidence suggests that low-order αS oligomers are neurotoxic and critical species in the pathogenesis of α-synucleinopathies. To develop disease modifying therapies for α-synucleinopathies, we examined effects of phenolic compounds (myricetin (Myr), curcumin, rosmarinic acid (RA), nordihydroguaiaretic acid, and ferulic acid) on αS oligomerization. Using methods such as photo-induced cross-linking of unmodified proteins, circular dichroism spectroscopy, the electron microscope, and the atomic force microscope, we showed that Myr and RA inhibited αS oligomerization and secondary structure conversion. The nuclear magnetic resonance analysis revealed that Myr directly bound to the N-terminal region of αS, whereas direct binding of RA to monomeric αS was not detected. Electrophysiological assays for long-term potentiation in mouse hippocampal slices revealed that Myr and RA ameliorated αS synaptic toxicity by inhibition of αS oligomerization. These results suggest that Myr and RA prevent the αS aggregation process, reducing the neurotoxicity of αS oligomers. To develop disease modifying therapies for α-synucleinopathies, we examined effects of phenolic compounds on α-synuclein (αS) oligomerization. Phenolic compounds, especially Myricetin (Myr) and Rosmarinic acid (RA), inhibited αS oligomerization and secondary structure conversion. Myr and RA ameliorated αS synaptic toxicity on the experiment of long-term potentiation. Our results suggest that Myr and RA prevent αS aggregation process and reduce the neurotoxicity of αS oligomers. Phenolic compounds are good

  8. Endocytic pathways mediating oligomeric Aβ42 neurotoxicity

    Directory of Open Access Journals (Sweden)

    Laxton Kevin

    2010-05-01

    Full Text Available Abstract Background One pathological hallmark of Alzheimer's disease (AD is amyloid plaques, composed primarily of amyloid-β peptide (Aβ. Over-production or diminished clearance of the 42 amino acid form of Aβ (Aβ42 in the brain leads to accumulation of soluble Aβ and plaque formation. Soluble oligomeric Aβ (oAβ has recently emerged to be as a likely proximal cause of AD. Results Here we demonstrate that endocytosis is critical in mediating oAβ42-induced neurotoxicity and intraneuronal accumulation of Aβ. Inhibition of clathrin function either with a pharmacological inhibitor, knock-down of clathrin heavy chain expression, or expression of the dominant-negative mutant of clathrin-assembly protein AP180 did not block oAβ42-induced neurotoxicity or intraneuronal accumulation of Aβ. However, inhibition of dynamin and RhoA by expression of dominant negative mutants reduced neurotoxicity and intraneuronal Aβ accumulation. Pharmacologic inhibition of the dynamin-mediated endocytic pathway by genistein also reduced neurotoxicity. Conclusions These data suggest that dynamin-mediated and RhoA-regulated endocytosis are integral steps for oligomeric Aβ42-induced neurotoxicity and intraneuronal Aβ accumulation.

  9. Molecular basis for oligomeric-DNA binding and episome maintenance by KSHV LANA.

    Directory of Open Access Journals (Sweden)

    John F Domsic

    Full Text Available LANA is the KSHV-encoded terminal repeat binding protein essential for viral replication and episome maintenance during latency. We have determined the X-ray crystal structure of LANA C-terminal DNA binding domain (LANADBD to reveal its capacity to form a decameric ring with an exterior DNA binding surface. The dimeric core is structurally similar to EBV EBNA1 with an N-terminal arm that regulates DNA binding and is required for replication function. The oligomeric interface between LANA dimers is dispensable for single site DNA binding, but is required for cooperative DNA binding, replication function, and episome maintenance. We also identify a basic patch opposite of the DNA binding surface that is responsible for the interaction with BRD proteins and contributes to episome maintenance function. The structural features of LANADBD suggest a novel mechanism of episome maintenance through DNA-binding induced oligomeric assembly.

  10. Regulation of complement by cartilage oligomeric matrix protein allows for a novel molecular diagnostic principle in rheumatoid arthritis

    DEFF Research Database (Denmark)

    Happonen, Kaisa E; Saxne, Tore; Aspberg, Anders;

    2010-01-01

    Cartilage oligomeric matrix protein (COMP) is a structural component of cartilage, where it catalyzes collagen fibrillogenesis. Elevated amounts of COMP are found in serum during increased turnover of cartilage associated with active joint disease, such as rheumatoid arthritis (RA) and osteoarthr......Cartilage oligomeric matrix protein (COMP) is a structural component of cartilage, where it catalyzes collagen fibrillogenesis. Elevated amounts of COMP are found in serum during increased turnover of cartilage associated with active joint disease, such as rheumatoid arthritis (RA......) and osteoarthritis (OA). This study was undertaken to investigate the ability of COMP to regulate complement, a capacity that has previously been shown for some other cartilage proteins....

  11. Systematic comparison of the effects of alpha-synuclein mutations on its oligomerization and aggregation.

    Directory of Open Access Journals (Sweden)

    Diana F Lázaro

    2014-11-01

    Full Text Available Aggregation of alpha-synuclein (ASYN in Lewy bodies and Lewy neurites is the typical pathological hallmark of Parkinson's disease (PD and other synucleinopathies. Furthermore, mutations in the gene encoding for ASYN are associated with familial and sporadic forms of PD, suggesting this protein plays a central role in the disease. However, the precise contribution of ASYN to neuronal dysfunction and death is unclear. There is intense debate about the nature of the toxic species of ASYN and little is known about the molecular determinants of oligomerization and aggregation of ASYN in the cell. In order to clarify the effects of different mutations on the propensity of ASYN to oligomerize and aggregate, we assembled a panel of 19 ASYN variants and compared their behaviour. We found that familial mutants linked to PD (A30P, E46K, H50Q, G51D and A53T exhibited identical propensities to oligomerize in living cells, but had distinct abilities to form inclusions. While the A30P mutant reduced the percentage of cells with inclusions, the E46K mutant had the opposite effect. Interestingly, artificial proline mutants designed to interfere with the helical structure of the N-terminal domain, showed increased propensity to form oligomeric species rather than inclusions. Moreover, lysine substitution mutants increased oligomerization and altered the pattern of aggregation. Altogether, our data shed light into the molecular effects of ASYN mutations in a cellular context, and established a common ground for the study of genetic and pharmacological modulators of the aggregation process, opening new perspectives for therapeutic intervention in PD and other synucleinopathies.

  12. Near-Field Optical Microscopy of Defects in Cholesteric Oligomeric Liquid Crystal Films

    Energy Technology Data Exchange (ETDEWEB)

    Lukishova, S.G.; Schmid, A.W.

    2006-08-18

    This paper describes formation of 2-D hexagonal structures with a periodicity ~0.5-0.8 um in the defects of thin films of cholesteric oligomeric liquid crystals prepared by the evaporation of the solvent from the oligomer solution on the substrate. These regular arrays were observed by scanning near-field optical and concurrent atomic force microscopy. The mechanisms considered are both Benard-Marangoni and buoyancy conventions induced by solvent evaporation and air-bubble creation around the condensed water droplets from the air during evaporative cooling. Hexagonal structures prepared by this method can be used in photonic devices for emission enhancement, for instance, in liquid crystal lasers and single photon sources with oligomeric liquid crystal hosts.

  13. Genetic noise control via protein oligomerization

    Energy Technology Data Exchange (ETDEWEB)

    Ghim, C; Almaas, E

    2008-06-12

    Gene expression in a cell entails random reaction events occurring over disparate time scales. Thus, molecular noise that often results in phenotypic and population-dynamic consequences sets a fundamental limit to biochemical signaling. While there have been numerous studies correlating the architecture of cellular reaction networks with noise tolerance, only a limited effort has been made to understand the dynamical role of protein-protein associations. We have developed a fully stochastic model for the positive feedback control of a single gene, as well as a pair of genes (toggle switch), integrating quantitative results from previous in vivo and in vitro studies. In particular, we explicitly account for the fast protein binding-unbinding kinetics, RNA polymerases, and the promoter/operator sequences of DNA. We find that the overall noise-level is reduced and the frequency content of the noise is dramatically shifted to the physiologically irrelevant high-frequency regime in the presence of protein dimerization. This is independent of the choice of monomer or dimer as transcription factor and persists throughout the multiple model topologies considered. For the toggle switch, we additionally find that the presence of a protein dimer, either homodimer or heterodimer, may significantly reduce its intrinsic switching rate. Hence, the dimer promotes the robust function of bistable switches by preventing the uninduced (induced) state from randomly being induced (uninduced). The specific binding between regulatory proteins provides a buffer that may prevent the propagation of fluctuations in genetic activity. The capacity of the buffer is a non-monotonic function of association-dissociation rates. Since the protein oligomerization per se does not require extra protein components to be expressed, it provides a basis for the rapid control of intrinsic or extrinsic noise. The stabilization of phenotypically important toggle switches, and nested positive feedback loops in

  14. Zn(2+) site engineering at the oligomeric interface of the dopamine transporter

    DEFF Research Database (Denmark)

    Norgaard-Nielsen, Kristine; Norregaard, Lene; Hastrup, Hanne

    2002-01-01

    of the dopamine transporter (DAT) corresponding to the external end of transmembrane segment 6. Upon binding to this site, which involves a histidine inserted in position 310 (V310H) and the endogenous Cys306 within the same DAT molecule, Zn(2+) potently inhibits [(3)H]dopamine uptake. These data provide indirect...... evidence that conformational changes critical for the translocation process may occur at the interface between two transporter molecules in the oligomeric structure....

  15. Phosphomimetic mutations enhance oligomerization of phospholemman and modulate its interaction with the Na/K-ATPase.

    Science.gov (United States)

    Song, Qiujing; Pallikkuth, Sandeep; Bossuyt, Julie; Bers, Donald M; Robia, Seth L

    2011-03-18

    Na/K-ATPase (NKA) activity is dynamically regulated by an inhibitory interaction with a small transmembrane protein, phospholemman (PLM). Inhibition is relieved upon PLM phosphorylation. Phosphorylation may alter how PLM interacts with NKA and/or itself, but details of these interactions are unknown. To address this, we quantified FRET between PLM and its regulatory target NKA in live cells. Phosphorylation of PLM was mimicked by mutation S63E (PKC site), S68E (PKA/PKC site), or S63E/S68E. The dependence of FRET on protein expression in live cells yielded information about the structure and binding affinity of the PLM-NKA regulatory complex. PLM phosphomimetic mutations altered the quaternary structure of the regulatory complex and reduced the apparent affinity of the PLM-NKA interaction. The latter effect was likely due to increased oligomerization of PLM phosphomimetic mutants, as suggested by PLM-PLM FRET measurements. Distance constraints obtained by FRET suggest that phosphomimetic mutations slightly alter the oligomer quaternary conformation. Photon-counting histogram measurements revealed that the major PLM oligomeric species is a tetramer. We conclude that phosphorylation of PLM increases its oligomerization into tetramers, decreases its binding to NKA, and alters the structures of both the tetramer and NKA regulatory complex.

  16. Phosphomimetic Mutations Enhance Oligomerization of Phospholemman and Modulate Its Interaction with the Na/K-ATPase*

    Science.gov (United States)

    Song, Qiujing; Pallikkuth, Sandeep; Bossuyt, Julie; Bers, Donald M.; Robia, Seth L.

    2011-01-01

    Na/K-ATPase (NKA) activity is dynamically regulated by an inhibitory interaction with a small transmembrane protein, phospholemman (PLM). Inhibition is relieved upon PLM phosphorylation. Phosphorylation may alter how PLM interacts with NKA and/or itself, but details of these interactions are unknown. To address this, we quantified FRET between PLM and its regulatory target NKA in live cells. Phosphorylation of PLM was mimicked by mutation S63E (PKC site), S68E (PKA/PKC site), or S63E/S68E. The dependence of FRET on protein expression in live cells yielded information about the structure and binding affinity of the PLM-NKA regulatory complex. PLM phosphomimetic mutations altered the quaternary structure of the regulatory complex and reduced the apparent affinity of the PLM-NKA interaction. The latter effect was likely due to increased oligomerization of PLM phosphomimetic mutants, as suggested by PLM-PLM FRET measurements. Distance constraints obtained by FRET suggest that phosphomimetic mutations slightly alter the oligomer quaternary conformation. Photon-counting histogram measurements revealed that the major PLM oligomeric species is a tetramer. We conclude that phosphorylation of PLM increases its oligomerization into tetramers, decreases its binding to NKA, and alters the structures of both the tetramer and NKA regulatory complex. PMID:21220422

  17. Uncovering the Mechanism of Forkhead-Associated Domain-Mediated TIFA Oligomerization That Plays a Central Role in Immune Responses.

    Science.gov (United States)

    Weng, Jui-Hung; Hsieh, Yin-Cheng; Huang, Chia-Chi Flora; Wei, Tong-You Wade; Lim, Liang-Hin; Chen, Yu-Hou; Ho, Meng-Ru; Wang, Iren; Huang, Kai-Fa; Chen, Chun-Jung; Tsai, Ming-Daw

    2015-10-13

    Forkhead-associated (FHA) domain is the only signaling domain that recognizes phosphothreonine (pThr) specifically. TRAF-interacting protein with an FHA domain (TIFA) was shown to be involved in immune responses by binding with TRAF2 and TRAF6. We recently reported that TIFA is a dimer in solution and that, upon stimulation by TNF-α, TIFA is phosphorylated at Thr9, which triggers TIFA oligomerization via pThr9-FHA domain binding and activates nuclear factor κB (NF-κB). However, the structural mechanism for the functionally important TIFA oligomerization remains to be established. While FHA domain-pThr binding is known to mediate protein dimerization, its role in oligomerization has not been demonstrated at the structural level. Here we report the crystal structures of TIFA (residues 1-150, with the unstructured C-terminal tail truncated) and its complex with the N-terminal pThr9 peptide (residues 1-15), which show unique features in the FHA structure (intrinsic dimer and extra β-strand) and in its interaction with the pThr peptide (with residues preceding rather than following pThr). These structural features support previous and additional functional analyses. Furthermore, the structure of the complex suggests that the pThr9-FHA domain interaction can occur only between different sets of dimers rather than between the two protomers within a dimer, providing the structural mechanism for TIFA oligomerization. Our results uncover the mechanism of FHA domain-mediated oligomerization in a key step of immune responses and expand the paradigm of FHA domain structure and function.

  18. Comparative analyses of quaternary arrangements in homo-oligomeric proteins in superfamilies: Functional implications.

    Science.gov (United States)

    Sudha, Govindarajan; Srinivasan, Narayanaswamy

    2016-09-01

    A comprehensive analysis of the quaternary features of distantly related homo-oligomeric proteins is the focus of the current study. This study has been performed at the levels of quaternary state, symmetry, and quaternary structure. Quaternary state and quaternary structure refers to the number of subunits and spatial arrangements of subunits, respectively. Using a large dataset of available 3D structures of biologically relevant assemblies, we show that only 53% of the distantly related homo-oligomeric proteins have the same quaternary state. Considering these homologous homo-oligomers with the same quaternary state, conservation of quaternary structures is observed only in 38% of the pairs. In 36% of the pairs of distantly related homo-oligomers with different quaternary states the larger assembly in a pair shows high structural similarity with the entire quaternary structure of the related protein with lower quaternary state and it is referred as "Russian doll effect." The differences in quaternary state and structure have been suggested to contribute to the functional diversity. Detailed investigations show that even though the gross functions of many distantly related homo-oligomers are the same, finer level differences in molecular functions are manifested by differences in quaternary states and structures. Comparison of structures of biological assemblies in distantly and closely related homo-oligomeric proteins throughout the study differentiates the effects of sequence divergence on the quaternary structures and function. Knowledge inferred from this study can provide insights for improved protein structure classification and function prediction of homo-oligomers. Proteins 2016; 84:1190-1202. © 2016 Wiley Periodicals, Inc.

  19. Ordering properties of oligomeric columnar discotic liquid crystals

    NARCIS (Netherlands)

    Umesh, C.P.

    2015-01-01

    The synthesis and liquid crystalline ordering properties of oligomeric discotic liquid crystals were investigated. The phase behaviour and surface ordering properties are dependent on among others core type, spacer length and fluorination.    

  20. Cartilage oligomeric matrix protein in patients with juvenile idiopathic arthritis

    DEFF Research Database (Denmark)

    Bjørnhart, Birgitte; Juul, Anders; Nielsen, Susan

    2009-01-01

    Cartilage oligomeric matrix protein (COMP) has been identified as a prognostic marker of progressive joint destruction in rheumatoid arthritis. In this population based study we evaluated associations between plasma concentrations of COMP, disease activity, and growth velocity in patients...

  1. Organization of the mitochondrial apoptotic BAK pore: oligomerization of the BAK homodimers.

    Science.gov (United States)

    Aluvila, Sreevidya; Mandal, Tirtha; Hustedt, Eric; Fajer, Peter; Choe, Jun Yong; Oh, Kyoung Joon

    2014-01-31

    The multidomain pro-apoptotic Bcl-2 family proteins BAK and BAX are believed to form large oligomeric pores in the mitochondrial outer membrane during apoptosis. Formation of these pores results in the release of apoptotic factors including cytochrome c from the intermembrane space into the cytoplasm, where they initiate the cascade of events that lead to cell death. Using the site-directed spin labeling method of electron paramagnetic resonance (EPR) spectroscopy, we have determined the conformational changes that occur in BAK when the protein targets to the membrane and forms pores. The data showed that helices α1 and α6 disengage from the rest of the domain, leaving helices α2-α5 as a folded unit. Helices α2-α5 were shown to form a dimeric structure, which is structurally homologous to the recently reported BAX "BH3-in-groove homodimer." Furthermore, the EPR data and a chemical cross-linking study demonstrated the existence of a hitherto unknown interface between BAK BH3-in-groove homodimers in the oligomeric BAK. This novel interface involves the C termini of α3 and α5 helices. The results provide further insights into the organization of the BAK oligomeric pores by the BAK homodimers during mitochondrial apoptosis, enabling the proposal of a BAK-induced lipidic pore with the topography of a "worm hole."

  2. Hydrodynamic and functional analysis of HIV-1 Vif oligomerization.

    Science.gov (United States)

    Techtmann, Stephen M; Ghirlando, Rodolfo; Kao, Sandra; Strebel, Klaus; Maynard, Ernest L

    2012-03-13

    HIV-1 Vif is an accessory protein that induces the proteasomal degradation of the host restriction factor, apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G). The N-terminal half of Vif binds to APOBEC3G, and the C-terminal half binds to subunits of a cullin 5-based ubiquitin ligase. This Vif-directed ubiquitin ligase induces the degradation of APOBEC3G (a cytidine deaminase) and thereby protects the viral genome from mutation. A conserved PPLP motif near the C-terminus of Vif is essential for Vif function and is also involved in Vif oligomerization. However, the mechanism and functional significance of Vif oligomerization is unclear. We employed analytical ultracentrifugation to examine the oligomeric properties of Vif in solution. Contrary to previous reports, we find that Vif oligomerization does not require the conserved PPLP motif. Instead, our data suggest a more complex mechanism involving interactions among the HCCH motif, the BC box, and downstream residues in Vif. Mutation of residues near the PPLP motif (S165 and V166) affected the oligomeric properties of Vif and weakened the ability of Vif to bind and induce the degradation of APOBEC3G. We propose that Vif oligomerization may represent a mechanism for regulating interactions with APOBEC3G.

  3. Oligomerization paths of the nucleoprotein of influenza A virus.

    Science.gov (United States)

    Tarus, B; Bakowiez, O; Chenavas, S; Duchemin, L; Estrozi, L F; Bourdieu, C; Lejal, N; Bernard, J; Moudjou, M; Chevalier, C; Delmas, B; Ruigrok, R W H; Di Primo, C; Slama-Schwok, A

    2012-03-01

    The influenza viruses contain a segmented, negative strand RNA genome. Each RNA segment is covered by multiple copies of the nucleoprotein (NP) and is associated with the polymerase complex into ribonucleoprotein (RNP) particles. Despite its importance in the virus life cycle, the interactions between the NP and the genome are not well understood. Here, we studied the assembly process of NP-RNA oligomers and analyzed how the oligomeric/monomeric status of RNA-free NP affects RNA binding and oligomerization. Recombinant wild-type NP purified in low salt concentrations and a derived mutant engineered for oligomerization deficiency (R416A) were mainly monomeric in RNA-free solutions as shown by biochemical and electron microscopy techniques. NP monomer formed with RNA a fast 1/1 complex characterized by surface plasmon resonance. In a subsequent and slow process that depended on the RNA length, oligomerization of NP was mediated by RNA binding. In contrast, preparations of wild-type NP purified in high salt concentrations as well as mutant Y148A engineered for deficiency in nucleic acid binding were partly or totally oligomeric in RNA-free solutions. These trimer/tetramer NP oligomers bind directly as oligomers to RNA with a higher affinity than that of the monomers. Both oligomerization routes we characterized could be exploited by cellular or viral factors to modulate or control viral RNA encapsidation by NP. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  4. Lanthanide Complexes for Oligomerization of Phenyl Isocyanate

    Institute of Scientific and Technical Information of China (English)

    DENG,Ming-Yu; YAO,Ying-Ming; ZHOU,Yu-Fang; ZHANG,Li-Fen; SHEN,Qi

    2003-01-01

    A series of lanthanide complexes including (Ind)3Sm(THF)(1),[(MeCp)2Sm(μ-SPh)(THF)]2(2),[(MeCp)2Y(μ-O-i-Pr)]2(3),(MeCp)3Sm·THF(4),Sm(SPh)3(hmpa)3(5),[(MeCp)2Y-(μ-OCH2CF3)2(6)and (CF3CH2O)3Y(THF)3(7) were synthesized and they have good activity for the oligomerization of phenylisocyanate.Among them 5 shows the highest activity.The conversion is as high as 96.2%,with 1/2500 of the molar ratio of cat./PhNCO.The main components in oligomer were characterized to be a cycdlodimer and a cyclotrimer.The ratio of cyclodimer to cyclotrimer depends on the lanthanide complexes used.7 gave 85.2%cyclotrimer with 1/300 of the molar ratio of cat./PhNCO at 40℃ for 0.5h,while 5 gave 77.6% cyclodimer with 1/300 of the molar ratio of cat./PhNCO at 40℃ for 4h.

  5. Functional foods effective for hepatitis C: Identification of oligomeric proanthocyanidin and its action mechanism

    Institute of Scientific and Technical Information of China (English)

    Yo-ichi; Ishida; Masahiko; Takeshita; Hiroaki; Kataoka

    2014-01-01

    Hepatitis C virus(HCV)is a major cause of viral hepatitis and currently infects approximately 170 million people worldwide.An infection by HCV causes high rates of chronic hepatitis(>75%)and progresses to liver cirrhosis and hepatocellular carcinoma ultimately.HCV can be eliminated by a combination of pegylatedα-interferon and the broad-spectrum antiviral drug ribavirin;however,this treatment is still associated with poor efficacy and tolerability and is often accompanied by serious side-effects.While some novel direct-actingantivirals against HCV have been developed recently,high medical costs limit the access to the therapy in cost-sensitive countries.To search for new natural anti-HCV agents,we screened local agricultural products for their suppressive activities against HCV replication using the HCV replicon cell system in vitro.We found a potent inhibitor of HCV RNA expression in the extracts of blueberry leaves and then identified oligomeric proanthocyanidin as the active ingredient.Further investigations into the action mechanism of oligomeric proanthocyanidin suggested that it is an inhibitor of heterogeneous nuclear ribonucleoproteins(hn RNPs)such as hn RNP A2/B1.In this review,we presented an overview of functional foods and ingredients efficient for HCV infection,the chemical structural characteristics of oligomeric proanthocyanidin,and its action mechanism.

  6. Non-Enzymatic Oligomerization of 3’, 5’ Cyclic AMP

    Science.gov (United States)

    Costanzo, Giovanna; Pino, Samanta; Timperio, Anna Maria; Šponer, Judit E.; Šponer, Jiří; Nováková, Olga; Šedo, Ondrej; Zdráhal, Zbyněk; Di Mauro, Ernesto

    2016-01-01

    Recent studies illustrate that short oligonucleotide sequences can be easily produced from nucleotide precursors in a template-free non-enzymatic way under dehydrating conditions, i.e. using essentially dry materials. Here we report that 3’,5’ cyclic AMP may also serve as a substrate of the reaction, which proceeds under moderate conditions yet with a lower efficiency than the previously reported oligomerization of 3’,5’ cyclic GMP. Optimally the oligomerization requires (i) a temperature of 80°C, (ii) a neutral to alkaline environment and (iii) a time on the order of weeks. Differences in the yield and required reaction conditions of the oligomerizations utilizing 3’,5’ cGMP and cAMP are discussed in terms of the crystal structures of the compounds. Polymerization of 3’,5’ cyclic nucleotides, whose paramount relevance in a prebiotic chemistry context has been widely accepted for decades, supports the possibility that the origin of extant genetic materials might have followed a direct uninterrupted path since its very beginning, starting from non-elaborately pre-activated monomer compounds and simple reactions. PMID:27802310

  7. Gelatin-based biomaterial engineering with anhydride-containing oligomeric cross-linkers.

    Science.gov (United States)

    Loth, Tina; Hötzel, Rudi; Kascholke, Christian; Anderegg, Ulf; Schulz-Siegmund, Michaela; Hacker, Michael C

    2014-06-01

    Chemically cross-linked gelatin hydrogels are versatile cell-adhesive hydrogel materials that have been established for a variety of biomedical applications. The most prominent cross-linker is glutaraldehyde, which, however, has been described to cause compatibility problems and loss of microscopic but relevant structural features. A recently developed oligomeric cross-linker that contains anhydride functionalities was evaluated as cross-linker for the fabrication of gelatin-based hydrogels and microparticles. In a fast curing reaction, hydrogels composed of gelatin and oligomeric cross-linker were fabricated with good conversion over a wide concentration range of constituents and with cross-linkers of different anhydride contents. Hydrogel properties, such as dry weight and mechanics, could be controlled by hydrogel composition and rheological properties correlated to elastic moduli from 1 to 10 kPa. The gels were shown to be cytocompatible and promoted cell adhesion. In soft formulations, cells migrated into the hydrogel bulk. Gelatin microparticles prepared by a standard water-in-oil emulsion technique were also treated with the novel oligomers, and cross-linking degrees matching those obtained with glutaraldehyde were obtained. At the same time, fewer interparticular cross-links were observed. Fluorescein-derivatized cross-linkers yielded labeled microparticles in a concentration-dependent manner. The oligomeric cross-linkers are presented as an efficient and possibly more functional and compatible alternative to glutaraldehyde. The engineered hydrogel materials hold potential for various biomedical applications.

  8. Disulphide bond restrains the C-terminal region of thermostable direct hemolysin during folding to promote oligomerization.

    Science.gov (United States)

    Kundu, Nidhi; Tichkule, Swapnil; Pandit, Shashi Bhushan; Chattopadhyay, Kausik

    2017-01-15

    Pore-forming toxins (PFTs) are typically produced as water-soluble monomers, which upon interacting with target cells assemble into transmembrane oligomeric pores. Vibrio parahaemolyticus thermostable direct hemolysin (TDH) is an atypical PFT that exists as a tetramer in solution, prior to membrane binding. The TDH structure highlights a core β-sandwich domain similar to those found in the eukaryotic actinoporin family of PFTs. However, the TDH structure harbors an extended C-terminal region (CTR) that is not documented in the actinoporins. This CTR remains tethered to the β-sandwich domain through an intra-molecular disulphide bond. Part of the CTR is positioned at the inter-protomer interface in the TDH tetramer. Here we show that the truncation, as well as mutation, of the CTR compromise tetrameric assembly, and the membrane-damaging activity of TDH. Our study also reveals that intra-protomer disulphide bond formation during the folding/assembly process of TDH restrains the CTR to mediate its participation in the formation of inter-protomer contact, thus facilitating TDH oligomerization. However, once tetramerization is achieved, disruption of the disulphide bond does not affect oligomeric assembly. Our study provides critical insights regarding the regulation of the oligomerization mechanism of TDH, which has not been previously documented in the PFT family.

  9. Controlling Protein Oligomerization with Surface Curvature on the Nanoscale

    Science.gov (United States)

    Kurylowicz, Marty; Dutcher, John

    2011-03-01

    We investigate the effect of surface curvature on the conformation of beta-lactoglobulin (β LG) using Single Molecule Force Spectroscopy. β LG is a model interfacial protein which stabilizes oil droplets in milk and is known to undergo structural rearrangement when adsorbed onto a surface. We reliably control nanoscale surface curvature by creating close-packed monolayers of monodisperse polystyrene (PS) nanoparticles with diameters of 20, 40, 60, 80 and 140 nm, which are stable in aqueous buffer. By adsorbing β LG onto these hydrophobic surfaces and collecting force-extension curves in the fluid phase we can compare the conformation of β LG on 5 different surface curvatures with that on a flat PS film. We demonstrate a transition from oligomeric to monomeric β LG as the surface curvature is increased. Histograms of contour length from fits to peaks in the force-extension curves show a single maximum near 30 nm for β LG adsorbed onto nanoparticles with diameters less than 80 nm. For the larger nanoparticles, the histogram approaches that observed for β LG adsorbed onto a flat PS film, with maxima indicative of β LG dimers and trimers.

  10. Characterization and preparation of oligomeric procyanidins from Litchi chinensis pericarp.

    Science.gov (United States)

    Sui, Yong; Zheng, Yan; Li, Xiaopeng; Li, Shuyi; Xie, Bijun; Sun, Zhida

    2016-07-01

    The main purpose of this study is to characterize and prepare A-type oligomeric procyanidins from litchi pericarp (Litchi chinensis Baila). The variety of oligomeric procyanidins was characterized by LC-ESI-MS analysis. There were (+)-catechin, (-)-epicatechin, twelve dimers and six trimers of procyanidins were found in litchi pericarp extracts, and A-type procyanidins were much more abundant than B-type procyanidins. The main flavan-3-ol monomer and oligomeric procyanidins in litchi pericarp were (-)-epicatechin, A-type dimers (A1 and A2) and trimer (epicatechin-(4β-8, 2β-O-7)-epicatechin- (4β-8)-epicatechin). Procyanidin A1 (epicatechin-(4β-8, 2β-O-7)-catechin) was identified by NMR in litchi pericarp for the first time. (-)-Epicatechin and oligomeric procyanidins were prepared by the combination of AB-8 column chromatography and Toyopearl HW-40S column chromatography. The results showed that each fraction predominantly owned a single compound and gave a high yield with (-)-epicatechin, A-type dimers (A1 and A2) and trimer, suggesting a useful method to obtain pure (-)-epicatechin and A-type oligomeric procyanidins.

  11. Mapping the conformational dynamics and pathways of spontaneous steric zipper Peptide oligomerization.

    Directory of Open Access Journals (Sweden)

    Dirk Matthes

    Full Text Available The process of protein misfolding and self-assembly into various, polymorphic aggregates is associated with a number of important neurodegenerative diseases. Only recently, crystal structures of several short peptides have provided detailed structural insights into -sheet rich aggregates, known as amyloid fibrils. Knowledge about early events of the formation and interconversion of small oligomeric states, an inevitable step in the cascade of peptide self-assembly, however, remains still limited. We employ molecular dynamics simulations in explicit solvent to study the spontaneous aggregation process of steric zipper peptide segments from the tau protein and insulin in atomistic detail. Starting from separated chains with random conformations, we find a rapid formation of structurally heterogeneous, -sheet rich oligomers, emerging from multiple bimolecular association steps and diverse assembly pathways. Furthermore, our study provides evidence that aggregate intermediates as small as dimers can be kinetically trapped and thus affect the structural evolution of larger oligomers. Alternative aggregate structures are found for both peptide sequences in the different independent simulations, some of which feature characteristics of the known steric zipper conformation (e.g., -sheet bilayers with a dry interface. The final aggregates interconvert with topologically distinct oligomeric states exclusively via internal rearrangements. The peptide oligomerization was analyzed through the perspective of a minimal oligomer, i.e., the dimer. Thereby all observed multimeric aggregates can be consistently mapped onto a space of reduced dimensionality. This novel method of conformational mapping reveals heterogeneous association and reorganization dynamics that are governed by the characteristics of peptide sequence and oligomer size.

  12. Three dimensional complex plasma structures in a combined radio frequency and direct current discharge

    CERN Document Server

    Mitic, S; Khrapak, S A; Morfill, G E; 10.1063/1.4798418

    2013-01-01

    We report on the first detailed analysis of large three dimensional (3D) complex plasma structures in experiments performed in pure rf and combined rf+dc discharge modes. Inductively coupled plasma (ICP) is generated by an rf coil wrapped around the vertically positioned cylindrical glass tube at a pressure of 0.3 mbar. In addition, dc plasma can be generated by applying voltage to the electrodes at the ends of the tube far from the rf coil. The injected monodisperse particles are levitated in the plasma below the coil. A scanning laser sheet and a high resolution camera are used to determine the 3D positions of about $10^5$ particles. The observed bowl-shaped particle clouds reveal coexistence of various structures, including well-distinguished solid-like, less ordered liquid-like, and pronounced string-like phases. New criteria to identify string-like structures are proposed.

  13. Uncoupling of allosteric and oligomeric regulation in a functional hybrid enzyme constructed from Escherichia coli and human ribonucleotide reductase.

    Science.gov (United States)

    Fu, Yuan; Long, Marcus J C; Rigney, Mike; Parvez, Saba; Blessing, William A; Aye, Yimon

    2013-10-08

    An N-terminal-domain (NTD) and adjacent catalytic body (CB) make up subunit-α of ribonucleotide reductase (RNR), the rate-limiting enzyme for de novo dNTP biosynthesis. A strong linkage exists between ligand binding at the NTD and oligomerization-coupled RNR inhibition, inducible by both dATP and nucleotide chemotherapeutics. These observations have distinguished the NTD as an oligomeric regulation domain dictating the assembly of inactive RNR oligomers. Inactive states of RNR differ between eukaryotes and prokaryotes (α6 in human versus α4β4 in Escherichia coli , wherein β is RNR's other subunit); however, the NTD structurally interconnects individual α2 or α2 and β2 dimeric motifs within the respective α6 or α4β4 complexes. To elucidate the influence of NTD ligand binding on RNR allosteric and oligomeric regulation, we engineered a human- E. coli hybrid enzyme (HE) where human-NTD is fused to E. coli -CB. Both the NTD and the CB of the HE bind dATP. The HE specifically partners with E. coli -β to form an active holocomplex. However, although the NTD is the sole physical tether to support α2 and/or β2 associations in the dATP-bound α6 or α4β4 fully inhibited RNR complexes, the binding of dATP to the HE NTD only partially suppresses HE activity and fully precludes formation of higher-order HE oligomers. We postulate that oligomeric regulation is the ultimate mechanism for potent RNR inhibition, requiring species-specific NTD-CB interactions. Such interdomain cooperativity in RNR oligomerization is unexpected from structural studies alone or biochemical studies of point mutants.

  14. Genetic noise control via protein oligomerization

    Directory of Open Access Journals (Sweden)

    Almaas Eivind

    2008-11-01

    Full Text Available Abstract Background Gene expression in a cell entails random reaction events occurring over disparate time scales. Thus, molecular noise that often results in phenotypic and population-dynamic consequences sets a fundamental limit to biochemical signaling. While there have been numerous studies correlating the architecture of cellular reaction networks with noise tolerance, only a limited effort has been made to understand the dynamic role of protein-protein interactions. Results We have developed a fully stochastic model for the positive feedback control of a single gene, as well as a pair of genes (toggle switch, integrating quantitative results from previous in vivo and in vitro studies. In particular, we explicitly account for the fast binding-unbinding kinetics among proteins, RNA polymerases, and the promoter/operator sequences of DNA. We find that the overall noise-level is reduced and the frequency content of the noise is dramatically shifted to the physiologically irrelevant high-frequency regime in the presence of protein dimerization. This is independent of the choice of monomer or dimer as transcription factor and persists throughout the multiple model topologies considered. For the toggle switch, we additionally find that the presence of a protein dimer, either homodimer or heterodimer, may significantly reduce its random switching rate. Hence, the dimer promotes the robust function of bistable switches by preventing the uninduced (induced state from randomly being induced (uninduced. Conclusion The specific binding between regulatory proteins provides a buffer that may prevent the propagation of fluctuations in genetic activity. The capacity of the buffer is a non-monotonic function of association-dissociation rates. Since the protein oligomerization per se does not require extra protein components to be expressed, it provides a basis for the rapid control of intrinsic or extrinsic noise. The stabilization of regulatory circuits

  15. Cartilage oligomeric matrix protein enhances the vascularization of acellular nerves

    Directory of Open Access Journals (Sweden)

    Wei-ling Cui

    2016-01-01

    Full Text Available Vascularization of acellular nerves has been shown to contribute to nerve bridging. In this study, we used a 10-mm sciatic nerve defect model in rats to determine whether cartilage oligomeric matrix protein enhances the vascularization of injured acellular nerves. The rat nerve defects were treated with acellular nerve grafting (control group alone or acellular nerve grafting combined with intraperitoneal injection of cartilage oligomeric matrix protein (experimental group. As shown through two-dimensional imaging, the vessels began to invade into the acellular nerve graft from both anastomotic ends at day 7 post-operation, and gradually covered the entire graft at day 21. The vascular density, vascular area, and the velocity of revascularization in the experimental group were all higher than those in the control group. These results indicate that cartilage oligomeric matrix protein enhances the vascularization of acellular nerves.

  16. Cartilage oligomeric matrix protein enhances the vascularization of acellular nerves

    Institute of Scientific and Technical Information of China (English)

    Wei-ling Cui; Long-hai Qiu; Jia-yan Lian; Jia-chun Li; Jun Hu; Xiao-lin Liu

    2016-01-01

    Vascularization of acellular nerves has been shown to contribute to nerve bridging. In this study, we used a 10-mm sciatic nerve defect model in rats to determine whether cartilage oligomeric matrix protein enhances the vascularization of injured acellular nerves. The rat nerve defects were treated with acellular nerve grafting (control group) alone or acellular nerve grafting combined with intraperitoneal injection of cartilage oligomeric matrix protein (experimental group). As shown through two-dimensional imaging, the vessels began to invade into the acellular nerve graft from both anastomotic ends at day 7 post-operation, and gradually covered the entire graft at day 21. The vascular density, vascular area, and the velocity of revascularization in the experimental group were all higher than those in the control group. These results indicate that cartilage oligomeric matrix protein enhances the vascularization of acellular nerves.

  17. Multi-PAS domain-mediated protein oligomerization of PpsR from Rhodobacter sphaeroides

    Energy Technology Data Exchange (ETDEWEB)

    Heintz, Udo; Meinhart, Anton; Winkler, Andreas, E-mail: andreas.winkler@mpimf-heidelberg.mpg.de [Max Planck Institute for Medical Research, Heidelberg (Germany)

    2014-03-01

    Crystal structures of two truncated variants of the transcription factor PpsR from R. sphaeroides are presented that enabled the phasing of a triple PAS domain construct. Together, these structures reveal the importance of α-helical PAS extensions for multi-PAS domain-mediated protein oligomerization and function. Per–ARNT–Sim (PAS) domains are essential modules of many multi-domain signalling proteins that mediate protein interaction and/or sense environmental stimuli. Frequently, multiple PAS domains are present within single polypeptide chains, where their interplay is required for protein function. Although many isolated PAS domain structures have been reported over the last decades, only a few structures of multi-PAS proteins are known. Therefore, the molecular mechanism of multi-PAS domain-mediated protein oligomerization and function is poorly understood. The transcription factor PpsR from Rhodobacter sphaeroides is such a multi-PAS domain protein that, in addition to its three PAS domains, contains a glutamine-rich linker and a C-terminal helix–turn–helix DNA-binding motif. Here, crystal structures of two N-terminally and C-terminally truncated PpsR variants that comprise a single (PpsR{sub Q-PAS1}) and two (PpsR{sub N-Q-PAS1}) PAS domains, respectively, are presented and the multi-step strategy required for the phasing of a triple PAS domain construct (PpsR{sub ΔHTH}) is illustrated. While parts of the biologically relevant dimerization interface can already be observed in the two shorter constructs, the PpsR{sub ΔHTH} structure reveals how three PAS domains enable the formation of multiple oligomeric states (dimer, tetramer and octamer), highlighting that not only the PAS cores but also their α-helical extensions are essential for protein oligomerization. The results demonstrate that the long helical glutamine-rich linker of PpsR results from a direct fusion of the N-cap of the PAS1 domain with the C-terminal extension of the N-domain that

  18. [Theory of V.A. dogiel on polymerization and oligomerization as a general integration concept].

    Science.gov (United States)

    Makmaev, Iu V

    2010-01-01

    The theory of V.A. Dogiel on the significance of polymerization and ligomerization processes in the evolution of Protozoa and Metazoa is compared with the paper of I.I. Schmalhauisen (1972) on factors and steps of aromorph evolution. Dogiel's theory is considered as a general integration conception. Four steps are distinguished in the evolution of biological systems: (1) formation of morphofunctional system by units of the lower structural level, (2) polymerization of morphofunctional units of a system, (3) oligomerization of morphofunctional units of system by means of their reduction, uniting, or differentiation, (4) integration and stabilization of a system owing to development of morphofunctional connections between its parts.

  19. The N-terminus of TDP-43 promotes its oligomerization and enhances DNA binding affinity

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Chung-ke [Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan (China); Wu, Tzong-Huah [Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan (China); Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Institute of Biochemistry, Academia Sinica, Taipei 115, Taiwan (China); Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 300, Taiwan (China); Wu, Chu-Ya [Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan (China); Graduate Institute of Engineering, National Taiwan University of Science and Technology, Taipei 106, Taiwan (China); Chiang, Ming-hui; Toh, Elsie Khai-Woon [Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan (China); Hsu, Yin-Chih; Lin, Ku-Feng [Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan (China); Liao, Yu-heng [Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan (China); Huang, Tai-huang, E-mail: bmthh@gate.sinica.edu.tw [Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan (China); Department of Physics, National Taiwan Normal University, Taipei 106, Taiwan (China); Huang, Joseph Jen-Tse, E-mail: jthuang@chem.sinica.edu.tw [Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan (China)

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer The N-terminus of TDP-43 contains an independently folded structural domain (NTD). Black-Right-Pointing-Pointer The structural domains of TDP-43 are arranged in a beads-on-a-string fashion. Black-Right-Pointing-Pointer The NTD promotes TDP-43 oligomerization in a concentration-dependent manner. Black-Right-Pointing-Pointer The NTD may assist nucleic acid-binding activity of TDP-43. -- Abstract: TDP-43 is a DNA/RNA-binding protein associated with different neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-U). Here, the structural and physical properties of the N-terminus on TDP-43 have been carefully characterized through a combination of nuclear magnetic resonance (NMR), circular dichroism (CD) and fluorescence anisotropy studies. We demonstrate for the first time the importance of the N-terminus in promoting TDP-43 oligomerization and enhancing its DNA-binding affinity. An unidentified structural domain in the N-terminus is also disclosed. Our findings provide insights into the N-terminal domain function of TDP-43.

  20. Oligomerization of Mannan-binding Lectin Dictates Binding Properties and Complement Activation.

    Science.gov (United States)

    Kjaer, T R; Jensen, L; Hansen, A; Dani, R; Jensenius, J C; Dobó, J; Gál, P; Thiel, S

    2016-07-01

    The complement system is a part of the innate immune system and is involved in recognition and clearance of pathogens and altered-self structures. The lectin pathway of the complement system is initiated when soluble pattern recognition molecules (PRMs) with collagen-like regions bind to foreign or altered self-surfaces. Associated with the collagen-like stems of these PRMs are three mannan-binding lectin (MBL)-associated serine proteases (MASPs) and two MBL-associated proteins (MAps). The most studied of the PRMs, MBL, is present in serum mainly as trimeric and tetrameric oligomers of the structural subunit. We hypothesized that oligomerization of MBL may influence both the potential to bind to micro organisms and the interaction with the MASPs and MAps, thus influencing the ability to initiate complement activation. When testing binding at 37 °C, we found higher binding of tetrameric MBL to Staphylococcus aureus (S. aureus) than trimeric and dimeric MBL. In serum, we found that tetrameric MBL was the main oligomeric form present in complexes with the MASPs and MAp44. Such preference was confirmed using purified forms of recombinant MBL (rMBL) oligomers, where tetrameric rMBL interacted stronger with all of the MASPs and MAp44, compared to trimeric MBL. As a direct consequence of the weaker interaction with the MASPs, we found that trimeric rMBL was inferior to tetrameric rMBL in activating the complement system. Our data suggest that the oligomeric state of MBL is crucial both for the binding properties and the effector function of MBL.

  1. Cartilage oligomeric matrix protein specific antibodies are pathogenic

    DEFF Research Database (Denmark)

    Geng, Hui; Nandakumar, Kutty Selva; Pramhed, Anna

    2012-01-01

    ABSTRACT: INTRODUCTION: Cartilage oligomeric matrix protein (COMP) is a major non-collagenous component of cartilage. Earlier, we developed a new mouse model for rheumatoid arthritis using COMP. This study was undertaken to investigate the epitope specificity and immunopathogenicity of COMP...

  2. Protic Cationic Oligomeric Ionic Liquids of the Urethane Type

    DEFF Research Database (Denmark)

    Shevchenko, V. V.; Stryutsky, A. V.; Klymenko, N. S.

    2014-01-01

    Protic oligomeric cationic ionic liquids of the oligo(ether urethane) type are synthesized via the reaction of an isocyanate prepolymer based on oligo(oxy ethylene)glycol with M = 1000 with hexamethylene-diisocyanate followed by blocking of the terminal isocyanate groups with the use of amine...

  3. Polyhedral oligomeric silsesquioxane grafted polymer in polymeric foam

    Energy Technology Data Exchange (ETDEWEB)

    King, Bruce A.; Patankar, Kshitish A.; Costeux, Stephane; Jeon, Hyun K.

    2017-01-17

    A polymeric foam article with a polymer matrix defining multiple cells therein has a polymer component with a first polymer that is a polyhedral oligomeric silsesquioxane grafted polymer that has a weight-average molecular weight of two kilograms per mole or higher and 200 kilograms per mole or lower.

  4. Polyhedral oligomeric silsesquioxane grafted polymer in polymeric foam

    Science.gov (United States)

    King, Bruce A.; Patankar, Kshitish A.; Costeux, Stephane; Jeon, Hyun K.

    2017-01-17

    A polymeric foam article with a polymer matrix defining multiple cells therein has a polymer component with a first polymer that is a polyhedral oligomeric silsesquioxane grafted polymer that has a weight-average molecular weight of two kilograms per mole or higher and 200 kilograms per mole or lower.

  5. Synthesis, Characterization of Neutral Nickel Complexes Bearing N-Fluorophenylsalicylaldimine Chelate Ligands and Their Catalytic Activity to Ethylene Oligomerization

    Institute of Scientific and Technical Information of China (English)

    赵蔚; 黄吉玲; 朱仕正

    2005-01-01

    A series of neutral nickel complexes featuring N-fluorophenylsalicylaldimine chelate ligands was synthesized,and the molecular structure of complex 4 was further confirmed by X-ray crystallographic analysis. The neutral nickel complexes showed high activity up to 4.22×105 g oligomers/(mol Nioh) and high selectivity to C6 olefins in catalyzing ethylene oligomerization using methylaluminoxane (MAO) as cocatalyst.

  6. [THE COMPARATIVE ANALYSIS OF LEVEL OF OLIGOMERIC MATRIX PROTEIN OF CARTILAGE IN BLOOD SERUM OF PATIENTS WITH DISEASES OF MUSCULO-SKELETAL SYSTEM].

    Science.gov (United States)

    Starodubtseva, L A; Vasilieva, L V

    2016-02-01

    The osteoarthritis and rheumatoid arthritis are considered as the most prevalent diseases in the structure of diseases of musculoskeletal system. The higher social significance of these nosologies dictates necessity of searching reliable cartilage biomarkers having diagnostic validity both in discerning degenerative alterations at early stage of disease of joints and in monitoring of treatment effectiveness. The content of oligomeric matrix protein of cartilage using ELISA was evaluated in blood serum ofpatients with secondary osteoarthritis under rheumatoid arthritis (n=248). The comparison of derived results was carried out using control groups. Within the framework of study relationship was evaluated between level of oligomeric matrix protein of cartilage in patients with secondary osteoarthritis under rheumatoid arthritis with values offunctional KOOS index. The analysis of derived results established trend to increasing of level of oligomeric matrix protein of cartilage in blood serum ofpatients with secondary osteoarthritis under rheumatoid arthritis as compared with control groups. The moderate correlation interdependence between cartilage biomarker and KOOS index.

  7. LDRD final report on new homogeneous and supported oligomerization catalysts (LDRD 42461).

    Energy Technology Data Exchange (ETDEWEB)

    Hascall, Anthony G.; Kemp, Richard Alan

    2004-11-01

    The overall purpose of this LDRD is multifold. First, we are interested in preparing new homogeneous catalysts that can be used in the oligomerization of ethylene and in understanding commercially important systems better. Second, we are interested in attempting to support these new homogeneous catalysts in the pores of nano- or mesoporous materials in order to force new and unusual distributions of a-olefins to be formed during the oligomerization. Thus the overall purpose is to try to prepare new catalytic species and to possibly control the active site architecture in order to yield certain desired products during a catalytic reaction, much like nature does with enzymes. In order to rationally synthesize catalysts it is imperative to comprehend the function of the various components of the catalyst. In heterogeneous systems, it is of utmost importance to know how a support interacts with the active site of the catalyst. In fact, in the catalysis world this lack of fundamental understanding of the relationship between active site and support is the single largest reason catalysis is considered an 'empirical' or 'black box' science rather than a well-understood one. In this work we will be preparing novel ethylene oligomerization catalysts, which are normally P-O chelated homogeneous complexes, with new ligands that replace P with a stable carbene. We will also examine a commercially catalyst system and investigate the active site in it via X-ray crystallography. We will also attempt to support these materials inside the pores of nano- and mesoporous materials. Essentially, we will be tailoring the size and scale of the catalyst active site and its surrounding environment to match the size of the molecular product(s) we wish to make. The overall purpose of the study will be to prepare new homogeneous catalysts, and if successful in supporting them to examine the effects that steric constraints and pore structures can have on growing oligomer

  8. APOBEC3G oligomerization is associated with the inhibition of both Alu and LINE-1 retrotransposition.

    Directory of Open Access Journals (Sweden)

    Takayoshi Koyama

    Full Text Available Alu and LINE-1 (L1, which constitute ~11% and ~17% of the human genome, respectively, are transposable non-LTR retroelements. They transpose not only in germ cells but also in somatic cells, occasionally causing cancer. We have previously demonstrated that antiretroviral restriction factors, human APOBEC3 (hA3 proteins (A-H, differentially inhibit L1 retrotransposition. In this present study, we found that hA3 members also restrict Alu retrotransposition at differential levels that correlate with those observed previously for L1 inhibition. Through deletion analyses based on the best-characterized hA3 member human APOBEC3G (hA3G, its N-terminal 30 amino acids were required for its inhibitory activity against Alu retrotransposition. The inhibitory effect of hA3G on Alu retrotransposition was associated with its oligomerization that was affected by the deletion of its N-terminal 30 amino acids. Through structural modeling, the amino acids 24 to 28 of hA3G were predicted to be located at the interface of the dimer. The mutation of these residues resulted in abrogated hA3G oligomerization, and consistently abolished the inhibitory activity of hA3G against Alu retrotransposition. Importantly, the anti-L1 activity of hA3G was also associated with hA3G oligomerization. These results suggest that the inhibitory activities of hA3G against Alu and L1 retrotransposition might involve a common mechanism.

  9. Modulation of the oligomerization of myelin proteolipid protein by transmembrane helix interaction motifs.

    Science.gov (United States)

    Ng, Derek P; Deber, Charles M

    2010-08-17

    Proteolipid protein (PLP) is a highly hydrophobic 276-residue integral membrane protein that constitutes more than 50% of the total protein in central nervous system myelin. Previous studies have shown that this protein exists in myelin as an oligomer rather than as a monomer, and mutations in PLP that lead to neurological disorders such as Pelizaeus-Merzbacher disease and spastic paraplegia type 2 have been reported to affect its normal oligomerization. Here we employ peptide-based and in vivo approaches to examine the role of the TM domain in the formation of PLP quaternary structure through homo-oligomeric helix-helix interactions. Focusing on the TM4 alpha-helix (sequence (239)FIAAFVGAAATLVSLLTFMIAATY(262)), the site of several disease-causing point mutations that involve putative small residue helix-helix interaction motifs in the TM4 sequence, we used SDS-PAGE, fluorescence resonance energy transfer, size-exclusion chromatography, and TOXCAT assays in an Escherichia coli membrane to show that the PLP TM4 helix readily assembles into varying oligomeric states. In addition, through targeted studies of the PLP TM4 alpha-helix with point mutations that selectively eliminate these small residue motifs via substitution of Gly, Ala, or Ser residues with Ile residues, we describe a potential mechanism through which disease-causing point mutations can lead to aberrant PLP assembly. The overall results suggest that TM segments in misfolded PLP monomers that expose and/or create surface-exposed helix-helix interaction sites that are normally masked may have consequences for disease.

  10. Oligomerization states of Bowman-Birk inhibitor by atomic force microscopy and computational approaches.

    Science.gov (United States)

    Silva, Luciano P; Azevedo, Ricardo B; Morais, Paulo C; Ventura, Manuel M; Freitas, Sonia M

    2005-11-15

    Several methods have been applied to study protein-protein interaction from structural and thermodynamic point of view. The present study reveals that atomic force microscopy (AFM), molecular modeling, and docking approaches represent alternative methods offering new strategy to investigate structural aspects in oligomerization process of proteinase inhibitors. The topography of the black-eyed pea trypsin/chymotrypsin inhibitor (BTCI) was recorded by AFM and compared with computational rigid-bodies docking approaches. Multimeric states of BTCI identified from AFM analysis showed globular-ellipsoidal shapes. Monomers, dimers, trimers, and hexamers were the most prominent molecular arrays observed in AFM images as evaluated by molecular volume calculations and corroborated by in silico docking and theoretical approaches. We therefore propose that BTCI adopts stable and well-packed self-assembled states in monomer-dimer-trimer-hexamer equilibrium. Although there are no correlation between specificity and packing efficiency among proteinases and proteinase inhibitors, the AFM and docked BTCI analyses suggest that these assemblies may exist in situ to play their potential function in oligomerization process.

  11. Influence of oligomeric silsesquioxane coating treatment on interfacial properties of CF/PAA composites

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xuezhong; HUANG Yudong; WANG Tianyu; HU Lijiang

    2007-01-01

    Carbon fibres (CF) were modified with different oligomeric silsesquioxane (SSO) coatings to improve the interfacial property of carbon fibres/polyarylacetylene (CF/PAA).The interlaminar shear strength (ILSS) of CF/PAA was tested to determine the effect of the treatment.Atomic force microscopy (AFM) in force modulation mode was adopted to study the cross-section surface of unidirectional CF/PAA composites and the relative stiffness of various phases, including CF,interphase and resin.The probability histogram and line distribution of CF/PAA cross-section surface relative stiffness,obtained from the statistical analysis of relative stiffness image,were used to compare and study the interface characterizations of composites.The results show that the ILSS increases effectively and the effects on interfacial characterizations are distinguished from each other in accor-dance with the CF surface modified with different SSO coatings owing to the various structures.Cage oligomeric silsesquioxane,including large organic groups (methacryl isobutyl-POSS),has better treatment result.AFM observa-tions lead to the conclusion that an interfacial transition layer with different morphology and stiffness appears in CF/PAA composites after being treated by the SSO coatings of different structures.It can be inferred that the appearance of the transition layer may contribute to the improvement of fibre/matrix adhesion.

  12. Binding of an Oligomeric Ellagitannin Series to Bovine Serum Albumin (BSA): Analysis by Isothermal Titration Calorimetry (ITC).

    Science.gov (United States)

    Karonen, Maarit; Oraviita, Marianne; Mueller-Harvey, Irene; Salminen, Juha-Pekka; Green, Rebecca J

    2015-12-16

    A unique series of oligomeric ellagitannins was used to study their interactions with bovine serum albumin (BSA) by isothermal titration calorimetry. Oligomeric ellagitannins, ranging from monomer to heptamer and a mixture of octamer-undecamers, were isolated as individual pure compounds. This series allowed studying the effects of oligomer size and other structural features. The monomeric to trimeric ellagitannins deviated most from the overall trends. The interactions of ellagitannin oligomers from tetramers to octa-undecamers with BSA revealed strong similarities. In contrast to the equilibrium binding constant, enthalpy showed an increasing trend from the dimer to larger oligomers. It is likely that first the macrocyclic part of the ellagitannin binds to the defined binding sites on the protein surface and then the "flexible tail" of the ellagitannin coats the protein surface. The results highlight the importance of molecular flexibility to maximize binding between the ellagitannin and protein surfaces.

  13. Oligomerization and Polymerization of Ethylene Initiated by a Novel Ni(Ⅱ)-Based Acetyliminopyridine Complexes as Single-Site Catalysts

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Novel Ni(Ⅱ)-based acetyliminopyridine complexes 1b, 2b, 3b (1-3b), which are synthesized from ligands 1a, 2a, 3a (1-3a) and [NiCl2(DME)], are suitable precursors for the catalysts that are necessary for ethylene oligomerization and polymerization reactions, activated by methylaluminoxane (MAO).The MAO-treated 1-3b presents an active catalytic center, which may oligomerize and polymerize ethylene to produce linear α-olefins and polyethylene, respectively. The molecular weight distributions of oligomers that are obtained are in good agreement with the Schulz-Flory rules for oligomers>C4. The activity of oligomers show significant reliance on the structures of catalyst precursors.

  14. Synthesis, Characterization and Ethylene Oligomerization Behavior of Neutral Nickel Complexes Bearing N-Fluorinated Phenyl Salicylaldiminato Chelate Ligands

    Institute of Scientific and Technical Information of China (English)

    宋力平; 宋绍迪; 黄吉玲

    2012-01-01

    A series of neutral nickel complexes featuring N-fluorinated phenyl salicylaldiminato chelate ligands was syn- thesized, and the novel molecular structure of complex C14 was further confirmed by X-ray crystallographic analy- sis. The neutral nickel complexes showed high activity up to 9.96× 10^5 g oligomers/(mol Nioh) and high selectivity of C6 in catalyzing ethylene oligomerization using methylaluminoxane (MAO) as cocatalyst. It was observed that the strong electron-withdrawing effect of the fluorinated salicylaldiminato ligand was able to significantly increase the catalytic activity for oligomerization of ethylene. In addition, the influence of reaction parameters such as A1/Ni molar ratio, reaction temperature, a variety of cocatalyst and ethylene pressure on catalytic activity was investigated.

  15. Moyamoya disease-associated protein mysterin/RNF213 is a novel AAA+ ATPase, which dynamically changes its oligomeric state

    Science.gov (United States)

    Morito, Daisuke; Nishikawa, Kouki; Hoseki, Jun; Kitamura, Akira; Kotani, Yuri; Kiso, Kazumi; Kinjo, Masataka; Fujiyoshi, Yoshinori; Nagata, Kazuhiro

    2014-03-01

    Moyamoya disease is an idiopathic human cerebrovascular disorder that is characterized by progressive stenosis and abnormal collateral vessels. We recently identified mysterin/RNF213 as its first susceptibility gene, which encodes a 591-kDa protein containing enzymatically active P-loop ATPase and ubiquitin ligase domains and is involved in proper vascular development in zebrafish. Here we demonstrate that mysterin further contains two tandem AAA+ ATPase modules and forms huge ring-shaped oligomeric complex. AAA+ ATPases are known to generally mediate various biophysical and mechanical processes with the characteristic ring-shaped structure. Fluorescence correlation spectroscopy and biochemical evaluation suggested that mysterin dynamically changes its oligomeric forms through ATP/ADP binding and hydrolysis cycles. Thus, the moyamoya disease-associated gene product is a unique protein that functions as ubiquitin ligase and AAA+ ATPase, which possibly contributes to vascular development through mechanical processes in the cell.

  16. Understanding Peptide Oligomeric State in Langmuir Monolayers of Amphiphilic 3-Helix Bundle-Forming Peptide-PEG Conjugates

    Science.gov (United States)

    Shu, Jessica Y.; Xu, Ting

    2016-01-01

    Coiled-coil peptide–polymer conjugates are an emerging class of biomaterials. Fundamental understanding of the coiled-coil oligomeric state and assembly process of these hybrid building blocks is necessary to exert control over their assembly into well-defined structures. Here, we studied the effect of peptide structure and PEGylation on the self-assembly process and oligomeric state of a Langmuir monolayer of amphiphilic coiled-coil peptide–polymer conjugates using X-ray reflectivity (XR) and grazing-incidence X-ray diffraction (GIXD). Our results show that the oligomeric state of PEGylated amphiphiles based on 3-helix bundle-forming peptide is surface pressure dependent, a mixture of dimers and trimers was formed at intermediate surface pressure but transitions into trimers completely upon increasing surface pressure. Moreover, the interhelical distance within the coiled-coil bundle of 3-helix peptide-PEG conjugate amphiphiles was not perturbed under high surface pressure. Present studies provide valuable insights into the self-assembly process of hybrid peptide–polymer conjugates and guidance to develop biomaterials with controlled multivalency of ligand presentation. PMID:27784156

  17. Tunable Gravimetric and Volumetric Hydrogen Storage Capacities in Polyhedral Oligomeric Silsesquioxane Frameworks.

    Science.gov (United States)

    Deshmukh, Amol; Chiu, Cheng-Chau; Chen, Yun-Wen; Kuo, Jer-Lai

    2016-09-28

    We study the hydrogen adsorption in porous frameworks composed of silsesquioxane cages linked via boron substituted aromatic structures by first-principles modeling. Such polyhedral oligomeric silsesquioxane (POSS) frameworks can be further modified by decorating them with metal atoms binding to the ring structures of the linkers. We have considered Sc- and Ti-doped frameworks which bind H2 via so-called Kubas interaction between hydrogen molecules and transition metal atoms. It will be demonstrated that the maximum H2 gravimetric capacity can be improved to more than 7.5 wt % by using longer linkers with more ring structures. However, the maximum H2 volumetric capacity can be tuned to more than 70 g/L by varying the size of silsesquioxane cages. We are optimistic that by varying the building blocks, POSS frameworks can be modified to meet the targets for the gravimetric and volumetric capacities set by the U.S. Department of Energy.

  18. Pharmacological Profiles of Oligomerized μ-Opioid Receptors

    OpenAIRE

    Ing-Kang Ho; Cynthia Wei-Sheng Lee

    2013-01-01

    Opioids are widely prescribed pain relievers with multiple side effects and potential complications. They produce analgesia via G-protein-protein coupled receptors: μ-, δ-, κ-opioid and opioid receptor-like 1 receptors. Bivalent ligands targeted to the oligomerized opioid receptors might be the key to developing analgesics without undesired side effects and obtaining effective treatment for opioid addicts. In this review we will update the biological effects of μ-opioids on homo- or hetero-ol...

  19. Receptor Oligomerization as a Process Modulating Cellular Semiotics

    DEFF Research Database (Denmark)

    Giorgi, Franco; Bruni, Luis Emilio; Maggio, Roberto

    2010-01-01

    The majority of G protein-coupled receptors (GPCRs) self-assemble in the form dimeric/oligomeric complexes along the plasma membrane. Due to the molecular interactions they participate, GPCRs can potentially provide the framework for discriminating a wide variety of intercellular signals, as based...... and degeneracy may appear as the required feature to integrate the cell system into functional units of progressively higher hierarchical levels....

  20. DNA display of glycoconjugates to emulate oligomeric interactions of glycans

    Directory of Open Access Journals (Sweden)

    Alexandre Novoa

    2015-05-01

    Full Text Available Glycans (carbohydrate portion of glycoproteins and glycolipids frequently exert their function through oligomeric interactions involving multiple carbohydrate units. In efforts to recapitulate the diverse spatial arrangements of the carbohydrate units, assemblies based on hybridization of nucleic acid conjugates have been used to display simplified ligands with tailored interligand distances and valences. The programmability of the assemblies lends itself to a combinatorial display of multiple ligands. Recent efforts in the synthesis and applications of such conjugates are discussed.

  1. Copper-induced oligomerization of peptides: a model study.

    Science.gov (United States)

    Schlosser, Gitta; Stefanescu, Raluca; Przybylski, Michael; Murariu, Manuela; Hudecz, Ferenc; Drochioiu, Gabi

    2007-01-01

    In this work, copper-binding of the tetraglycine peptide (Gly-Gly-Gly-Gly) was studied by electrospray ionization mass spectrometry. Experiments were performed under alkaline conditions, in the presence of ethanolamine (pH 10.95). We observed that the presence of copper(II) ions induces the aggregation of the peptide and the formation of copper-bound complexes with higher molecular mass is favored, such as the oligomer complexes [3M+2Cu-3H](+) and [4M+3Cu-5H](+). At 1:1 peptide-copper(II) ion ratio, the singly charged [3M+2Cu-3H](+) oligomer complex is the base peak in the mass spectrum. Metal ion-induced oligomer-ization of neurotoxic peptides is well known in the literature; however, there are very few examples in which such oligomerization was directly observed by mass spectrometry. Our results show that application of short peptides can be useful to study the -mechanism of metal ion binding and metal ion-induced oligomerization of peptides.

  2. Characterization of Lassa virus glycoprotein oligomerization and influence of cholesterol on virus replication.

    Science.gov (United States)

    Schlie, Katrin; Maisa, Anna; Lennartz, Frank; Ströher, Ute; Garten, Wolfgang; Strecker, Thomas

    2010-01-01

    Mature glycoprotein spikes are inserted in the Lassa virus envelope and consist of the distal subunit GP-1, the transmembrane-spanning subunit GP-2, and the signal peptide, which originate from the precursor glycoprotein pre-GP-C by proteolytic processing. In this study, we analyzed the oligomeric structure of the viral surface glycoprotein. Chemical cross-linking studies of mature glycoprotein spikes from purified virus revealed the formation of trimers. Interestingly, sucrose density gradient analysis of cellularly expressed glycoprotein showed that in contrast to trimeric mature glycoprotein complexes, the noncleaved glycoprotein forms monomers and oligomers spanning a wide size range, indicating that maturation cleavage of GP by the cellular subtilase SKI-1/S1P is critical for formation of the correct oligomeric state. To shed light on a potential relation between cholesterol and GP trimer stability, we performed cholesterol depletion experiments. Although depletion of cholesterol had no effect on trimerization of the glycoprotein spike complex, our studies revealed that the cholesterol content of the viral envelope is important for the infectivity of Lassa virus. Analyses of the distribution of viral proteins in cholesterol-rich detergent-resistant membrane areas showed that Lassa virus buds from membrane areas other than those responsible for impaired infectivity due to cholesterol depletion of lipid rafts. Thus, derivation of the viral envelope from cholesterol-rich membrane areas is not a prerequisite for the impact of cholesterol on virus infectivity.

  3. Multicoil2: predicting coiled coils and their oligomerization states from sequence in the twilight zone.

    Directory of Open Access Journals (Sweden)

    Jason Trigg

    Full Text Available The alpha-helical coiled coil can adopt a variety of topologies, among the most common of which are parallel and antiparallel dimers and trimers. We present Multicoil2, an algorithm that predicts both the location and oligomerization state (two versus three helices of coiled coils in protein sequences. Multicoil2 combines the pairwise correlations of the previous Multicoil method with the flexibility of Hidden Markov Models (HMMs in a Markov Random Field (MRF. The resulting algorithm integrates sequence features, including pairwise interactions, through multinomial logistic regression to devise an optimized scoring function for distinguishing dimer, trimer and non-coiled-coil oligomerization states; this scoring function is used to produce Markov Random Field potentials that incorporate pairwise correlations localized in sequence. Multicoil2 significantly improves both coiled-coil detection and dimer versus trimer state prediction over the original Multicoil algorithm retrained on a newly-constructed database of coiled-coil sequences. The new database, comprised of 2,105 sequences containing 124,088 residues, includes reliable structural annotations based on experimental data in the literature. Notably, the enhanced performance of Multicoil2 is evident when tested in stringent leave-family-out cross-validation on the new database, reflecting expected performance on challenging new prediction targets that have minimal sequence similarity to known coiled-coil families. The Multicoil2 program and training database are available for download from http://multicoil2.csail.mit.edu.

  4. SYNTHESIS AND CHARACTERIZATION OF A NOVEL REACTIVE OLIGOMERIC LADDERLIKE COPOLYMETHYL-AMINOPROPYL SILSESQUIOXANE

    Institute of Scientific and Technical Information of China (English)

    Qing-hua Duan; Tao-yi Zhang; Kui-lin Deng; Ping Xie; Rong-ben Zhang

    2005-01-01

    A novel ordered reactive oligomeric ladderlike copolymethyl-aminopropylsilsesquioxane (Me-AP-T), was successfully synthesized by an efficient hydrosilylation graft reaction of 1,1,1-trimethyl-N-2-propenyl-silanamine with a reactive oligomeric ladderlike copolymethyl-hydrosilsesquioxane (Me-H-T), which was prepared by "supramolecular chemistry-dictated stepwise coupling polymerization" process. FT-IR, 1H- and 29Si-NMR spectra show that hydrosilylation reaction proceeds completely. Moreover, 1H-NMR spectra also indicate that both the molar ratios of ≡SiMe group to Si-H and ≡ SiMe group to ≡ SiPrNH2 are approximately 1:1 respectively based on the ratio of integrated resonance areas of the different groups. XRD analysis and 29Si-NMR spectra are used for investigating the ladderlike structure of the polymers. A high Tg (128.38℃) of Me-AP-T shown in DSC measurement implies that the ordered reactive Me-AP-T macromolecules have much stiff chains.

  5. Redox and metal-regulated oligomeric state for human porphobilinogen synthase activation.

    Science.gov (United States)

    Sawada, N; Nagahara, N; Arisaka, F; Mitsuoka, K; Minami, M

    2011-06-01

    The oligomeric state of human porphobilinogen synthase (PBGS) [EC.4.2.1.24] is homooctamer, which consists of conformationally heterogenous subunits in the tertiary structure under air-saturated conditions. When PBGS is activated by reducing agent with zinc ion, a reservoir zinc ion coordinated by Cys(223) is transferred in the active center to be coordinated by Cys(122), Cys(124), and Cys(132) (Sawada et al. in J Biol Inorg Chem 10:199-207, 2005). The latter zinc ion serves as an electrophilic catalysis. In this study, we investigated a conformational change associated with the PBGS activation by reducing agent and zinc ion using analytical ultracentrifugation, negative staining electron microscopy, native PAGE, and enzyme activity staining. The results are in good agreement with our notion that the main component of PBGS is octamer with a few percent of hexamer and that the octamer changes spatial subunit arrangement upon reduction and further addition of zinc ion, accompanying decrease in f/f (0). It is concluded that redox-regulated PBGS activation via cleavage of disulfide bonds among Cys(122), Cys(124), and Cys(132) and coordination with zinc ion is closely linked to change in the oligomeric state.

  6. Synthesis and Characterization of A Polynuclear Yttrium Trifluoroethoxide and Its Activity for Oligomerization of Phenyl Isocyanate

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    A polynuclear yttrium trifluoroethoxide was synthesized and its activity for the oligomerization of phenyl isocyanate was tested. Reaction of anhydrous YCl3 with CF3CH2ONa in a 1:3 molar ratio in THF at room temperature, after workup, gave complex 1 as colorless crystals. The composition of complex 1 is [Y(OCH2CF3)3]6ONa2(THF)12, which was identified by elemental analysis and 1H NMR. Further X-ray structure determination reveals that complex 1 is a polynuclear ion pair compound, [Y6(μ6-O)(OCH2CF3)18]2-[Na(THF)6]+2 (1). The crystal data of complex 1 are trigonal, P-3 space group, a=1.78440(14) nm, b=1.78440(14) nm, c=1.27395(12) nm, γ=120°, V=3.5129(5) nm3, Z=1, Dc=1.607 mg·m-3, μ=2.591 mm-1 (Mo Kα), F(000)=1710, R=0.067, wR=0.182. Preliminary results demonstrated that complex 1 is a highly effective catalyst for the oligomerization of phenyl isocyanate.

  7. The ESR Characterization of Oligomeric Thiophene Materials

    Science.gov (United States)

    1994-06-28

    1955). 16 Table 1. Summary of cyclic voltametry results. Table 2. Summary of ESR results. 17 Figure 1. Structures of molecules studied. Figure 2...H20 removal system). The cyclic voltammetry experiments were performed with a potentiostat/galvanostat EG&G model 273 and recorded on an X-Y IBM...Results of cyclic voltammetry measurements are given in Table 1 and representative voltammograms are shown in Figures 2-3. The cyclic voltammetry of 7

  8. Metal-dependent SpoIIE oligomerization stabilizes FtsZ during asymmetric division in Bacillus subtilis

    Science.gov (United States)

    Król, Ewa; de Sousa Borges, Anabela; Kopacz, Malgorzata

    2017-01-01

    SpoIIE is a bifunctional protein involved in asymmetric septum formation and in activation of the forespore compartment-specific transcription factor σF through dephosphorylation of SpoIIAA-P. The phosphatase activity of SpoIIE requires Mn2+ as a metal cofactor. Here, we show that the presence of a metal cofactor also influences SpoIIE oligomerization and asymmetric septum formation. Absence of Mn2+ from sporulation medium results in a delay of the formation of polar FtsZ-rings, similar to a spoIIE null mutant. We purified the entire cytoplasmic part of the SpoIIE protein, and show that the protein copurifies with bound metals. Metal binding both stimulates SpoIIE oligomerization, and results in the formation of larger oligomeric structures. The presence of SpoIIE oligomers reduces FtsZ GTP hydrolysis activity and stabilizes FtsZ polymers in a light scattering assay. Combined, these results indicate that metal binding is not just required for SpoIIE phosphatase activity but also is important for SpoIIE's role in asymmetric septum formation. PMID:28358838

  9. Protective role of Cys-178 against the inactivation and oligomerization of human insulin-degrading enzyme by oxidation and nitrosylation.

    Science.gov (United States)

    Ralat, Luis A; Ren, Min; Schilling, Alexander B; Tang, Wei-Jen

    2009-12-01

    Insulin-degrading enzyme (IDE), a 110-kDa metalloendopeptidase, hydrolyzes several physiologically relevant peptides, including insulin and amyloid-beta (Abeta). Human IDE has 13 cysteines and is inhibited by hydrogen peroxide and S-nitrosoglutathione (GSNO), donors of reactive oxygen and nitrogen species, respectively. Here, we report that the oxidative burst of BV-2 microglial cells leads to oxidation or nitrosylation of secreted IDE, leading to the reduced activity. Hydrogen peroxide and GSNO treatment of IDE reduces the V(max) for Abeta degradation, increases IDE oligomerization, and decreases IDE thermostability. Additionally, this inhibitory response of IDE is substrate-dependent, biphasic for Abeta degradation but monophasic for a shorter bradykinin-mimetic substrate. Our mutational analysis of IDE and peptide mass fingerprinting of GSNO-treated IDE using Fourier transform-ion cyclotron resonance mass spectrometer reveal a surprising interplay of Cys-178 with Cys-110 and Cys-819 for catalytic activity and with Cys-789 and Cys-966 for oligomerization. Cys-110 is near the zinc-binding catalytic center and is normally buried. The oxidation and nitrosylation of Cys-819 allow Cys-110 to be oxidized or nitrosylated, leading to complete inactivation of IDE. Cys-789 is spatially adjacent to Cys-966, and their nitrosylation and oxidation together trigger the oligomerization and inhibition of IDE. Interestingly, the Cys-178 modification buffers the inhibition caused by Cys-819 modification and prevents the oxidation or nitrosylation of Cys-110. The Cys-178 modification can also prevent the oligomerization-mediated inhibition. Thus, IDE can be intricately regulated by reactive oxygen or nitrogen species. The structure of IDE reveals the molecular basis for the long distance interactions of these cysteines and how they regulate IDE function.

  10. Transmembrane oligomeric form of Vibrio cholerae cytolysin triggers TLR2/TLR6-dependent proinflammatory responses in monocytes and macrophages.

    Science.gov (United States)

    Khilwani, Barkha; Mukhopadhaya, Arunika; Chattopadhyay, Kausik

    2015-02-15

    Vibrio cholerae cytolysin (VCC) kills target eukaryotic cells by forming transmembrane oligomeric β-barrel pores. Once irreversibly converted into the transmembrane oligomeric form, VCC acquires an unusual structural stability and loses its cytotoxic property. It is therefore possible that, on exertion of its cytotoxic activity, the oligomeric form of VCC retained in the disintegrated membrane fractions of the lysed cells would survive within the host cellular milieu for a long period, without causing any further cytotoxicity. Under such circumstances, VCC oligomers may potentially be recognized by the host immune cells. Based on such a hypothesis, in the present study we explored the interaction of the transmembrane oligomeric form of VCC with the monocytes and macrophages of the innate immune system. Our study shows that the VCC oligomers assembled in the liposome membranes elicit potent proinflammatory responses in monocytes and macrophages, via stimulation of the toll-like receptor (TLR)2/TLR6-dependent signalling cascades that involve myeloid differentiation factor 88 (MyD88)/interleukin-1-receptor-associated kinase (IRAK)1/tumour-necrosis-factor-receptor-associated factor (TRAF)6. VCC oligomer-mediated proinflammatory responses critically depend on the activation of the transcription factor nuclear factor-κB. Proinflammatory responses induced by the VCC oligomers also require activation of the mitogen-activated protein kinase (MAPK) family member c-Jun N-terminal kinase, which presumably acts via stimulation of the transcription factor activator protein-1. Notably, the role of the MAPK p38 could not be documented in the process.

  11. Properties of PMR Polyimides Improved by Preparation of Polyhedral Oligomeric Silsesquioxane (POSS) Nanocomposites

    Science.gov (United States)

    Campbell, Sandi G.; Lee, Andre

    2005-01-01

    The field of hybrid organic-inorganic materials has grown drastically over the last several years. This interest stems from our ever-increasing ability to custom-build and control molecular structure at several length scales. This ability to control both the composition and structure of hybrid materials is sometimes broadly referred to as nanocomposite systems. One class of hybrid (organic-inorganic) nanostructured material is polyhedral oligomeric silsesquioxane (POSS), shown in the preceding diagram. The hybrid composition gives POSS materials dramatically enhanced properties relative to traditional hydrocarbons and inorganics. An important benefit of this technology is that it makes possible the formulations of nanostructured chemicals with excellent thermal and oxidative stability. This is largely due to the inorganic component.

  12. Functional Perfluoroalkyl Polyhedral Oligomeric Silsesquioxane (F-POSS): Building Blocks for Low Surface Energy Materials

    Science.gov (United States)

    2010-10-21

    Technical Paper 3. DATES COVERED (From - To) 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Functional Perfluoroalkyl Polyhedral Oligomeric Silsesquioxane (F...long chain fluorinated alkyl groups ranging from 6-12 carbon atoms in length. Herein, a disilanol perfluoroalkyl polyhedral oligomeric...FUNCTIONAL PERFLUOROALKYL POLYHEDRAL OLIGOMERIC SILSESQUIOXANES (F-POSS): BUILDING BLOCKS FOR LOW SURFACE ENERGY MATERIA LS Sean M Rami,.e:, Yvonne Dia

  13. hA molecular switch in amyloid assembly: Met35 and amyloid beta-protein oligomerization

    OpenAIRE

    Bitan, Gal; Tarus, Bogdan; Vollers, Sabrina S.; Lashuel, Hilal A.; Condron, Margaret M.; Straub, John E.; Teplow, David B.

    2003-01-01

    Aberrant protein oligomerization is an important pathogenetic process in vivo. In Alzheimer's disease (AD), the amyloid beta-protein (Abeta) forms neurotoxic oligomers. The predominant in vivo Abeta alloforms, Abeta40 and Abeta42, have distinct oligomerization pathways. Abeta42 monomers oligomerize into pentamer/hexamer units (paranuclei) which self-associate to form larger oligomers. Abeta40 does not form these paranuclei, a fact which may explain the particularly strong linkage of Abeta42 w...

  14. Large-scale identification of coevolution signals across homo-oligomeric protein interfaces by direct coupling analysis.

    Science.gov (United States)

    Uguzzoni, Guido; John Lovis, Shalini; Oteri, Francesco; Schug, Alexander; Szurmant, Hendrik; Weigt, Martin

    2017-03-28

    Proteins have evolved to perform diverse cellular functions, from serving as reaction catalysts to coordinating cellular propagation and development. Frequently, proteins do not exert their full potential as monomers but rather undergo concerted interactions as either homo-oligomers or with other proteins as hetero-oligomers. The experimental study of such protein complexes and interactions has been arduous. Theoretical structure prediction methods are an attractive alternative. Here, we investigate homo-oligomeric interfaces by tracing residue coevolution via the global statistical direct coupling analysis (DCA). DCA can accurately infer spatial adjacencies between residues. These adjacencies can be included as constraints in structure prediction techniques to predict high-resolution models. By taking advantage of the ongoing exponential growth of sequence databases, we go significantly beyond anecdotal cases of a few protein families and apply DCA to a systematic large-scale study of nearly 2,000 Pfam protein families with sufficient sequence information and structurally resolved homo-oligomeric interfaces. We find that large interfaces are commonly identified by DCA. We further demonstrate that DCA can differentiate between subfamilies with different binding modes within one large Pfam family. Sequence-derived contact information for the subfamilies proves sufficient to assemble accurate structural models of the diverse protein-oligomers. Thus, we provide an approach to investigate oligomerization for arbitrary protein families leading to structural models complementary to often-difficult experimental methods. Combined with ever more abundant sequential data, we anticipate that this study will be instrumental to allow the structural description of many heteroprotein complexes in the future.

  15. Key residues for the oligomerization of A{beta}42 protein in Alzheimer's disease

    Energy Technology Data Exchange (ETDEWEB)

    Ngo, Sam [Department of Neurology, Brain Research Institute, Molecular Biology Institute, University of California, Los Angeles, CA 90095 (United States); Guo, Zhefeng, E-mail: zhefeng@ucla.edu [Department of Neurology, Brain Research Institute, Molecular Biology Institute, University of California, Los Angeles, CA 90095 (United States)

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer A{beta} oligomers are neurotoxins and likely the causing agents for Alzheimer's disease. Black-Right-Pointing-Pointer A{beta}42 fusion protein form globular oligomers. Black-Right-Pointing-Pointer A{beta}42 fusion protein oligomers contain SDS-resistant tetramers and hexamers. Black-Right-Pointing-Pointer Cysteine substitutions at residues 31, 32, 34, 39-41 disrupt A{beta}42 oligomerization. -- Abstract: Deposition of amyloid fibrils consisting of amyloid {beta} (A{beta}) protein as senile plaques in the brain is a pathological hallmark of Alzheimer's disease. However, a growing body of evidence shows that soluble A{beta} oligomers correlate better with dementia than fibrils, suggesting that A{beta} oligomers may be the primary toxic species. The structure and oligomerization mechanism of these A{beta} oligomers are crucial for developing effective therapeutics. Here we investigated the oligomerization of A{beta}42 in the context of a fusion protein containing GroES and ubiquitin fused to the N-terminus of A{beta} sequence. The presence of fusion protein partners, in combination with a denaturing buffer containing 8 M urea at pH 10, is unfavorable for A{beta}42 aggregation, thus allowing only the most stable structures to be observed. Transmission electron microscopy showed that A{beta}42 fusion protein formed globular oligomers, which bound weakly to thioflavin T and Congo red. SDS-PAGE shows that A{beta}42 fusion protein formed SDS-resistant hexamers and tetramers. In contrast, A{beta}40 fusion protein remained as monomers on SDS gel, suggesting that the oligomerization of A{beta}42 fusion protein is not due to the fusion protein partners. Cysteine scanning mutagenesis at 22 residue positions further revealed that single cysteine substitutions of the C-terminal hydrophobic residues (I31, I32, L34, V39, V40, and I41) led to disruption of hexamer and tetramer formation, suggesting that hydrophobic interactions

  16. Functional role of oligomerization for bacterial and plant SWEET sugar transporter family.

    Science.gov (United States)

    Xuan, Yuan Hu; Hu, Yi Bing; Chen, Li-Qing; Sosso, Davide; Ducat, Daniel C; Hou, Bi-Huei; Frommer, Wolf B

    2013-09-24

    Eukaryotic sugar transporters of the MFS and SWEET superfamilies consist of 12 and 7 α-helical transmembrane domains (TMs), respectively. Structural analyses indicate that MFS transporters evolved from a series of tandem duplications of an ancestral 3-TM unit. SWEETs are heptahelical proteins carrying a tandem repeat of 3-TM separated by a single TM. Here, we show that prokaryotes have ancestral SWEET homologs with only 3-TM and that the Bradyrhizobium japonicum SemiSWEET1, like Arabidopsis SWEET11, mediates sucrose transport. Eukaryotic SWEETs most likely evolved by internal duplication of the 3-TM, suggesting that SemiSWEETs form oligomers to create a functional pore. However, it remains elusive whether the 7-TM SWEETs are the functional unit or require oligomerization to form a pore sufficiently large to allow for sucrose passage. Split ubiquitin yeast two-hybrid and split GFP assays indicate that Arabidopsis SWEETs homo- and heterooligomerize. We examined mutant SWEET variants for negative dominance to test if oligomerization is necessary for function. Mutation of the conserved Y57 or G58 in SWEET1 led to loss of activity. Coexpression of the defective mutants with functional A. thaliana SWEET1 inhibited glucose transport, indicating that homooligomerization is necessary for function. Collectively, these data imply that the basic unit of SWEETs, similar to MFS sugar transporters, is a 3-TM unit and that a functional transporter contains at least four such domains. We hypothesize that the functional unit of the SWEET family of transporters possesses a structure resembling the 12-TM MFS structure, however, with a parallel orientation of the 3-TM unit.

  17. Spontaneous Oligomerization of Nucleotide Alternatives in Aqueous Solutions

    Science.gov (United States)

    Smith, Karen E.; House, Christopher H.; Dworkin, Jason P.; Callahan, Michael P.

    2017-03-01

    On early Earth, a primitive polymer that could spontaneously form from likely available precursors may have preceded both RNA and DNA as the first genetic material. Here, we report that heated aqueous solutions containing 5-hydroxymethyluracil (HMU) result in oligomers of uracil, heated solutions containing 5-hydroxymethylcytosine (HMC) result in oligomers of cytosine, and heated solutions containing both HMU and HMC result in mixed oligomers of uracil and cytosine. Oligomerization of hydroxymethylated pyrimidines, which may have been abundant on the primitive Earth, might have been important in the development of simple informational polymers.

  18. The sigma-1 receptors are present in monomeric and oligomeric forms in living cells in the presence and absence of ligands

    Science.gov (United States)

    Singh, Deo R.; Biener, Gabriel; Yang, Jay; Oliver, Julie A.; Ruoho, Arnold; Raicu, Valerică

    2015-01-01

    The sigma-1 receptor (S1R) is a 223-amino-acid membrane protein that resides in the endoplasmic reticulum and the plasma membrane of some mammalian cells. The S1R is regulated by various synthetic molecules including (+)-pentazocine, cocaine and haloperidol and endogenous molecules such as sphingosine, dimethyltryptamine and dehydroepiandrosterone. Ligand-regulated protein chaperone functions linked to oxidative stress and neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) and neuropathic pain have been attributed to the S1R. Several client proteins that interact with S1R have been identified including various types of ion channels and G-protein coupled receptors (GPCRs). When S1R constructs containing C-terminal monomeric GFP2 and YFP fusions were co-expressed in COS-7 cells and subjected to FRET spectrometry analysis, monomers, dimers and higher oligomeric forms of S1R were identified under non-liganded conditions. In the presence of the prototypic S1R agonist, (+)-pentazocine, however, monomers and dimers were the prevailing forms of S1R. The prototypic antagonist, haloperidol, on the other hand, favoured higher order S1R oligomers. These data, in sum, indicate that heterologously expressed S1Rs occur in vivo in COS-7 cells in multiple oligomeric forms and that S1R ligands alter these oligomeric structures. We suggest that the S1R oligomerization states may regulate its function(s). PMID:25510962

  19. Assembly of the intrinsic factor domains and oligomerization of the protein in the presence of cobalamin

    DEFF Research Database (Denmark)

    Fedosov, Sergey N; Fedosova, Natalya U; Berglund, Lars;

    2004-01-01

    oligomerized. A mixture of two fragments IF(30) + IF(20) and Cbl produced a firm complex, IF(30+20).Cbl, which could not associate to dimers. In contrast to IF(30+20).Cbl, the saturated full-length monomers IF(50).Cbl dimerized with K(d) approximately 1 microM. We suggest a two-domain organization of the full......-length protein, where two distant units, IF(30) and IF(20), can be assembled only by Cbl. They are connected by a protease-sensitive link, whose native structure is likely to be important for dimerization. However, linkage between two domains is not compulsory for Cbl binding. Advantages of the two...

  20. Synthesis and characterization of luminescent organic-inorganic hybrid nanocomposite from polyhedral oligomeric silsesquioxane

    Institute of Scientific and Technical Information of China (English)

    Yan

    2010-01-01

    A novel polyhedral oligomeric silsesquioxane (POSS)-based organic-inorganic hybrid nanocomposite (EF-POSS) was prepared by Pt-catalyzed hydrosilylation reaction of octahydridosilsesquioxane (T8H8, POSS) with a luminescent substituted acetylene (2-ethynyl-7-(4-(4-methylstyryl)styryl)-9,9-dioctyl-9H-fluorene (EF)) in high yield. The hybrid nanocomposite was soluble in common solvents such as CH2Cl2, CHCl3, THF and 1,4-dioxane. Its structure and property were characterized by FTIR, NMR, TGA, UV and PL, respectively. The results show that the hybrid nanocomposite with high thermal stability emits stable blue light as a result of photo excitation and possesses high photoluminescence quantum efficiency (φfL).

  1. Chalcane-stilbene conjugates and oligomeric flavonoids from Chinese Dragon's Blood produced from Dracaena cochinchinensis.

    Science.gov (United States)

    Hao, Qian; Saito, Yoshinori; Matsuo, Yosuke; Li, Hai-Zhou; Tanaka, Takashi

    2015-11-01

    A detailed chemical investigation of Chinese Dragon's Blood, which is a traditional medicine produced form the red resin of Dracaena cochinchinensis, yielded two chalcane-stilbene conjugates, named cochinchinenenes G and H, together with 25 known compounds. The structures of these compounds were determined by spectroscopic examination. HPLC analysis of the resin indicated that the major constituents were a complex mixture of oligomeric polyphenols, which were detected as a broad hump on the base line of a HPLC chromatogram. (13)C NMR analysis indicated that the oligomers were mainly composed of oxygenated chalcane units. This suggestion was supported by the results of a thiol degradation experiment with mercaptoethanol, which yielded a thioether of 4-[(4-hydroxyphenyl)propyl]-3-methoxyphenol. Furthermore, methylation followed by electrospray ionization mass spectroscopic analysis of the resulting fractions established the presence of at least one heptamer of chalcane units. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Auto-oligomerization and hydration of pyrrole revealed by x-ray absorption spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Advanced Light Source; Schwartz, Craig P.; Uejio, Janel S.; Duffin, Andrew M.; England, Alice H.; Prendergast, David; Saykally, Richard J

    2009-05-29

    Near edge x-ray absorption fine structure (NEXAFS) spectra have been measured at the carbon and nitrogen K-edges of the prototypical aromatic molecule, pyrrole, both in the gas phase and when solvated in water, and compared with spectra simulated using a combination of classical molecular dynamics and first principles density functional theory in the excited state core hole approximation. The excellent agreement enabled detailed assignments. Pyrrole is highly reactive, particularly in water, and reaction products formed by the auto-oligomerization of pyrrole are identified. The solvated spectra have been measured at two different temperatures, indicating that the final states remain largely unaffected by both hydration and temperature. This is somewhat unexpected, since the nitrogen in pyrrole can donate a hydrogen bond to water.

  3. Reaction Mechanisms of the Initial Oligomerization of Aluminophosphate.

    Science.gov (United States)

    Xiang, Yan; Xin, Liang; Deetz, Joshua D; Sun, Huai

    2016-05-12

    The mechanisms of aluminophosphate oligomerization were investigated using density functional theory with the SMD solvation model. Two aluminum species, Al(OH)4(-) and Al(H2O)6(3+), and four phosphorus species, H3PO4, H2PO4(-), HPO4(2-), and PO4(3-), were considered as the monomers for polycondensation reactions. It was found that the most favorable pathway to dimerization was a Lewis acid-base reaction: the aprotic oxygen of phosphoric acid (P═O) performs a nucleophilic attack on the central aluminum atom of Al(OH)4(-). Using this mechanism as a pattern, plausible dimerization mechanisms were investigated by varying the proticity and hydration of the phosphorus and aluminum monomers, respectively. The relative reaction rates of each mechanism were estimated under different pH conditions. The chain growth of aluminophosphates to trimers, tetramers, and pentamers and the cyclization of a linear tetramer were also investigated. For oligomerization reactions beyond dimer formation, it is found that cluster growth favors the addition of the phosphoric monomers rather than aluminum monomers.

  4. Modulation of Innate Immune Signalling by Lipid-Mediated MAVS Transmembrane Domain Oligomerization.

    Directory of Open Access Journals (Sweden)

    Luis Nobre

    Full Text Available RIG-I-like receptors detect viral RNA in infected cells and promote oligomerization of the outer mitochondrial membrane protein MAVS to induce innate immunity to viral infection through type I interferon production. Mitochondrial reactive oxygen species (mROS have been shown to enhance anti-viral MAVS signalling, but the mechanisms have remained obscure. Using a biochemical oligomerization-reporter fused to the transmembrane domain of MAVS, we found that mROS inducers promoted lipid-dependent MAVS transmembrane domain oligomerization in the plane of the outer mitochondrial membrane. These events were mirrored by Sendai virus infection, which similarly induced lipid peroxidation and promoted lipid-dependent MAVS transmembrane domain oligomerization. Our observations point to a role for mROS-induced changes in lipid bilayer properties in modulating antiviral innate signalling by favouring the oligomerization of MAVS transmembrane domain in the outer-mitochondrial membrane.

  5. Effect of leaving group on the oligomerization of 5'-AMP on montmorillonite. [Abstract only

    Science.gov (United States)

    Prabahar, K. Joseph; Ferris, James P.

    1994-01-01

    The oligomerization of imidazole derivative of 5'-AMP (ImpA) in the presence of montmorillonite clay yields oligomers containing up to 10 monomer units. In these reactions, the heterocyclic base, imidazole is the leaving group. In our present study, we synthesized a series of activated nucleotides of 5'AMP using other leaving groups such as pyrazole, 1,2,4-triazole, piperidine, morpholine, 4-aminopyridine, 4-methylaminopyridine, 4-dimethylaminopyridine, 2-aminobenzimidazole etc. to determine the effect of amine leaving group on the products of the oligomerization reaction. Earlier results from our laboratory showed that the presence AppA in the clay reaction of ImpA enhances the oligomerization reaction to yield higher oligomers. We also studied the effect of AppA in the clay mediated oligomerization reaction of the activated nucleotides. Oligomerization of 2-amino-benzimidazole derivative of 5'-AMP gave higher oligomers containing up to nine monomer units in the presence of AppA.

  6. 40 CFR 721.3100 - Oligomeric silicic acid ester compound with a hy-droxyl-al-kyla-mine.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Oligomeric silicic acid ester compound... Significant New Uses for Specific Chemical Substances § 721.3100 Oligomeric silicic acid ester compound with a... chemical substance identified generically as oligomeric silicic acid ester compound with...

  7. Core-6 fucose and the oligomerization of the 1918 pandemic influenza viral neuraminidase

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Zhengliang L., E-mail: Leon.wu@bio-techne.com [Bio-Techne Inc., 614 McKinley Place NE, Minneapolis, MN 55413 (United States); Zhou, Hui [Gregg Hall, UNH Glycomics Center, University of New Hampshire (United States); Ethen, Cheryl M. [Bio-Techne Inc., 614 McKinley Place NE, Minneapolis, MN 55413 (United States); Reinhold, Vernon N., E-mail: Vernon.Reinhold@unh.edu [Gregg Hall, UNH Glycomics Center, University of New Hampshire (United States)

    2016-04-29

    The 1918 H1N1 influenza virus was responsible for one of the most deadly pandemics in human history. Yet to date, the structure component responsible for its virulence is still a mystery. In order to search for such a component, the neuraminidase (NA) antigen of the virus was expressed, which led to the discovery of an active form (tetramer) and an inactive form (dimer and monomer) of the protein due to different glycosylation. In this report, the N-glycans from both forms were released and characterized by mass spectrometry. It was found that the glycans from the active form had 26% core-6 fucosylated, while the glycans from the inactive form had 82% core-6 fucosylated. Even more surprisingly, the stalk region of the active form was almost completely devoid of core-6-linked fucose. These findings were further supported by the results obtained from in vitro incorporation of azido fucose and {sup 3}H-labeled fucose using core-6 fucosyltransferase, FUT8. In addition, the incorporation of fucose did not change the enzymatic activity of the active form, implying that core-6 fucose is not directly involved in the enzymatic activity. It is postulated that core-6 fucose prohibits the oligomerization and subsequent activation of the enzyme. - Graphical abstract: Proposed mechanism for how core-fucose prohibits the tetramerization of the 1918 pandemic viral neuraminidase. Only the cross section of the stalk region with two N-linked glycans are depicted for clarity. (A) Carbohydrate–carbohydrate interaction on non-fucosylated monomer allows tetramerization. (B) Core-fucosylation disrupts the interaction and prevents the tetramerization. - Highlights: • Expressed 1918 pandemic influenza viral neuraminidase has inactive and active forms. • The inactive form contains high level of core-6 fucose, while the active form lacks such modification. • Core fucose could interfere the oligomerization of the neuraminidase and thus its activation. • This discovery may explain

  8. Antioxidant activity of oligomeric acylphloroglucinols from Myrtus communis L.

    Science.gov (United States)

    Rosa, Antonella; Deiana, Monica; Casu, Viviana; Corona, Giulia; Appendino, Giovanni; Bianchi, Federica; Ballero, Mauro; Dessì, M Assunta

    2003-09-01

    The use of myrtle (Myrtus communis L.) as a culinary spice and as a flavoring agent for alcoholic beverages is widespread in the Mediterranean area, and especially in Sardinia. Myrtle contains unique oligomeric non-prenylated acylphloroglucinols, whose antioxidant activity was investigated in various systems. Both semimyrtucommulone (1) and myrtucommulone A (2) showed powerful antioxidant properties, protecting linoleic acid against free radical attack in simple in vitro systems, inhibiting its autoxidation and its FeCl3- and EDTA-mediated oxidation. While both compounds lacked pro-oxidant activity, semimyrtucommulone was more powerful than myrtucommulone A, and was further evaluated in rat liver homogenates for activity against lipid peroxidation induced by ferric-nitrilotriacetate, and in cell cultures for cytotoxicity and the inhibition of TBH- or FeCl3-induced oxidation. The results of these studies established semimyrtucommulone as a novel dietary antioxidant lead.

  9. Does oligomerization in fused thiophene affect reactivity and aromaticity?

    Indian Academy of Sciences (India)

    Siddhartha Kr Purkayastha; Pradip Kr Bhattacharyya

    2016-02-01

    Reactivity and aromaticity of a few fused thiophene oligomers and their conformers are discussed in the light of density functional theory (DFT) and conceptual density functional theory. Reactivity parameters, such as hardness () and electrophilicity (), chemical potential () and energy of the HOMO (highest occupied molecular orbital) have been studied. Oligomerization raises the HOMO of the species, which in turn increases the reactivity of the oligomers. The absorption spectra of the species are analysed using TDDFT (time dependent density functional theory). The absorption peaks show red shift with increasing size of the oligomers. Aromaticity of the species is gauged by nucleus independent chemical shift (NICS). The out-of-plane component, (NICSzz) values advocate higher aromatic character at longer distance whereas, NICS supports the reverse.

  10. Cartilage oligomeric matrix protein specific antibodies are pathogenic

    DEFF Research Database (Denmark)

    Geng, Hui; Nandakumar, Kutty Selva; Pramhed, Anna;

    2012-01-01

    ABSTRACT: INTRODUCTION: Cartilage oligomeric matrix protein (COMP) is a major non-collagenous component of cartilage. Earlier, we developed a new mouse model for rheumatoid arthritis using COMP. This study was undertaken to investigate the epitope specificity and immunopathogenicity of COMP...... and the pathogenicity of mAbs was investigated by passive transfer experiments. RESULTS: B cell immunodominant epitopes were localized within 4 antigenic domains of the COMP but with preferential response to the epidermal growth factor (EGF)-like domain. Some of our anti-COMP mAbs showed interactions with the native...... form of COMP, which is present in cartilage and synovium. Passive transfer of COMP-specific mAbs enhanced arthritis when co-administrated with a sub-arthritogenic dose of a mAb specific to collagen type II. Interestingly, we found that a combination of 5 COMP mAbs was capable of inducing arthritis...

  11. Oligomeric rare-earth metal cluster complexes with endohedral transition metal atoms

    Energy Technology Data Exchange (ETDEWEB)

    Steinberg, Simon; Zimmermann, Sina; Brühmann, Matthias; Meyer, Eva; Rustige, Christian; Wolberg, Marike; Daub, Kathrin; Bell, Thomas; Meyer, Gerd, E-mail: gerd.meyer@uni-koeln.de

    2014-11-15

    Comproportionation reactions of rare-earth metal trihalides (RX{sub 3}) with the respective rare-earth metals (R) and transition metals (T) led to the formation of 22 oligomeric R cluster halides encapsulating T, in 19 cases for the first time. The structures of these compounds were determined by single-crystal X-ray diffraction and are composed of trimers ((T{sub 3}R{sub 11})X{sub 15}-type, P6{sub 3}/m), tetramers ((T{sub 4}R{sub 16})X{sub 28}(R{sub 4}) (P-43m), (T{sub 4}R{sub 16})X{sub 20} (P4{sub 2}/nnm), (T{sub 4}R{sub 16})X{sub 24}(RX{sub 3}){sub 4} (I4{sub 1}/a) and (T{sub 4}R{sub 16})X{sub 23} (C2/m) types of structure) and pentamers ((Ru{sub 5}La{sub 14}){sub 2}Br{sub 39}, Cc) of (TR{sub r}){sub n} (n=2–5) clusters. These oligomers are further enveloped by inner (X{sup i}) as well as outer (X{sup a}) halido ligands, which possess diverse functionalities and interconnect like oligomers through i–i, i–a and/or a–i bridges. The general features of the crystal structures for these new compounds are discussed and compared to literature entries as well as different structure types with oligomeric T centered R clusters. Dimers and tetramers originating from the aggregation of (TR{sub 6}) octahedra via common edges are more frequent than trimers and pentamers, in which the (TR{sub r}) clusters share common faces. - Graphical abstract: Rare earth-metal cluster complexes with endohedral transition metal atoms (TR{sub 6}) may connect via common edges or faces to form dimers, trimers, tetramers and pentamers of which the tetramers are the most prolific. Packing effects and electron counts play an important role. - Highlights: • Rare-earth metal cluster complexes encapsulate transition metal atoms. • Oligomers are built via connection of octahedral clusters via common edges or faces. • Dimers through pentamers with closed structures are known. • Tetramers including a tetrahedron of endohedral atoms are the most prolific.

  12. An enigmatic peptide ligation reaction: protease-catalyzed oligomerization of a native protein segment in neat aqueous solution.

    Science.gov (United States)

    Kumaran, S; Datta, D; Roy, R P

    2000-04-01

    We report an enigmatic peptide ligation reaction catalyzed by Glu-specific Staphylococcus aureus V8 protease that occurs in neat aqueous solution around neutral pH utilizing a totally unprotected peptide substrate containing free alpha-carboxyl and alpha-amino groups. V8 protease catalyzed a chain of ligation steps between pH 6 and 8 at 4 degrees C, producing a gamut of covalent oligomers (dimer through octamer or higher) of a native protein segment TAAAKFE (S39) derived from ribonuclease A (RNAse A). Size-exclusion chromatography suggested the absence of strong interaction between the reacting peptides. The circular dichroism spectra of monomer through pentamer showed length-dependent enhancement of secondary structure in the oligomers, suggesting that protease-catalyzed ligation of a monomer to an oligomer resulted in a product that was more structured than its precursor. The relative conformational stability of the oligomers was reflected in their ability to resist proteolysis, indicating that the oligomerization reaction was facilitated as a consequence of the "conformational trapping" of the product. The ligation reaction proceeded in two phases-slow formation and accumulation of the dimer followed by a fast phase of oligomerization, implying that the conformational trap encountered in the oligomerization reaction was a two-step process. The Gly substitution at any position of the TAAAKFE sequence was deleterious, suggesting that the first step of the conformational trap, namely the dimerization reaction, that proceeded very slowly even with the parent peptide, was quite sensitive to amino acid sequence. In contrast, the oligomerization reaction of an Ala analog, AAAAKFE, occurred in much the same way as S39, albeit with faster rate, suggesting that Ala substitution stabilized the overall conformational trapping process. The results suggest the viability of the product-directed "conformational trap" as a mechanism to achieve peptide ligation of totally

  13. In situ oligomerization of 2-(thiophen-3-yl)acetate intercalated into Zn{sub 2}Al layered double hydroxide

    Energy Technology Data Exchange (ETDEWEB)

    Tronto, Jairo, E-mail: jairotronto@ufv.br [Universidade Federal de Viçosa, Instituto de Ciências Exatas e Tecnológicas, Campus de Rio Parsanaíba, Rodovia BR 354 km 310, Cx. Postal 22, CEP, 38.810-000 Rio Paranaíba, MG (Brazil); Pinto, Frederico G.; Costa, Liovando M. da [Universidade Federal de Viçosa, Instituto de Ciências Exatas e Tecnológicas, Campus de Rio Parsanaíba, Rodovia BR 354 km 310, Cx. Postal 22, CEP, 38.810-000 Rio Paranaíba, MG (Brazil); Leroux, Fabrice; Dubois, Marc [Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, BP 10448, F-63000 Clermont-Ferrand (France); CNRS, UMR 6296, ICCF, BP 80026, F-6317 Clermont-Ferrand (France); Valim, João B. [Universidade de São Paulo, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Departamento de Química, Av. dos Bandeirantes 3900, CEP 14.040-901, Ribeirão Preto, SP (Brazil)

    2015-01-15

    A layered double hydroxide (LDH) with cation composition Zn{sub 2}Al was intercalated with 2-(thiophen-3-yl)acetate (3-TA) monomers. To achieve in situ polymerization and/or oligomerization of the intercalated monomers, soft thermal treatments were carried out, and subsequent hybrid LDH materials were analyzed by means of several characterization techniques using powder X-ray diffraction (PXRD), Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), {sup 13}C CP–MAS nuclear magnetic resonance (NMR), electron spin resonance (EPR), scanning electron microscopy (SEM), and transmission electron microscopy (TEM), inductively coupled plasma optical emission spectroscopy (ICP–OES), and elemental analysis. PXRD analysis suggested that the intercalated monomers formed a bilayer. Thermal treatment of the hybrid LDH assembly above 120 °C provokes partially the breakdown of the layered structure, generating the phase zincite. EPR results indicated that vicinal monomers (oligomerization) were bound to each other after hydrothermal or thermal treatment, leading to a polaron response characteristic of electron conductivity localized on a restricted number of thiophene-based monomer segments. Localized unpaired electrons exist in the material and interact with the {sup 27}Al nuclei of the LDH layers by superhyperfine coupling. These unpaired electrons also interact with the surface of ZnO (O{sup 2−} vacancies), formed during the thermal treatments. - Graphical abstract: We synthesized a layered double hydroxide (LDH) with cation composition Zn{sub 2}Al, intercalated with 2-(thiophen-3-yl)acetate (3-TA) monomers, by coprecipitation at constant pH. We thermally treated the material, to achieve in situ polymerization and/or oligomerization of the intercalated monomers. - Highlights: • A Zn{sub 2}Al–LDH was intercalated with 2-(thiophen-3-yl)acetate monomers. • To achieve in situ oligomerization of the monomers, thermal treatments were made.

  14. Effects of Glycine, Water, Ammonia, and Ammonium Bicarbonate on the Oligomerization of Methionine

    Science.gov (United States)

    Huang, Rui; Furukawa, Yoshihiro; Otake, Tsubasa; Kakegawa, Takeshi

    2017-06-01

    The abiotic oligomerization of amino acids may have created primordial, protein-like biological catalysts on the early Earth. Previous studies have proposed and evaluated the potential of diagenesis for the amino acid oligomerization, simulating the formation of peptides that include glycine, alanine, and valine, separately. However, whether such conditions can promote the formation of peptides composed of multiple amino acids remains unclear. Furthermore, the chemistry of pore water in sediments should affect the oligomerization and degradation of amino acids and oligomers, but these effects have not been studied extensively. In this study, we investigated the effects of water, ammonia, ammonium bicarbonate, pH, and glycine on the oligomerization and degradation of methionine under high pressure (150 MPa) and high temperature conditions (175 °C) for 96 h. Methionine is more difficult to oligomerize than glycine and methionine dimer was formed in the incubation of dry powder of methionine. Methionine oligomers as long as trimers, as well as methionylglycine and glycylmethionine, were formed under every condition with these additional compounds. Among the compounds tested, the oligomerization reaction rate was accelerated by the presence of water and by an increase in pH. Ammonia also increased the oligomerization rate but consumed methionine by side reactions and resulted in the rapid degradation of methionine and its peptides. Similarly, glycine accelerated the oligomerization rate of methionine and the degradation of methionine, producing water, ammonia, and bicarbonate through its decomposition. With Gly, heterogeneous dimers (methionylglycine and glycylmethionine) were formed in greater amounts than with other additional compounds although smaller amount of these heterogeneous dimers were formed with other additional compounds. These results suggest that accelerated reaction rates induced by water and co-existing reactive compounds promote the oligomerization

  15. Effects of Glycine, Water, Ammonia, and Ammonium Bicarbonate on the Oligomerization of Methionine

    Science.gov (United States)

    Huang, Rui; Furukawa, Yoshihiro; Otake, Tsubasa; Kakegawa, Takeshi

    2016-09-01

    The abiotic oligomerization of amino acids may have created primordial, protein-like biological catalysts on the early Earth. Previous studies have proposed and evaluated the potential of diagenesis for the amino acid oligomerization, simulating the formation of peptides that include glycine, alanine, and valine, separately. However, whether such conditions can promote the formation of peptides composed of multiple amino acids remains unclear. Furthermore, the chemistry of pore water in sediments should affect the oligomerization and degradation of amino acids and oligomers, but these effects have not been studied extensively. In this study, we investigated the effects of water, ammonia, ammonium bicarbonate, pH, and glycine on the oligomerization and degradation of methionine under high pressure (150 MPa) and high temperature conditions (175 °C) for 96 h. Methionine is more difficult to oligomerize than glycine and methionine dimer was formed in the incubation of dry powder of methionine. Methionine oligomers as long as trimers, as well as methionylglycine and glycylmethionine, were formed under every condition with these additional compounds. Among the compounds tested, the oligomerization reaction rate was accelerated by the presence of water and by an increase in pH. Ammonia also increased the oligomerization rate but consumed methionine by side reactions and resulted in the rapid degradation of methionine and its peptides. Similarly, glycine accelerated the oligomerization rate of methionine and the degradation of methionine, producing water, ammonia, and bicarbonate through its decomposition. With Gly, heterogeneous dimers (methionylglycine and glycylmethionine) were formed in greater amounts than with other additional compounds although smaller amount of these heterogeneous dimers were formed with other additional compounds. These results suggest that accelerated reaction rates induced by water and co-existing reactive compounds promote the oligomerization

  16. Molecular basis of the STIL coiled coil oligomerization explains its requirement for de-novo formation of centrosomes in mammalian cells.

    Science.gov (United States)

    David, Ahuvit; Amartely, Hadar; Rabinowicz, Noa; Shamir, Mai; Friedler, Assaf; Izraeli, Shai

    2016-04-14

    The STIL protein is essential for centriole replication and for the non-templated, de novo centriole biogenesis that is required for mammalian embryogenesis. Here we performed quantitative biophysical and structural analysis of the central short coiled coil domain (CCD) of STIL that is critical for its function. Using biophysical, biochemical and cell biology approaches, we identified the specific residues in the CCD that mediate the oligomerization, centrosomal localization and protein interactions of STIL. We characterized the structural properties of the coiled coil peptide using circular dichroism spectroscopy and size exclusion chromatography. We identified two regions in this domain, containing eight hydrophobic residues, which mediate the coiled coil oligomerization. Mutations in these residues destabilized the coiled coil thermodynamically but in most cases did not affect its secondary structure. Reconstituting mouse embryonic fibroblasts lacking endogenous Stil, we show that STIL oligomerization mediated by these residues is not only important for the centrosomal functions of STIL during the canonical duplication process but also for de-novo formation of centrosomes.

  17. Penicillin-binding protein 5 can form a homo-oligomeric complex in the inner membrane of Escherichia coli.

    Science.gov (United States)

    Skoog, Karl; Bruzell, Filippa Stenberg; Ducroux, Aurélie; Hellberg, Mårten; Johansson, Henrik; Lehtiö, Janne; Högbom, Martin; Daley, Daniel O

    2011-09-01

    Penicillin-binding protein 5 (PBP5) is a DD-carboxypeptidase, which cleaves the terminal D-alanine from the muramyl pentapeptide in the peptidoglycan layer of Escherichia coli and other bacteria. In doing so, it varies the substrates for transpeptidation and plays a key role in maintaining cell shape. In this study, we have analyzed the oligomeric state of PBP5 in detergent and in its native environment, the inner membrane. Both approaches indicate that PBP5 exists as a homo-oligomeric complex, most likely as a homo-dimer. As the crystal structure of the soluble domain of PBP5 (i.e., lacking the membrane anchor) shows a monomer, we used our experimental data to generate a model of the homo-dimer. This model extends our understanding of PBP5 function as it suggests how PBP5 can interact with the peptidoglycan layer. It suggests that the stem domains interact and the catalytic domains have freedom to move from the position observed in the crystal structure. This would allow the catalytic domain to have access to pentapeptides at different distances from the membrane. Copyright © 2011 The Protein Society.

  18. Application of MALDI-TOF mass spectrometry for study on fibrillar and oligomeric aggregates of alpha-synuclein

    Directory of Open Access Journals (Sweden)

    Severinovskaya O. V.

    2014-05-01

    Full Text Available Aim. To study the -synuclein (ASN aggregates of different structural origin, namely amyloid fibrils and spherical oligomers, in comparison with a native protein. Methods. MALDI TOF mass spectrometry and atomic for- ce microscopy (AFM. Results. The mass spectra of native and fibrillar ASN have similar character, i. e. they are characterized by the well pronounced peak of protein molecular ion, the low molecular weight associates, and rather low contain of fragmentation products. The spectrum of oligomeric aggregate is characterized by the high contain of fragmentation products, low intensity of protein molecular ion and the absence of peaks of associates. Conclusions. The difference between the spectra of fibrillar and oligomeric ASN could be explained, first, by the different content of the «residual» monomeric ASN and the protein degradation products in the studied samples, and, second, by the different structure-depended mechanisms of the protein degradation induced by the laser ionization. We suggested that the MALDI-TOF mass spectroscopy is a method useful for the investigation of ASN aggregation and characterization of its high order self-associates; besides, there is an interest in estimating the potency of the MALDI-TOF for the analysis of aggregation of various amyloidogenic proteins.

  19. Oncogenic Mutations Differentially Affect Bax Monomer, Dimer, and Oligomeric Pore Formation in the Membrane

    Science.gov (United States)

    Zhang, Mingzhen; Zheng, Jie; Nussinov, Ruth; Ma, Buyong

    2016-09-01

    Dysfunction of Bax, a pro-apoptotic regulator of cellular metabolism is implicated in neurodegenerative diseases and cancer. We have constructed the first atomistic models of the Bax oligomeric pore consisting with experimental residue-residue distances. The models are stable, capturing well double electron-electron resonance (DEER) spectroscopy measurements and provide structural details in line with the DEER data. Comparison with the latest experimental results revealed that our models agree well with both Bax and Bak pores, pointed to a converged structural arrangement for Bax and Bak pore formation. Using multi-scale molecular dynamics simulations, we probed mutational effects on Bax transformation from monomer → dimer → membrane pore formation at atomic resolution. We observe that two cancer-related mutations, G40E and S118I, allosterically destabilize the monomer and stabilize an off-pathway swapped dimer, preventing productive pore formation. This observation suggests a mechanism whereby the mutations may work mainly by over-stabilizing the monomer → dimer transformation toward an unproductive off-pathway swapped-dimer state. Our observations point to misfolded Bax states, shedding light on the molecular mechanism of Bax mutation-elicited cancer. Most importantly, the structure of the Bax pore facilitates future study of releases cytochrome C in atomic detail.

  20. Structure–property relationships in hybrid dental nanocomposite resins containing monofunctional and multifunctional polyhedral oligomeric silsesquioxanes

    Science.gov (United States)

    Wang, Weiguo; Sun, Xiang; Huang, Li; Gao, Yu; Ban, Jinghao; Shen, Lijuan; Chen, Jihua

    2014-01-01

    Organic-inorganic hybrid materials, such as polyhedral oligomeric silsesquioxanes (POSS), have the potential to improve the mechanical properties of the methacrylate-based composites and resins used in dentistry. In this article, nanocomposites of methacryl isobutyl POSS (MI-POSS [bears only one methacrylate functional group]) and methacryl POSS (MA-POSS [bears eight methacrylate functional groups]) were investigated to determine the effect of structures on the properties of dental resin. The structures of the POSS-containing networks were determined by scanning electron microscopy, transmission electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy. Monofunctional POSS showed a strong tendency toward aggregation and crystallization, while multifunctional POSS showed higher miscibility with the dimethacrylate monomer. The mechanical properties and wear resistance decreased with increasing amounts of MI-POSS, indicating that the MI-POSS agglomerates act as the mechanical weak point in the dental resins. The addition of small amounts of MA-POSS improved the mechanical and shrinkage properties. However, samples with a higher MA-POSS concentration showed lower flexural strength and flexural modulus, indicating that there is a limited range in which the reinforcement properties of MA-POSS can operate. This concentration dependence is attributed to phase separation at higher concentrations of POSS, which affects the structural integrity, and thus, the mechanical and shrinkage properties of the dental resin. Our results show that resin with 3% MA-POSS is a potential candidate for resin-based dental materials. PMID:24550674

  1. Staphylococcus aureus thiaminase II: oligomerization warrants proteolytic protection against serine proteases.

    Science.gov (United States)

    Begum, Afshan; Drebes, Julia; Kikhney, Alexey; Müller, Ingrid B; Perbandt, Markus; Svergun, Dmitri; Wrenger, Carsten; Betzel, Christian

    2013-12-01

    Staphylococcus aureus TenA (SaTenA) is a thiaminase type II enzyme that catalyzes the deamination of aminopyrimidine, as well as the cleavage of thiamine into 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) and 5-(2-hydroxyethyl)-4-methylthiazole (THZ), within thiamine (vitamin B1) metabolism. Further, by analogy with studies of Bacillus subtilis TenA, SaTenA may act as a regulator controlling the secretion of extracellular proteases such as the subtilisin type of enzymes in bacteria. Thiamine biosynthesis has been identified as a potential drug target of the multi-resistant pathogen S. aureus and therefore all enzymes involved in the S. aureus thiamine pathway are presently being investigated in detail. Here, the structure of SaTenA, determined by molecular replacement and refined at 2.7 Å resolution to an R factor of 21.6% with one homotetramer in the asymmetric unit in the orthorhombic space group P212121, is presented. The tetrameric state of wild-type (WT) SaTenA was postulated to be the functional biological unit and was confirmed by small-angle X-ray scattering (SAXS) experiments in solution. To obtain insights into structural and functional features of the oligomeric SaTenA, comparative kinetic investigations as well as experiments analyzing the structural stability of the WT SaTenA tetramer versus a monomeric SaTenA mutant were performed.

  2. Cartilage oligomeric matrix protein in patients with juvenile idiopathic arthritis: relation to growth and disease activity

    DEFF Research Database (Denmark)

    Bjørnhart, Birgitte; Juul, Anders; Nielsen, Susan

    2009-01-01

    OBJECTIVE: Cartilage oligomeric matrix protein (COMP) has been identified as a prognostic marker of progressive joint destruction in rheumatoid arthritis. In this population based study we evaluated associations between plasma concentrations of COMP, disease activity, and growth velocity in patie......OBJECTIVE: Cartilage oligomeric matrix protein (COMP) has been identified as a prognostic marker of progressive joint destruction in rheumatoid arthritis. In this population based study we evaluated associations between plasma concentrations of COMP, disease activity, and growth velocity...

  3. Self-assembly of iron coordination polymer of bowl-shaped N-ligand and dodecamolybdosilicate anion

    Science.gov (United States)

    Tang, Qun; Chen, Ya-Guang

    2014-06-01

    [FeII2(ttmb)2(H2O)6][SiMo12O40]·4H2O (1) (ttmb = 1,3,5-tris(triazol-1-ylmethyl) benzene) was synthesized under hydrothermal conditions and characterized by IR spectroscopy, TG analysis and X-ray diffraction method. In 1 the bowl-like tridentate ligand, ttmb, bridges Fe2+ ions forming a coordination polymeric layer, [FeII2(ttmb)2(H2O)6]n4n+, with a large dimensional grid of 7.00 × 7.11 Å, in the presence of Keggin-type polyoxoanion as a template. The coordination polymeric layers and the polyoxoanions are fused into an inorganic-organic hybrid by hydrogen bonds. The photoluminescent spectrum of 1 in solid state at room temperature shows that the coordination of ttmb to Fe2+ ion results in a quench of the ttmb intramolecular fluorescence and a weak O-Mo LMCT fluorescent emission was observed at 520 nm.

  4. The oligomeric integrity of toposome is essential for its morphogenetic function.

    Science.gov (United States)

    Scaturro, G; Zito, F; Matranga, V

    1998-01-01

    Sea urchin embryos are uniquely suitable for the study of morphogenetic cell interactions. Efforts to identify the molecules responsible for morphogenetic cell adhesion led to the isolation of a 22S glycoprotein complex from Paracentrotus lividus sea urchin embryo, that has been called toposome. The biological activity of toposome in mediating cellular adhesion has been fully documented. Its function in determining positional guidance during the development of the sea urchin embryo has been proposed. Here studies on the molecular structure of toposome are reported showing that, under non-reducing conditions, it is resolved in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in a major band with an apparent molecular weight of 260 kDa, a doublet of 180-160 kDa and a lower band of 80 kDa. Digestion with EndoH endoglycosidase reduced the molecular sizes of the bands of 10%, 20% and 40%, respectively. In order to establish if the oligomeric integrity of toposome was essential for its function, the biological activity of each subunit on cells dissociated from sea urchin blastula embryos was tested. The resulting swimming embryoids were lacking skeleton, while reaggregating cells supplemented with native toposome developed into pluteus-like structures with skeletal elements.

  5. Polyhedral Oligomeric Silsesquioxane Functionalized Carbon Dots for Cell Imaging.

    Science.gov (United States)

    Wang, Wen-Jing; Hai, Xin; Mao, Quan-Xing; Chen, Ming-Li; Wang, Jian-Hua

    2015-08-05

    In the present study, octa-aminopropyl polyhedral oligomeric silsesquioxane hydrochloride salt (OA-POSS) functionalized carbon dots (CDs/POSS) are prepared by a one-pot approach with glycerol as carbon source and solvent medium. OA-POSS serves as a passivation agent, and it is obtained via hydrolytic condensation of 3-aminopropyltriethoxysilane (APTES). During the functionalization process, the amino groups on OA-POSS combine with carboxylic groups on the bare CDs via formation of amide bond to construct organic-inorganic hybrid carbon dots. The obtained CDs/POSS are well dispersed in aqueous medium with a diameter of ca. 3.6 nm. It is demonstrated that CDs/POSS provide favorable photoluminescent property with a quantum yield of 24.0%. They also exhibit resistance to photobleaching and excellent photoluminescence stability in the presence of biological sample matrix (characterized by heavy metals and organic molecules), which facilitate cell imaging in biological systems. Both the photoluminescent emission wavelength and the fluorescence intensity depend closely on the excitation wavelength, and thus, it provides a potential for multicolor imaging as demonstrated with HeLa cells and MCF-7 cells.

  6. Accurate length control of supramolecular oligomerization: Vernier assemblies.

    Science.gov (United States)

    Hunter, Christopher A; Tomas, Salvador

    2006-07-12

    Linear oligomeric supramolecular assemblies of defined length have been generated using the Vernier principle. Two molecules, containing a different number (n and m) of mutually complementary binding sites, separated by the same distance, interact with each other to form an assembly of length (n x m). The assembly grows in the same way as simple supramolecular polymers, but at a molecular stop signal, when the binding sites come into register, the assembly terminates giving an oligomer of defined length. This strategy has been realized using tin and zinc porphyrin oligomers as the molecular building blocks. In the presence of isonicotinic acid, a zinc porphyrin trimer and a tin porphyrin dimer form a 3:4 triple stranded Vernier assembly six porphyrins long. The triple strand Vernier architecture introduced here adds an additional level of cooperativity, yielding a stability and selectivity that cannot be achieved via a simple Vernier approach. The assembly properties of the system were characterized using fluorescence titrations and size-exclusion chromatography (SEC). Assembly of the Vernier complex is efficient at micromolar concentrations in nonpolar solvents, and under more competitive conditions, a variety of fragmentation assemblies can be detected, allowing determination of the stability constants for this system and detailed speciation profiles to be constructed.

  7. Oligomerization of optically active N-(4-hydroxyphenylmandelamide in the presence of β-cyclodextrin and the minor role of chirality

    Directory of Open Access Journals (Sweden)

    Helmut Ritter

    2014-10-01

    Full Text Available The oxidative oligomerization of a chiral mandelamide derivative (N-(4-hydroxyphenylmandelamide, 1 was performed in the presence of horseradish peroxidase, laccase and N,N'-bis(salicylideneethylenediamine-iron(II to obtain chiral oligophenols 2. The low enantioselectivity of the enzymatic catalyzed asymmetric enantiomer-differentiating oligomerizations was investigated. In addition, the poor influence of cyclodextrin on the enantioselectivity of enzymatic catalyzed asymmetric enantiomer-differentiating oligomerizations was studied.

  8. Core-6 fucose and the oligomerization of the 1918 pandemic influenza viral neuraminidase.

    Science.gov (United States)

    Wu, Zhengliang L; Zhou, Hui; Ethen, Cheryl M; N Reinhold, Vernon

    2016-04-29

    The 1918 H1N1 influenza virus was responsible for one of the most deadly pandemics in human history. Yet to date, the structure component responsible for its virulence is still a mystery. In order to search for such a component, the neuraminidase (NA) antigen of the virus was expressed, which led to the discovery of an active form (tetramer) and an inactive form (dimer and monomer) of the protein due to different glycosylation. In this report, the N-glycans from both forms were released and characterized by mass spectrometry. It was found that the glycans from the active form had 26% core-6 fucosylated, while the glycans from the inactive form had 82% core-6 fucosylated. Even more surprisingly, the stalk region of the active form was almost completely devoid of core-6-linked fucose. These findings were further supported by the results obtained from in vitro incorporation of azido fucose and (3)H-labeled fucose using core-6 fucosyltransferase, FUT8. In addition, the incorporation of fucose did not change the enzymatic activity of the active form, implying that core-6 fucose is not directly involved in the enzymatic activity. It is postulated that core-6 fucose prohibits the oligomerization and subsequent activation of the enzyme.

  9. Synthesis and Self-Assembly Behaviors of Polyhedral Oligomeric Silsesquioxane Based Giant Molecular Shape Amphiphiles

    Science.gov (United States)

    Yue, Kan; Yu, Xinfei; Liu, Chang; Zhang, Wen-Bin; Cheng, Stephen

    2013-03-01

    Recently, our group has focus on the synthesis and characterization of novel giant molecular shape amphiphiles (GMSAs) based on functionalized molecular nanoparticles (MNPs), such as polyhedral oligomeric silsesquioxane (POSS), tethered with polymeric tails. A general synthetic method via the combination of sequential ?click? reactions has been developed and several model GMSAs with various tail lengths and distinct molecular topologies, which can be referred as the ?giant surfactants?, ?giant lipids?, ?giant gemini surfactants?, and ?giant bolaform surfactants? etc., have been demonstrated. Studies on their self-assembly behaviors in the bulk have revealed the formation of different ordered mesophase structures with feature sizes around 10 nanometers, which have been investigated in detail by small angle X-ray scattering (SAXS) technique and transmission electron microscopy (TEM). These findings have general implications on understanding the underlying principles of self-assembly behaviors of GMSAs, and might have potential applications in nano-patterning technology. This work is supported by NSF (DMR-0906898) and the Joint-Hope Foundation.

  10. The substitution of Proline 168 favors Bax oligomerization and stimulates its interaction with LUVs and mitochondria.

    Science.gov (United States)

    Simonyan, Lilit; Légiot, Alexandre; Lascu, Ioan; Durand, Grégory; Giraud, Marie-France; Gonzalez, Cécile; Manon, Stéphen

    2017-06-01

    Bax is a major player in the apoptotic process, being at the core of the mitochondria permeabilization events. In spite of the major recent advances in the knowledge of Bax organization within the membrane, the precise behavior of the C-terminal helix α9 remains elusive, since it was absent from the resolved structure of active Bax. The Proline 168 (P168) residue, located in the short loop between α8 and α9, has been the target of site-directed mutagenesis experiments, with conflicting results. We have produced and purified a recombinant mutant Bax-P168A, and we have compared its behavior with that of wild-type Bax in a series of tests on Large Unilamellar Vesicles (LUVs) and isolated mitochondria. We conclude that Bax-P168A had a greater ability to oligomerize and bind to membranes. Bax-P168A was not more efficient than wild-type Bax to permeabilize liposomes to small molecules but was more prone to release cytochrome c from mitochondria. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Characterization of tau oligomeric seeds in progressive supranuclear palsy.

    Science.gov (United States)

    Gerson, Julia E; Sengupta, Urmi; Lasagna-Reeves, Cristian A; Guerrero-Muñoz, Marcos J; Troncoso, Juan; Kayed, Rakez

    2014-06-14

    Progressive supranuclear palsy (PSP) is a neurodegenerative tauopathy which is primarily defined by the deposition of tau into globose-type neurofibrillary tangles (NFT). Tau in its native form has important functions for microtubule dynamics. Tau undergoes alternative splicing in exons 2, 3, and 10 which results in six different isoforms. Products of splicing on exon 10 are the most prone to mutations. Three repeat (3R) and four repeat (4R) tau, like other disease-associated amyloids, can form oligomers which may then go on to further aggregate and form fibrils. Recent studies from our laboratory and others have provided evidence that tau oligomers, not NFTs, are the most toxic species in neurodegenerative tauopathies and seed the pathological spread of tau. Analysis of PSP brain sections revealed globose-type NFTs, as well as both phosphorylated and unphosphorylated tau oligomers. Analysis of PSP brains via Western blot and ELISA revealed the presence of increased levels of tau oligomers compared to age-matched control brains. Oligomers were immunoprecipitated from PSP brain and were capable of seeding the oligomerization of both 3R and 4R tau isoforms. This is the first time tau oligomers have been characterized in PSP. These results indicate that tau oligomers are an important component of PSP pathology, along with NFTs. The ability of PSP brain-derived tau oligomers to seed 3R and 4R tau suggests that these oligomers represent the pathological species responsible for disease propagation and the presence of oligomers in a pure neurodegenerative tauopathy implies a common neuropathological process for tau seen in diseases with other amyloid proteins.

  12. Interfacial behavior of cytoplasmic and mitochondrial creatine kinase oligomeric states.

    Science.gov (United States)

    Vernoux, Nathalie; Granjon, Thierry; Marcillat, Olivier; Besson, Françoise; Vial, Christian

    2006-03-01

    Adsorption to the air/water interface of isoenzymes of creatine kinase was investigated using surface pressure-area isotherms and Brewster angle microscopy (BAM) observations. Octameric mitochondrial creatine kinase (mtCK) exhibits a significant affinity for the air/water interface. Whatever the mode of formation of the interfacial film, i.e., injection of the protein in the subphase or spreading onto the buffer surface, the final arrangement and conformation adopted by mtCK molecules lead to a similar result. In contrast, the dimeric isoenzymes mtCK and cytosolic MMCK do not induce any surface pressure variation. However, when the subphase contains 0.3M NaCl, both isoenzymes adsorb to the interface. When treated with 0.8 or 3M GdnHCl, muscle creatine kinase (MMCK) becomes surface active and occupies a greater surface than mtCK. This result contrasts with previous observations, often derived from monomeric proteins, that their surface activity is increased upon unfolding. It underlines the possible influence exerted by the protein oligomeric state on its interfacial activity. At a subphase pH of 8.8, which corresponds to the pI of octameric mtCK, the profiles of the isotherms obtained with dimeric and octameric states and the resistance to compression of the protein monolayers are significantly affected when compared to those recorded at pH 7.4. These data suggest that the octamer is more hydrophobic than the dimer and may contribute to explaining why octamers bind to the inner mitochondrial membrane while dimers do not. Copyright 2005 Wiley Periodicals, Inc.

  13. Small Angle X-Ray Scattering Studies of Mitochondrial Glutaminase C Reveal Extended Flexible Regions, and Link Oligomeric State with Enzyme Activity

    DEFF Research Database (Denmark)

    Møller, M.; Nielsen, Søren Skou; Ramachandran, Siddharth

    2013-01-01

    , using a high-throughput microfluidic-mixing chip for the SAXS data collection, and we confirm that the oligomeric state correlates with activity. The in-depth solution behavior analysis further reveals the structural behavior of flexible regions of the protein in the dimeric, tetrameric and octameric...... state and investigates the C-terminal influence on the enzyme solution behavior. Our data enable SAXS-based rigid body modeling of the full-length tetramer states, thereby presenting the first ever experimentally derived structural model of mitochondrial glutaminase C including the N- and C...

  14. Bax phosphorylation association with nucleus and oligomerization after neonatal hypoxia-ischemia.

    Science.gov (United States)

    Infante, Smitha Krishna; Oberhauser, Andres F; Perez-Polo, J Regino

    2013-09-01

    Neonatal hypoxia-ischemia (HI) is a common occurrence in preterm and low-birth-weight infants, and the incidence of low-birth-weight and preterm births is increasing. Characterization of brain injury after HI is of critical importance in developing new treatments that more accurately target the injury. After severe HI, neuronal cells undergo necrosis and secondary apoptosis of the surrounding cells as a result of neuroinflammation. We sought to characterize the biochemical pathways associated with cell death after HI. Bax, a cell death signaling protein, is activated after HI and translocates to the nucleus, endoplasmic reticulum, and mitochondria. The translocation patterns of Bax affect the resultant cell death phenotype (necrotic or apoptotic) observed. Although Bax is known to oligomerize once it is activated, less is known about the factors that control its translocation and oligomerization. We hypothesize that Bax kinase-specific phosphorylation determines its oligomerization and intracellular localization. Using well-established in vivo and in vitro models of neonatal HI, we characterized Bax oligomerization and multiorganelle translocation. We found that HI-dependent phosphorylation of Bax determines its oligomerization status and multiorganelle localization, and, ultimately, the cell death phenotype observed. Understanding the mechanisms of Bax translocation will aid in the rational design of therapeutic strategies that decrease the trauma resulting from HI-associated inflammation.

  15. Tris-acetate polyacrylamide gradient gel electrophoresis for the analysis of protein oligomerization.

    Science.gov (United States)

    Cubillos-Rojas, Monica; Schneider, Taiane; Sánchez-Tena, Susana; Bartrons, Ramon; Ventura, Francesc; Rosa, Jose Luis

    2016-02-01

    Here we report a new approach for studying protein oligomerization in cells using a single electrophoresis gel. We combined the use of a crosslinking reagent for sample preparation, such as glutaraldehyde, with the analysis of oligomers by Tris-acetate polyacrylamide gel electrophoresis. The use of a 3-15% Tris-acetate polyacrylamide gradient gel allows for the simultaneous analysis of proteins of masses ranging from 10 to 500 kDa. We showed the usefulness of this method for analyzing endogenous p53 oligomerization with high resolution and sensitivity in human cells. Oligomerization analysis was dependent on the crosslinker concentration used. We also showed that this method could be used to study the regulation of oligomerization. In all experiments, Tris-acetate polyacrylamide gel electrophoresis proved to be a robust, manageable, and cost- and time-efficient method that provided excellent results using a single gel. This approach can be easily extrapolated to the study of other oligomers. All of these features make this method a highly useful tool for the analysis of protein oligomerization.

  16. A fluorimetric readout reporting the kinetics of nucleotide-induced human ribonucleotide reductase oligomerization.

    Science.gov (United States)

    Fu, Yuan; Lin, Hong-Yu; Wisitpitthaya, Somsinee; Blessing, William A; Aye, Yimon

    2014-11-24

    Human ribonucleotide reductase (hRNR) is a target of nucleotide chemotherapeutics in clinical use. The nucleotide-induced oligomeric regulation of hRNR subunit α is increasingly being recognized as an innate and drug-relevant mechanism for enzyme activity modulation. In the presence of negative feedback inhibitor dATP and leukemia drug clofarabine nucleotides, hRNR-α assembles into catalytically inert hexameric complexes, whereas nucleotide effectors that govern substrate specificity typically trigger α-dimerization. Currently, both knowledge of and tools to interrogate the oligomeric assembly pathway of RNR in any species in real time are lacking. We therefore developed a fluorimetric assay that reliably reports on oligomeric state changes of α with high sensitivity. The oligomerization-directed fluorescence quenching of hRNR-α, covalently labeled with two fluorophores, allows for direct readout of hRNR dimeric and hexameric states. We applied the newly developed platform to reveal the timescales of α self-assembly, driven by the feedback regulator dATP. This information is currently unavailable, despite the pharmaceutical relevance of hRNR oligomeric regulation.

  17. Biocatalytically Oligomerized Epicatechin with Potent and Specific Anti-proliferative Activity for Human Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Ramaswamy Nagarajan

    2008-11-01

    Full Text Available Catechins, naturally occurring flavonoids derived from wine and green tea, are known to exhibit multiple health benefits. Epigallocatechin gallate (EGCG is one of the most widely investigated catechins, but its efficacy in cancer therapy is still inconsistent and limited. The poor stability of EGCG has contributed to the disparity in the reported anti-cancer activity and other beneficial properties. Here we report an innovative enzymatic strategy for the oligomerization of catechins (specifically epicatechin that yields stable, water-soluble oligomerized epicatechins with enhanced and highly specific anti-proliferative activity for human breast cancer cells. This one-pot oxidative oligomerization is carried out in ambient conditions using Horseradish Peroxidase (HRP as a catalyst yielding water-soluble oligo(epicatechins. The oligomerized epicatechins obtained exhibit excellent growth inhibitory effects against human breast cancer cells with greater specificity towards growth-inhibiting cancer cells as opposed to normal cells, achieving a high therapeutic differential. Our studies indicate that water-soluble oligomeric epicatechins surpass EGCG in stability, selectivity and efficacy at lower doses.

  18. Conservation of the oligomeric state of native VDAC1 in detergent micelles.

    Science.gov (United States)

    Clémençon, Benjamin; Fine, Michael; Hediger, Matthias A

    2016-08-01

    The voltage-dependent anion-selective channel (VDAC) is an intrinsic β-barrel membrane protein located within the mitochondrial outer membrane where it serves as a pore, connecting the mitochondria to the cytosol. The high-resolution structures of both the human and murine VDACs have been resolved by X-ray diffraction and nuclear magnetic resonance spectroscopy (NMR) in 2008. However, the structural data are not completely in line with the findings that were obtained after decades of research on biochemical and functional analysis of VDAC. This discrepancy may be related to the fact that structural biology studies of membrane proteins reveal specific static conformations that may not necessarily represent the physiological state. For example, overexpression of membrane proteins in bacterial inclusion bodies or simply the extraction from the native lipid environment using harsh purification methods (i.e. chaotropic agents) can disturb the physiological conformations and the supramolecular assemblies. To address these potential issues, we have developed a method, allowing rapid one step purification of endogenous VDAC expressed in the native mitochondrial membrane without overexpression of recombinant protein or usage of harsh chaotropic extraction procedures. Using the Saccharomyces cerevisiae isoform 1 of VDAC as a model, this method yields efficient purification, preserving VDAC in a more physiological, native state following extraction from mitochondria. Single particle analysis using transmission electron microscopy (TEM) demonstrated conservation of oligomeric assembly after purification. Maintenance of the native state was evaluated using functional assessment that involves an ATP-binding assay by micro-scale thermophoresis (MST). Using this approach, we were able to determine for the first time the apparent KD for ATP of 1.2 mM.

  19. Structure–property relationships in hybrid dental nanocomposite resins containing monofunctional and multifunctional polyhedral oligomeric silsesquioxanes

    Directory of Open Access Journals (Sweden)

    Wang WG

    2014-02-01

    Full Text Available Weiguo Wang,1,* Xiang Sun,1,* Li Huang,2,* Yu Gao,1 Jinghao Ban,1 Lijuan Shen,1 Jihua Chen1 1Department of Prosthodontics, 2Department of General Dentistry and Emergency, School of Stomatology, Fourth Military Medical University, Xi'an, People's Republic of China*These authors contributed equally to this workAbstract: Organic-inorganic hybrid materials, such as polyhedral oligomeric silsesquioxanes (POSS, have the potential to improve the mechanical properties of the methacrylate-based composites and resins used in dentistry. In this article, nanocomposites of methacryl isobutyl POSS (MI-POSS [bears only one methacrylate functional group] and methacryl POSS (MA-POSS [bears eight methacrylate functional groups] were investigated to determine the effect of structures on the properties of dental resin. The structures of the POSS-containing networks were determined by scanning electron microscopy, transmission electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy. Monofunctional POSS showed a strong tendency toward aggregation and crystallization, while multifunctional POSS showed higher miscibility with the dimethacrylate monomer. The mechanical properties and wear resistance decreased with increasing amounts of MI-POSS, indicating that the MI-POSS agglomerates act as the mechanical weak point in the dental resins. The addition of small amounts of MA-POSS improved the mechanical and shrinkage properties. However, samples with a higher MA-POSS concentration showed lower flexural strength and flexural modulus, indicating that there is a limited range in which the reinforcement properties of MA-POSS can operate. This concentration dependence is attributed to phase separation at higher concentrations of POSS, which affects the structural integrity, and thus, the mechanical and shrinkage properties of the dental resin. Our results show that resin with 3% MA-POSS is a potential candidate for resin-based dental materials

  20. Protective effect of the oligomeric acylphloroglucinols from Myrtus communis on cholesterol and human low density lipoprotein oxidation.

    Science.gov (United States)

    Rosa, Antonella; Melis, M Paola; Deiana, Monica; Atzeri, Angela; Appendino, Giovanni; Corona, Giulia; Incani, Alessandra; Loru, Debora; Dessì, M Assunta

    2008-09-01

    Myrtle (Myrtus communis L.), a culinary spice and flavouring agent for alcoholic beverages widespread in the Mediterranean area and especially in Sardinia, contains the structurally unique oligomeric non-prenylated acylphloroglucinols, semimyrtucommulone and myrtucommulone A, whose antioxidant activity was investigated during the oxidative modification of lipid molecules implicated in the onset of cardiovascular diseases. Both acylphloroglucinols showed powerful antioxidant properties during the thermal (140 degrees C), solvent-free degradation of cholesterol. Moreover, the pre-treatment with semimyrtucommulone and myrtucommulone A significantly preserved LDL from oxidative damage induced by Cu(2+) ions at 2h of oxidation, and showed remarkable protective effect on the reduction of polyunsaturated fatty acids and cholesterol, inhibiting the increase of their oxidative products (conjugated dienes fatty acids hydroperoxides, 7beta-hydroxycholesterol, and 7-ketocholesterol). Taking into account the widespread culinary use of myrtle leaves, the results of the present work qualify the natural compounds semimyrtucommulone and myrtucommulone A as interesting dietary antioxidants with potential antiatherogenicity.

  1. Isolation of a hexanuclear chromium cluster with a tetrahedral hydridic core and its catalytic behavior for ethylene oligomerization.

    Science.gov (United States)

    Alzamly, Ahmed; Gambarotta, Sandro; Korobkov, Ilia; Murugesu, Muralee; Le Roy, Jennifer J H; Budzelaar, Peter H M

    2014-06-16

    A chromium complex [2-(NHCH2PPh2)C5H4N]CrCl3·THF2 (1) of the ligand PyNHCH2PPh2 has been synthesized, characterized, and examined for its catalytic behavior toward ethylene oligomerization. When complex 1 was treated with (i-Bu)3Al, an unprecedented divalent polyhydride chromium cluster μ,κ(1),κ(2),κ(3)-N,N,P-{[2-(NCH2PPh2)C5H4N]Cr(μ-H)}4[(μ-Cl)Cr(μ-Cl)Al(i-Bu)2Cl]2 (2) was obtained. The complex contains a Cr4H4 core, which is expected to be diamagnetic, and which remains coordinated to two additional divalent high-spin Cr atoms via bridging interactions. Two aluminate residues remain bonded to the peripheral chromium atoms. The structure, magnetism, and electronic configuration are herein discussed.

  2. Crystallization and mechanical properties of biodegradable poly(p-dioxanone)/octamethyl-polyhedral oligomeric silsesquioxanes nanocomposites via simple solution casting method

    Indian Academy of Sciences (India)

    Zhecun Wang; Chengdong Xiong; Qing Li

    2015-10-01

    In this study, biodegradable poly(p-dioxanone) (PPDO)/octamethyl-polyhedral oligomeric silsesquioxanes (ome-POSS) nanocomposites were fabricated by the simple solution casting method with various ome-POSS loadings. Scanning electron microscopic observations indicate that ome-POSS is well dispersed in the PPDO matrix. Effect of ome-POSS on the isothermal melt crystallization and dynamic mechanical properties of PPDO in the nanocomposites were studied in detail. It shows that the overall crystallization rates are faster in the nanocomposites than in neat PPDO and increase with the increase in ome-POSS loadings; however, X-ray diffraction patterns, POM and the Avrami exponent suggest that the crystal structure and the crystallization mechanism do not change despite the presence of ome-POSS. The mechanical property of PPDO/ome-POSS nanocomposites was enhanced with respect to neat PPDO.

  3. Size exclusion chromatography (XIII): Identification and quantitation of silicon hydride containing oligomeric components of Sylgard prepolymers. [Polydimethylsiloxane

    Energy Technology Data Exchange (ETDEWEB)

    Kohn, E.; Chisum, M.E.; Moore, K.L.

    1987-12-01

    Methods for the identification and quantitation of oligomeric, silicon hydride (SiH), containing components of Sylgard prepolymers (polydimethylsiloxane: PDMS) have been developed. They employ a combination of size exclusion chromatography (SEC), with infrared (ir) and differential refractometer (DR) detection and gas chromatography/mass spectrometry (GC/MS) verification. The identified oligomers were present at about 20% of total prepolymer and contained from one to six SiH linkages per molecule. Such lower molecular weight oligomers may contribute to the vapor pressure of resins prepared from the prepolymers since they may contain a significant number of molecules which have only one or two SiH groups and are not capable of forming large polymeric structures. 5 refs., 8 figs., 2 tabs.

  4. Identification of a novel oligomerization disrupting mutation in CRYΑA associated with congenital cataract in a South Australian family.

    Science.gov (United States)

    Laurie, Kate J; Dave, Alpana; Straga, Tania; Souzeau, Emmanuelle; Chataway, Timothy; Sykes, Matthew J; Casey, Theresa; Teo, Theodosia; Pater, John; Craig, Jamie E; Sharma, Shiwani; Burdon, Kathryn P

    2013-03-01

    Congenital cataract is a heterogeneous disorder causing severe visual impairment in affected children. We screened four South Australian families with autosomal dominant congenital cataract for mutations in 10 crystallin genes known to cause congenital cataract. We identified a novel segregating heterozygous mutation, c.62G>A (p.R21Q), in the CRYΑA gene in one family. Western blotting of proteins freshly extracted from cataractous lens material of the proband demonstrated a marked reduction in the amount of the high-molecular-weight oligomers seen in the lens material of an unaffected individual. We conclude that the p.R21Q mutation, which is located in the highly conserved and structurally significant N-terminal region of the protein, is responsible for the cataract phenotype observed in the family as this mutation likely reduces the formation of the functional oligomeric alpha-crystallin.

  5. The Effect of Detergent, Temperature, and Lipid on the Oligomeric State of MscL Constructs: Insights from Mass Spectrometry.

    Science.gov (United States)

    Reading, Eamonn; Walton, Troy A; Liko, Idlir; Marty, Michael T; Laganowsky, Arthur; Rees, Douglas C; Robinson, Carol V

    2015-05-21

    The mechanosensitive channel of large conductance (MscL) acts as an emergency release valve for osmotic shock of bacteria preventing cell lysis. The large pore size, essential for function, requires the formation of oligomers with tetramers, pentamers, or hexamers observed depending on the species and experimental approach. We applied non-denaturing (native) mass spectrometry to five different homologs of MscL to determine the oligomeric state under more than 50 different experimental conditions elucidating lipid binding and subunit stoichiometry. We found equilibrium between pentameric and tetrameric species, which can be altered by detergent, disrupted by binding specific lipids, and perturbed by increasing temperature (37°C). We also established the presence of lipopolysaccharide bound to MscL and other membrane proteins expressed in Escherichia coli, revealing a potential source of heterogeneity. More generally, we highlight the use of mass spectrometry in probing membrane proteins under a variety of detergent-lipid environments relevant to structural biology.

  6. N-(1-Pyrenyl Maleimide Induces Bak Oligomerization and Mitochondrial Dysfunction in Jurkat Cells

    Directory of Open Access Journals (Sweden)

    Pei-Rong Huang

    2015-01-01

    Full Text Available N-(1-pyrenyl maleimide (NPM is a fluorescent reagent that is frequently used as a derivatization agent for the detection of thio-containing compounds. NPM has been shown to display a great differential cytotoxicity against hematopoietic cancer cells. In this study, the molecular mechanism by which NPM induces apoptosis was examined. Here, we show that treatment of Jurkat cells with NPM leads to Bak oligomerization, loss of mitochondrial membrane potential (Δψm, and release of cytochrome C from mitochondria to cytosol. Induction of Bak oligomerization appears to play a critical role in NPM-induced apoptosis, as downregulation of Bak by shRNA significantly prevented NPM-induced apoptosis. Inhibition of caspase 8 by Z-IETD-FMK and/or depletion of Bid did not affect NPM-induced oligomerization of Bak. Taken together, these results suggest that NPM-induced apoptosis is mediated through a pathway that is independent of caspase-8 activation.

  7. Molecular Design of Squalene/Squalane Countertypes via the Controlled Oligomerization of Isoprene and Evaluation of Vaccine Adjuvant Applications.

    Science.gov (United States)

    Adlington, Kevin; El Harfi, Jaouad; Li, Jianing; Carmichael, Kim; Guderian, Jeffrey A; Fox, Christopher B; Irvine, Derek J

    2016-01-11

    The potential to replace shark-derived squalene in vaccine adjuvant applications with synthetic squalene/poly(isoprene) oligomers, synthesized by the controlled oligomerization of isoprene is demonstrated. Following on from our previous work regarding the synthesis of poly(isoprene) oligomers, we demonstrate the ability to tune the molecular weight of the synthetic poly(isoprene) material beyond that of natural squalene, while retaining a final backbone structure that contained a minimum of 75% of 1,4 addition product and an acceptable polydispersity. The synthesis was successfully scaled from the 2 g to the 40 g scale both in the bulk (i.e., solvent free) and with the aid of additional solvent by utilizing catalytic chain transfer polymerization (CCTP) as the control method, such that the target molecular weight, acceptable dispersity levels, and the desired level of 1,4 addition in the backbone structure and an acceptable yield (∼60%) are achieved. Moreover, the stability and in vitro bioactivity of nanoemulsion adjuvant formulations manufactured with the synthetic poly(isoprene) material are evaluated in comparison to emulsions made with shark-derived squalene. Emulsions containing the synthetic poly(isoprene) achieved smaller particle size and equivalent or enhanced bioactivity (stimulation of cytokine production in human whole blood) compared to corresponding shark squalene emulsions. However, as opposed to the shark squalene-based emulsions, the poly(isoprene) emulsions demonstrated reduced long-term size stability and induced hemolysis at high concentrations. Finally, we demonstrate that the synthetic oligomeric poly(isoprene) material could successfully be hydrogenated such that >95% of the double bonds were successfully removed to give a representative poly(isoprene)-derived squalane mimic.

  8. Phospholamban Modulates the Functional Coupling between Nucleotide Domains in Ca-ATPase Oligomeric Complexes in Cardiac Sarcoplasmic Reticulum

    Energy Technology Data Exchange (ETDEWEB)

    Chen, L.; Yao, Qing; Soares, Thereza A.; Squier, Thomas C.; Bigelow, Diana J.

    2009-03-24

    Oligomeric interactions between Ca-ATPase polypeptide chains and their modulation by phospholamban (PLB) were measured in native cardiac sarcoplasmic reticulum (SR) microsomes. Progressive modification of Lys514 with fluorescein-5-isothiocyanate (FITC), which physically blocks access to the nucleotide binding site by ATP, demonstrates that Ca-ATPase active sites function independently of one another prior to the phosphorylation of PLB. However, upon PKA-dependent phosphorylation of PLB, a second-order dependence between enzyme activity and the fraction of active sites is observed, consistent with a dimeric functional complex. Complementary distance measurements were made using FITC or 5-iodoacetamido-fluorescein (IAF) bound to Cys674 within the N- or P-domains respectively, to detect structural coupling within oligomeric complexes. Accompanying the phosphorylation of PLB, neighboring Ca-ATPase polypeptide chains exhibit a 4 ± 2 Å decrease in the proximity between FITC sites within the N-domain and a 9 ± 3 Å increase in the proximity between IAF sites within P-domains. Thus, the phosphorylation of PLB induces spatial rearrangements between the N- and P-domain elements of proximal Ca-ATPase polypeptide chains which restore functional interactions between neighboring polypeptide chains and, in turn, result in increased rates of catalytic turnover. These results are interpreted in terms of a structural model, calculated through optimization of shape complementarity, desolvation, and electrostatic energies, which suggests a dimeric arrangement of Ca-ATPase polypeptide chains through the proximal association of N-domains. We suggest that the phosphorylation of PLB acts to release constraints involving interdomain subunit interactions that enhance catalytically important N-domain motions.

  9. A domain in the N-terminal part of Hsp26 is essential for chaperone function and oligomerization.

    Science.gov (United States)

    Haslbeck, Martin; Ignatiou, Athanasios; Saibil, Helen; Helmich, Sonja; Frenzl, Elke; Stromer, Thusnelda; Buchner, Johannes

    2004-10-15

    Small heat-shock proteins (Hsps) are ubiquitous molecular chaperones which prevent the unspecific aggregation of non-native proteins. For Hsp26, a cytosolic sHsp from of Saccharomyces cerevisiae, it has been shown that, at elevated temperatures, the 24 subunit complex dissociates into dimers. This dissociation is required for the efficient interaction with non-native proteins. Deletion analysis of the protein showed that the N-terminal half of Hsp26 (amino acid residues 1-95) is required for the assembly of the oligomer. Limited proteolysis in combination with mass spectrometry suggested that this region can be divided in two parts, an N-terminal segment including amino acid residues 1-30 and a second part ranging from residues 31-95. To analyze the structure and function of the N-terminal part of Hsp26 we created a deletion mutant lacking amino acid residues 1-30. We show that the oligomeric state and the structure, as determined by size exclusion chromatography and electron microscopy, corresponds to that of the Hsp26 wild-type protein. Furthermore, this truncated version of Hsp26 is active as a chaperone. However, in contrast to full length Hsp26, the truncated version dissociates at lower temperatures and complexes with non-native proteins are less stable than those found with wild-type Hsp26. Our results suggest that the N-terminal segment of Hsp26 is involved in both, oligomerization and chaperone function and that the second part of the N-terminal region (amino acid residues 31-95) is essential for both functions.

  10. p53 oligomerization status modulates cell fate decisions between growth, arrest and apoptosis.

    Science.gov (United States)

    Fischer, Nicholas W; Prodeus, Aaron; Malkin, David; Gariépy, Jean

    2016-12-01

    Mutations in the oligomerization domain of p53 are genetically linked to cancer susceptibility in Li-Fraumeni Syndrome. These mutations typically alter the oligomeric state of p53 and impair its transcriptional activity. Activation of p53 through tetramerization is required for its tumor suppressive function by inducing transcriptional programs that lead to cell fate decisions such as cell cycle arrest or apoptosis. How p53 chooses between these cell fate outcomes remains unclear. Here, we use 5 oligomeric variants of p53, including 2 novel p53 constructs, that yield either monomeric, dimeric or tetrameric forms of p53 and demonstrate that they induce distinct cellular activities and gene expression profiles that lead to different cell fate outcomes. We report that dimeric p53 variants are cytostatic and can arrest cell growth, but lack the ability to trigger apoptosis in p53-null cells. In contrast, p53 tetramers induce rapid apoptosis and cell growth arrest, while a monomeric variant is functionally inactive, supporting cell growth. In particular, the expression of pro-arrest CDKN1A and pro-apoptotic P53AIP1 genes are important cell fate determinants that are differentially regulated by the oligomeric state of p53. This study suggests that the most abundant oligomeric species of p53 present in resting cells, namely p53 dimers, neither promote cell growth or cell death and that shifting the oligomeric state equilibrium of p53 in cells toward monomers or tetramers is a key parameter in p53-based cell fate decisions.

  11. A stable transcription factor complex nucleated by oligomeric AML1–ETO controls leukaemogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Xiao-Jian; Wang, Zhanxin; Wang, Lan; Jiang, Yanwen; Kost, Nils; Soong, T. David; Chen, Wei-Yi; Tang, Zhanyun; Nakadai, Tomoyoshi; Elemento, Olivier; Fischle, Wolfgang; Melnick, Ari; Patel, Dinshaw J.; Nimer, Stephen D.; Roeder, Robert G.

    2013-06-30

    Transcription factors are frequently altered in leukaemia through chromosomal translocation, mutation or aberrant expression. AML1–ETO, a fusion protein generated by the t(8;21) translocation in acute myeloid leukaemia, is a transcription factor implicated in both gene repression and activation. AML1–ETO oligomerization, mediated by the NHR2 domain, is critical for leukaemogenesis, making it important to identify co-regulatory factors that ‘read’ the NHR2 oligomerization and contribute to leukaemogenesis. Here we show that, in human leukaemic cells, AML1–ETO resides in and functions through a stable AML1–ETO-containing transcription factor complex (AETFC) that contains several haematopoietic transcription (co)factors. These AETFC components stabilize the complex through multivalent interactions, provide multiple DNA-binding domains for diverse target genes, co-localize genome wide, cooperatively regulate gene expression, and contribute to leukaemogenesis. Within the AETFC complex, AML1–ETO oligomerization is required for a specific interaction between the oligomerized NHR2 domain and a novel NHR2-binding (N2B) motif in E proteins. Crystallographic analysis of the NHR2–N2B complex reveals a unique interaction pattern in which an N2B peptide makes direct contact with side chains of two NHR2 domains as a dimer, providing a novel model of how dimeric/oligomeric transcription factors create a new protein-binding interface through dimerization/oligomerization. Intriguingly, disruption of this interaction by point mutations abrogates AML1–ETO-induced haematopoietic stem/progenitor cell self-renewal and leukaemogenesis. These results reveal new mechanisms of action of AML1–ETO, and provide a potential therapeutic target in t(8;21)-positive acute myeloid leukaemia.

  12. Bioactivity and properties of a dental adhesive functionalized with polyhedral oligomeric silsesquioxanes (POSS) and bioactive glass.

    Science.gov (United States)

    Rizk, Marta; Hohlfeld, Lisa; Thanh, Loan Tao; Biehl, Ralf; Lühmann, Nicole; Mohn, Dirk; Wiegand, Annette

    2017-09-01

    This study aimed to analyze the effect of infiltrating a commercial adhesive with nanosized bioactive glass (BG-Bi) particles or methacryl-functionalized polyhedral oligomeric silsesquioxanes (POSS) on material properties and bioactivity. An acetone-based dental adhesive (Solobond Plus adhesive, VOCO GmbH, Cuxhaven, Germany) was infiltrated with nanosized bioactive glass particles (0.1 or 1wt%), or with monofunctional or multifunctional POSS particles (10 or 20wt%). Unfilled adhesive served as control. Dispersion and hydrodynamic radius of the nanoparticles were studied by dynamic light scattering. Set specimens were immersed for 28days in artificial saliva at 37°C, and surfaces were mapped for the formation of calcium phospate (Ca/P) precipitates (scanning electron microscopy/energy-dispersive X-ray spectroscopy). Viscosity (rheometry) and the structural characteristic of the networks were studied, such as degree of conversion (FTIR spectroscopy), sol fraction and water sorption. POSS particles showed a good dispersion of the particles for both types of particles being smaller than 3nm, while the bioactive glass particles had a strong tendency to agglomerate. All nanoparticles induced the formation of Ca/P precipitates. The viscosity of the adhesive was not or only slightly increased by POSS particle addition but strongly increased by the bioactive glass particles. The degree of conversion, water sorption and sol fraction showed a maintained or improved network structure and properties when filled with BG-Bi and multifunctional POSS, however, less polymerization was found when loading a monofunctional POSS. Multifunctional POSS may be incorporated into dental adhesives to provide a bioactive potential without changing material properties adversely. Copyright © 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

  13. A "coiled-coil" motif is important for oligomerization and DNA binding properties of human cytomegalovirus protein UL77.

    Directory of Open Access Journals (Sweden)

    Christina Sylvia Meissner

    Full Text Available Human cytomegalovirus (HCMV UL77 gene encodes the essential protein UL77, its function is characterized in the present study. Immunoprecipitation identified monomeric and oligomeric pUL77 in HCMV infected cells. Immunostaining of purified virions and subviral fractions showed that pUL77 is a structural protein associated with capsids. In silico analysis revealed the presence of a coiled-coil motif (CCM at the N-terminus of pUL77. Chemical cross-linking of either wild-type pUL77 or CCM deletion mutant (pUL77ΔCCM implicated that CCM is critical for oligomerization of pUL77. Furthermore, co-immunoprecipitations of infected and transfected cells demonstrated that pUL77 interacts with the capsid-associated DNA packaging motor components, pUL56 and pUL104, as well as the major capsid protein. The ability of pUL77 to bind dsDNA was shown by an in vitro assay. Binding to certain DNA was further confirmed by an assay using biotinylated 36-, 250-, 500-, 1000-meric dsDNA and 966-meric HCMV-specific dsDNA designed for this study. The binding efficiency (BE was determined by image processing program defining values above 1.0 as positive. While the BE of the pUL56 binding to the 36-mer bio-pac1 containing a packaging signal was 10.0 ± 0.63, the one for pUL77 was only 0.2±0.03. In contrast to this observation the BE of pUL77 binding to bio-500 bp or bio-1000 bp was 2.2 ± 0.41 and 4.9 ± 0.71, respectively. By using pUL77ΔCCM it was demonstrated that this protein could not bind to dsDNA. These data indicated that pUL77 (i could form homodimers, (ii CCM of pUL77 is crucial for oligomerization and (iii could bind to dsDNA in a sequence independent manner.

  14. Hydrogen ions directly regulating the oligomerization state of Photosystem I in intact Spirulina platensis cells

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    H+ concentration induced-monomerization or trimerization of photosystem I (PSI) in cyanobacteria has never been directly observed. In this work, taking characteristic spectra for the trimers and monomers as the indicators, it was experimentally demonstrated that H+ could induce the oligomeric changes of PSI reaction centers in the intact Spirulina ,platensis cells and also in the isolated thylakoid membrane complexes. Especially, the higher concentration of H+ would induce the monomerization while the lower the trimerization, suggesting the electrostatic interaction should be mainly responsible forchanges in the oligomeric state of PSI in Spirulina platensis.

  15. Phosphate and HEPES buffers potently affect the fibrillation and oligomerization mechanism of Alzheimer's A{beta} peptide

    Energy Technology Data Exchange (ETDEWEB)

    Garvey, Megan; Tepper, Katharina [Max-Planck-Forschungsstelle fuer Enzymologie der Proteinfaltung, Weinbergweg 22, D-06120 Halle (Saale) (Germany); Haupt, Caroline [Institute fuer Physik, Biophysik, Martin-Luther Universitaet Halle-Wittenberg, Betty-Heimann-Str. 7, D-06120 Halle (Saale) (Germany); Knuepfer, Uwe [Leibniz-Institute for Infection Biology and Natural Product Research, Beutenbergstr. 11a, D-07745 Jena (Germany); Klement, Karolin; Meinhardt, Jessica [Leibniz-Institute for Age Research (FLI), Beutenbergstr. 11, D-07745 Jena (Germany); Horn, Uwe [Leibniz-Institute for Infection Biology and Natural Product Research, Beutenbergstr. 11a, D-07745 Jena (Germany); Balbach, Jochen [Institute fuer Physik, Biophysik, Martin-Luther Universitaet Halle-Wittenberg, Betty-Heimann-Str. 7, D-06120 Halle (Saale) (Germany); Faendrich, Marcus, E-mail: fandrich@enzyme-halle.mpg.de [Max-Planck-Forschungsstelle fuer Enzymologie der Proteinfaltung, Weinbergweg 22, D-06120 Halle (Saale) (Germany); Bio zentrum, Martin-Luther Universitaet Halle-Wittenberg, Weinbergweg 22, D-06120 Halle (Saale) (Germany)

    2011-06-10

    Highlights: {yields} Sodium phosphate buffer accelerated A{beta}(1-40) nucleation relative to HEPES. {yields} A{beta}(1-40) fibrils formed in the two buffers show only minor structural differences. {yields} NMR revealed that A{beta}(1-40) histidine residues mediate buffer dependent changes. -- Abstract: The oligomerization of A{beta} peptide into amyloid fibrils is a hallmark of Alzheimer's disease. Due to its biological relevance, phosphate is the most commonly used buffer system for studying the formation of A{beta} and other amyloid fibrils. Investigation into the characteristics and formation of amyloid fibrils frequently relies upon material formed in vitro, predominantly in phosphate buffers. Herein, we examine the effects on the fibrillation and oligomerization mechanism of A{beta} peptide that occur due solely to the influence of phosphate buffer. We reveal that significant differences in amyloid fibrillation are observed due to fibrillation being initiated in phosphate or HEPES buffer (at physiological pH and temperature). Except for the differing buffer ions, all experimental parameters were kept constant. Fibril formation was assessed using fluorescently monitored kinetic studies, microscopy, X-ray fiber diffraction and infrared and nuclear magnetic resonance spectroscopies. Based on this set up, we herein reveal profound effects on the mechanism and speed of A{beta} fibrillation. The three histidine residues at positions 6, 13 and 14 of A{beta}(1-40) are instrumental in these mechanistic changes. We conclude that buffer plays a more significant role in fibril formation than has been generally acknowledged.

  16. Assessment of Escherichia coli selenophosphate synthetase oligomeric states by analytical ultracentrifugation and small angle X-ray scattering

    Energy Technology Data Exchange (ETDEWEB)

    Silva, I.R.; Faim, F.M.; Oliveira Neto, M.; Thiemann, O.H. [Universidade de Sao Paulo (USP-SC), Sao Carlos, SP (Brazil); Borges, J.C. [Universidade de Sao Paulo (IQSC/USP), Sao Carlos, SP (Brazil). Inst. de Quimica

    2012-07-01

    Full text: Selenium is an essential micronutrient for many organisms and is present in selenium-containing proteins as selenocysteine (Sec) and RNAs as selenouridine. Specific selenium incorporation into selenoproteins and RNAs requires the generation of a biologically active selenium donor compound, selenophosphate, which is produced from the activation of selenide with adenosine 5-triphosphate (ATP) in a reaction catalyzed by Selenophosphate Synthetase (SELD). Therefore, SELD is a key enzyme of the selenium pathway in the cell. The Escherichia coli SELD open reading frame was cloned into pET28a (Novagen) expression vector and the recombinant protein was over expressed in Escherichia coli BL21(DE3) strain. In order to purify the protein, we used metal-chelate affinity chromatography followed by a gel filtration step. Analytical Ultracentrifugation (AUC) and Small Angle X-ray Scattering (SAXS) were employed to study the oligomeric states of the soluble protein. The results of AUC revealed dimer-tetramer and tetramer-octamer equilibrium at low concentrations of protein, with dissociation constants of 70 2 and 560 40 M, respectively. Moreover, the SAXS results pointed the oligomeric state of the protein at higher concentrations as predominantly dimeric and the p(r) and the SAXS envelope revealed the SELD as elongated. We also performed initial crystallization trials with protein samples at 7 mg/ml in 96-well sitting-drop crystallization plates at room temperature using a crystallization robot. Needle crystals appeared after some days. X-ray diffraction for these crystals were tested in the MX2 beamline at the Brazilian Synchrotron Laboratory (LNLS Campinas). We are now working to improve these crystals in order to obtain suitable crystals for structure determination. (author)

  17. Structure and function of Neisseria gonorrhoeae MtrF illuminates a class of antimetabolite efflux pumps.

    Science.gov (United States)

    Su, Chih-Chia; Bolla, Jani Reddy; Kumar, Nitin; Radhakrishnan, Abhijith; Long, Feng; Delmar, Jared A; Chou, Tsung-Han; Rajashankar, Kanagalaghatta R; Shafer, William M; Yu, Edward W

    2015-04-07

    Neisseria gonorrhoeae is an obligate human pathogen and the causative agent of the sexually transmitted disease gonorrhea. The control of this disease has been compromised by the increasing proportion of infections due to antibiotic-resistant strains, which are growing at an alarming rate. N. gonorrhoeae MtrF is an integral membrane protein that belongs to the AbgT family of transporters for which no structural information is available. Here, we describe the crystal structure of MtrF, revealing a dimeric molecule with architecture distinct from all other families of transporters. MtrF is a bowl-shaped dimer with a solvent-filled basin extending from the cytoplasm to halfway across the membrane bilayer. Each subunit of the transporter contains nine transmembrane helices and two hairpins, posing a plausible pathway for substrate transport. A combination of the crystal structure and biochemical functional assays suggests that MtrF is an antibiotic efflux pump mediating bacterial resistance to sulfonamide antimetabolite drugs.

  18. Structure and Function of Neisseria gonorrhoeae MtrF Illuminates a Class of Antimetabolite Efflux Pumps

    Directory of Open Access Journals (Sweden)

    Chih-Chia Su

    2015-04-01

    Full Text Available Neisseria gonorrhoeae is an obligate human pathogen and the causative agent of the sexually transmitted disease gonorrhea. The control of this disease has been compromised by the increasing proportion of infections due to antibiotic-resistant strains, which are growing at an alarming rate. N. gonorrhoeae MtrF is an integral membrane protein that belongs to the AbgT family of transporters for which no structural information is available. Here, we describe the crystal structure of MtrF, revealing a dimeric molecule with architecture distinct from all other families of transporters. MtrF is a bowl-shaped dimer with a solvent-filled basin extending from the cytoplasm to halfway across the membrane bilayer. Each subunit of the transporter contains nine transmembrane helices and two hairpins, posing a plausible pathway for substrate transport. A combination of the crystal structure and biochemical functional assays suggests that MtrF is an antibiotic efflux pump mediating bacterial resistance to sulfonamide antimetabolite drugs.

  19. Synthesis of polyhedral oligomeric silsesquioxane (POSS) functionalized carbon nanotubes for improved dispersion in polyurethane films

    Science.gov (United States)

    Kou, Xiaonan

    Carbon nanotube (CNT) polymer nanocomposites are promising advanced materials. These materials exhibit the advantages of traditional polymeric materials, such as being light weight and easy to process, combined with the potential to exhibit enhanced mechanical, thermal and electrical properties compared to pure polymers. To achieve substantial improvement of composite properties at low CNT loading, uniform dispersion of CNTs in the polymer matrix and strong CNT-polymer interfacial interaction are needed. However, it is difficult to achieve adequate dispersion and interfacial interactions due to the inert nature of CNTs. In this project, polyhedral oligomeric silsequioxane (POSS) will be used as a dispersing agent for multi-walled carbon nanotubes (MWNTs) in polyurethane (PU) matrices. This dissertation consists of six chapters. Chapter I provides a detailed introduction of the fundamental knowledge of CNTs, PU, and POSS. At the end of this chapter, the motivation and rationale of this research are given. Chapter II establishes the overall goal and specific objectives of this research. Chapter III describes the synthesis and characterization of three POSS modified CNTs and one organosilane modified CNT. Grafting efficiency of the different grafted molecules are calculated and compared. Chapter IV discusses the dispersion behavior of four covalently modified CNTs in both solvents and PU matrices. Differences in dispersion behaviors of the modified CNTs are correlated to the solubility parameters of the grafting molecules and the surface structures of modified CNTs. Chapter V provides further discussion of the dispersion of POSS and silane modified CNTs by reviewing the assessment of the physical properties of PU composites containing the modified CNTs. Morphological, thermal, mechanical and electrical properties are used to estimate the interactions of the modified CNTs with the PU matrix. Chapter VI explores the function of the trisilanolphenyl POSS lithium salt

  20. Organic-inorganic hybrid compounds containing polyhedral oligomeric silsesquioxane for conservation of stone heritage.

    Science.gov (United States)

    Son, Seunghwan; Won, Jongok; Kim, Jeong-Jin; Jang, Yun Deuk; Kang, Yong Soo; Kim, Sa Dug

    2009-02-01

    Alkoxysilane solutions based on tetraethoxysilane (TEOS) have been widely used for the consolidation of decaying heritage stone surfaces. TEOS-based products polymerize within the porous structure of the decaying stone, significantly increasing the cohesion of the grains of stone components. However, they suffer from practical drawbacks, such as crack formation of the gel during the drying phase due to the developing capillary force and dense gel fractures left inside of the stone. In this study, a TEOS-based stone consolidant containing functional (3-glycidoxypropyl)trimethoxysilane (GPTMS) and polyhedral oligomeric silsesquioxane (POSS) has been prepared in order to reduce gel crack formation during the drying phase. The addition of nanometer-sized POSS and/or GPTMS having a flexible segment reduces the capillary force developed during solvent evaporation. The properties of the TEOS/GPTMS/POSS composite solutions were compared with those of commercial products (Wacker OH and Unil sandsteinfestiger OH 1:1). The gelation time was similar to that of commercial consolidants, and the TEOS/GPTMS/POSS solution was stable over a period of up to 6 months. The addition of POSS and GPTMS provided a crack-free gel, while the gel from the commercial consolidants exhibited cracks after drying. The surface hydrophobicity of the treated decayed granite increased with the addition of POSS and GPTMS, and it was higher than that of the commercial product, implying the possibility of POSS and GPTMS as barriers to the penetration of water. This result implies that the TEOS/GPTMS/POSS solution showed a high suitability for the consolidation of granite heritage.

  1. Zinc-induced oligomerization of zinc α2 glycoprotein reveals multiple fatty acid-binding sites.

    Science.gov (United States)

    Zahid, Henna; Miah, Layeque; Lau, Andy M; Brochard, Lea; Hati, Debolina; Bui, Tam T T; Drake, Alex F; Gor, Jayesh; Perkins, Stephen J; McDermott, Lindsay C

    2016-01-01

    Zinc α2 glycoprotein (ZAG) is an adipokine with a class I MHC protein fold and is associated with obesity and diabetes. Although its intrinsic ligand remains unknown, ZAG binds the dansylated C11 fatty acid 11-(dansylamino)undecanoic acid (DAUDA) in the groove between the α1 and α2 domains. The surface of ZAG has approximately 15 weak zinc-binding sites deemed responsible for precipitation from human plasma. In the present study the functional significance of these metal sites was investigated. Analytical ultracentrifugation (AUC) and CD showed that zinc, but not other divalent metals, causes ZAG to oligomerize in solution. Thus ZAG dimers and trimers were observed in the presence of 1 and 2 mM zinc. Molecular modelling of X-ray scattering curves and sedimentation coefficients indicated a progressive stacking of ZAG monomers, suggesting that the ZAG groove may be occluded in these. Using fluorescence-detected sedimentation velocity, these ZAG-zinc oligomers were again observed in the presence of the fluorescent boron dipyrromethene fatty acid C16-BODIPY (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid). Fluorescence spectroscopy confirmed that ZAG binds C16-BODIPY. ZAG binding to C16-BODIPY, but not to DAUDA, was reduced by increased zinc concentrations. We conclude that the lipid-binding groove in ZAG contains at least two distinct fatty acid-binding sites for DAUDA and C16-BODIPY, similar to the multiple lipid binding seen in the structurally related immune protein CD1c. In addition, because high concentrations of zinc occur in the pancreas, the perturbation of these multiple lipid-binding sites by zinc may be significant in Type 2 diabetes where dysregulation of ZAG and zinc homoeostasis occurs.

  2. De novo polymerase activity and oligomerization of hepatitis C virus RNA-dependent RNA-polymerases from genotypes 1 to 5.

    Directory of Open Access Journals (Sweden)

    Pilar Clemente-Casares

    Full Text Available Hepatitis C virus (HCV shows a great geographical diversity reflected in the high number of circulating genotypes and subtypes. The response to HCV treatment is genotype specific, with the predominant genotype 1 showing the lowest rate of sustained virological response. Virally encoded enzymes are candidate targets for intervention. In particular, promising antiviral molecules are being developed to target the viral NS3/4A protease and NS5B polymerase. Most of the studies with the NS5B polymerase have been done with genotypes 1b and 2a, whilst information about other genotypes is scarce. Here, we have characterized the de novo activity of NS5B from genotypes 1 to 5, with emphasis on conditions for optimum activity and kinetic constants. Polymerase cooperativity was determined by calculating the Hill coefficient and oligomerization through a new FRET-based method. The V(max/K(m ratios were statistically different between genotype 1 and the other genotypes (p<0.001, mainly due to differences in V(max values, but differences in the Hill coefficient and NS5B oligomerization were noted. Analysis of sequence changes among the studied polymerases and crystal structures show the αF helix as a structural component probably involved in NS5B-NS5B interactions. The viability of the interaction of αF and αT helixes was confirmed by docking studies and calculation of electrostatic surface potentials for genotype 1 and point mutants corresponding to mutations from different genotypes. Results presented in this study reveal the existence of genotypic differences in NS5B de novo activity and oligomerization. Furthermore, these results allow us to define two regions, one consisting of residues Glu128, Asp129, and Glu248, and the other consisting of residues of αT helix possibly involved in NS5B-NS5B interactions.

  3. Styrene oligomerization as a molecular probe reaction for Brønsted acidity at the nanoscale

    NARCIS (Netherlands)

    Aramburo Corrales, Luis; Wirick, Sue; Miedema, P.S.; Buurmans, I.L.C.; de Groot, F.M.F.; Weckhuysen, B.M.

    2012-01-01

    The Brønsted acid-catalyzed oligomerization of 4-fluorostyrene has been studied on a series ofH-ZSM-5 zeolite powders, steamed under different conditions, with a combination of UV-Vismicro-spectroscopy and Scanning Transmission X-ray Microscopy (STXM). UV-Vis microspectroscopyand STXM have been used

  4. Selective Antimicrobial Activities and Action Mechanism of Micelles Self-Assembled by Cationic Oligomeric Surfactants.

    Science.gov (United States)

    Zhou, Chengcheng; Wang, Fengyan; Chen, Hui; Li, Meng; Qiao, Fulin; Liu, Zhang; Hou, Yanbo; Wu, Chunxian; Fan, Yaxun; Liu, Libing; Wang, Shu; Wang, Yilin

    2016-02-17

    This work reports that cationic micelles formed by cationic trimeric, tetrameric, and hexameric surfactants bearing amide moieties in spacers can efficiently kill Gram-negative E. coli with a very low minimum inhibitory concentration (1.70-0.93 μM), and do not cause obvious toxicity to mammalian cells at the concentrations used. With the increase of the oligomerization degree, the antibacterial activity of the oligomeric surfactants increases, i.e., hexameric surfactant > tetrameric surfactant > trimeric surfactant. Isothermal titration microcalorimetry, scanning electron microscopy, and zeta potential results reveal that the cationic micelles interact with the cell membrane of E. coli through two processes. First, the integrity of outer membrane of E. coli is disrupted by the electrostatic interaction of the cationic ammonium groups of the surfactants with anionic groups of E. coli, resulting in loss of the barrier function of the outer membrane. The inner membrane then is disintegrated by the hydrophobic interaction of the surfactant hydrocarbon chains with the hydrophobic domains of the inner membrane, leading to the cytoplast leakage. The formation of micelles of these cationic oligomeric surfactants at very low concentration enables more efficient interaction with bacterial cell membrane, which endows the oligomeric surfactants with high antibacterial activity.

  5. Selective lowering of synapsins induced by oligomeric α-synuclein exacerbates memory deficits.

    Science.gov (United States)

    Larson, Megan E; Greimel, Susan J; Amar, Fatou; LaCroix, Michael; Boyle, Gabriel; Sherman, Mathew A; Schley, Hallie; Miel, Camille; Schneider, Julie A; Kayed, Rakez; Benfenati, Fabio; Lee, Michael K; Bennett, David A; Lesné, Sylvain E

    2017-06-06

    Mounting evidence indicates that soluble oligomeric forms of amyloid proteins linked to neurodegenerative disorders, such as amyloid-β (Aβ), tau, or α-synuclein (αSyn) might be the major deleterious species for neuronal function in these diseases. Here, we found an abnormal accumulation of oligomeric αSyn species in AD brains by custom ELISA, size-exclusion chromatography, and nondenaturing/denaturing immunoblotting techniques. Importantly, the abundance of αSyn oligomers in human brain tissue correlated with cognitive impairment and reductions in synapsin expression. By overexpressing WT human αSyn in an AD mouse model, we artificially enhanced αSyn oligomerization. These bigenic mice displayed exacerbated Aβ-induced cognitive deficits and a selective decrease in synapsins. Following isolation of various soluble αSyn assemblies from transgenic mice, we found that in vitro delivery of exogenous oligomeric αSyn but not monomeric αSyn was causing a lowering in synapsin-I/II protein abundance. For a particular αSyn oligomer, these changes were either dependent or independent on endogenous αSyn expression. Finally, at a molecular level, the expression of synapsin genes SYN1 and SYN2 was down-regulated in vivo and in vitro by αSyn oligomers, which decreased two transcription factors, cAMP response element binding and Nurr1, controlling synapsin gene promoter activity. Overall, our results demonstrate that endogenous αSyn oligomers can impair memory by selectively lowering synapsin expression.

  6. Antitumor-promoting activity of oligomeric proanthocyanidins in mouse epidermis in vivo

    Science.gov (United States)

    Mei Xiao Gao; Elisabeth M. Perchellet; Hala U. Gali; Limarie Rodriguez; Richard W. Hemingway; Jean-Pierre Perchellet

    1994-01-01

    The flavanoid catechin and heterogenous samples of oligomeric proanthocyanidins extracted from various sources were compared for their ability to inhibit the biochemical and biological effects of 12-o-tertra-decanoylphorbol-13-acetate (TPA) in mouse epidermis in vivo. Topical applications of catechin fail to alter the hydroperoxide response to TPA but inhibit the...

  7. Monitoring receptor oligomerization by line-scan fluorescence cross-correlation spectroscopy

    NARCIS (Netherlands)

    Hink, M.A.; Postma, M.; Conn, P.M.

    2013-01-01

    Membrane-localized receptor proteins are involved in many signaling cascades, and diffusion and oligomerization are key processes controlling their activity. In order to study these processes in living cells, fluorescence fluctuation spectroscopy techniques have been developed that allow the quantif

  8. Concentration Dependent Ion-Protein Interaction Patterns Underlying Protein Oligomerization Behaviours

    Science.gov (United States)

    Batoulis, Helena; Schmidt, Thomas H.; Weber, Pascal; Schloetel, Jan-Gero; Kandt, Christian; Lang, Thorsten

    2016-04-01

    Salts and proteins comprise two of the basic molecular components of biological materials. Kosmotropic/chaotropic co-solvation and matching ion water affinities explain basic ionic effects on protein aggregation observed in simple solutions. However, it is unclear how these theories apply to proteins in complex biological environments and what the underlying ionic binding patterns are. Using the positive ion Ca2+ and the negatively charged membrane protein SNAP25, we studied ion effects on protein oligomerization in solution, in native membranes and in molecular dynamics (MD) simulations. We find that concentration-dependent ion-induced protein oligomerization is a fundamental chemico-physical principle applying not only to soluble but also to membrane-anchored proteins in their native environment. Oligomerization is driven by the interaction of Ca2+ ions with the carboxylate groups of aspartate and glutamate. From low up to middle concentrations, salt bridges between Ca2+ ions and two or more protein residues lead to increasingly larger oligomers, while at high concentrations oligomers disperse due to overcharging effects. The insights provide a conceptual framework at the interface of physics, chemistry and biology to explain binding of ions to charged protein surfaces on an atomistic scale, as occurring during protein solubilisation, aggregation and oligomerization both in simple solutions and membrane systems.

  9. Styrene oligomerization as a molecular probe reaction for Brønsted acidity at the nanoscale

    NARCIS (Netherlands)

    Aramburo Corrales, Luis; Wirick, Sue; Miedema, P.S.; Buurmans, I.L.C.; de Groot, F.M.F.; Weckhuysen, B.M.

    2012-01-01

    The Brønsted acid-catalyzed oligomerization of 4-fluorostyrene has been studied on a series ofH-ZSM-5 zeolite powders, steamed under different conditions, with a combination of UV-Vismicro-spectroscopy and Scanning Transmission X-ray Microscopy (STXM). UV-Vis microspectroscopyand STXM have been used

  10. Oligomerization of a Glucagon-like Peptide 1 Analog: Bridging Experiment and Simulations

    DEFF Research Database (Denmark)

    Frederiksen, Tine Maja; Sønderby, Pernille; Ryberg, Line A.

    2015-01-01

    The glucagon-like peptide 1 (GLP-1) analog, liraglutide, is a GLP-1 agonist and is used in the treatment of type-2 diabetes mellitus and obesity. From a pharmaceutical perspective, it is important to know the oligomerization state of liraglutide with respect to stability. Compared to GLP-1...

  11. Higher-order assemblies of oligomeric cargo receptor complexes form the membrane scaffold of the Cvt vesicle.

    Science.gov (United States)

    Bertipaglia, Chiara; Schneider, Sarah; Jakobi, Arjen J; Tarafder, Abul K; Bykov, Yury S; Picco, Andrea; Kukulski, Wanda; Kosinski, Jan; Hagen, Wim Jh; Ravichandran, Arvind C; Wilmanns, Matthias; Kaksonen, Marko; Briggs, John Ag; Sachse, Carsten

    2016-07-01

    Selective autophagy is the mechanism by which large cargos are specifically sequestered for degradation. The structural details of cargo and receptor assembly giving rise to autophagic vesicles remain to be elucidated. We utilize the yeast cytoplasm-to-vacuole targeting (Cvt) pathway, a prototype of selective autophagy, together with a multi-scale analysis approach to study the molecular structure of Cvt vesicles. We report the oligomeric nature of the major Cvt cargo Ape1 with a combined 2.8 Å X-ray and negative stain EM structure, as well as the secondary cargo Ams1 with a 6.3 Å cryo-EM structure. We show that the major dodecameric cargo prApe1 exhibits a tendency to form higher-order chain structures that are broken upon interaction with the receptor Atg19 in vitro The stoichiometry of these cargo-receptor complexes is key to maintaining the size of the Cvt aggregate in vivo Using correlative light and electron microscopy, we further visualize key stages of Cvt vesicle biogenesis. Our findings suggest that Atg19 interaction limits Ape1 aggregate size while serving as a vehicle for vacuolar delivery of tetrameric Ams1.

  12. Confocal Spectroscopy to Study Dimerization, Oligomerization and Aggregation of Proteins: A Practical Guide.

    Science.gov (United States)

    Gambin, Yann; Polinkovsky, Mark; Francois, Bill; Giles, Nichole; Bhumkar, Akshay; Sierecki, Emma

    2016-04-30

    Protein self-association is a key feature that can modulate the physiological role of proteins or lead to deleterious effects when uncontrolled. Protein oligomerization is a simple way to modify the activity of a protein, as the modulation of binding interfaces allows for self-activation or inhibition, or variation in the selectivity of binding partners. As such, dimerization and higher order oligomerization is a common feature in signaling proteins, for example, and more than 70% of enzymes have the potential to self-associate. On the other hand, protein aggregation can overcome the regulatory mechanisms of the cell and can have disastrous physiological effects. This is the case in a number of neurodegenerative diseases, where proteins, due to mutation or dysregulation later in life, start polymerizing and often fibrillate, leading to the creation of protein inclusion bodies in cells. Dimerization, well-defined oligomerization and random aggregation are often difficult to differentiate and characterize experimentally. Single molecule "counting" methods are particularly well suited to the study of self-oligomerization as they allow observation and quantification of behaviors in heterogeneous conditions. However, the extreme dilution of samples often causes weak complexes to dissociate, and rare events can be overlooked. Here, we discuss a straightforward alternative where the principles of single molecule detection are used at higher protein concentrations to quantify oligomers and aggregates in a background of monomers. We propose a practical guide for the use of confocal spectroscopy to quantify protein oligomerization status and also discuss about its use in monitoring changes in protein aggregation in drug screening assays.

  13. SLC30A3 (ZnT3 oligomerization by dityrosine bonds regulates its subcellular localization and metal transport capacity.

    Directory of Open Access Journals (Sweden)

    Gloria Salazar

    Full Text Available Non-covalent and covalent homo-oligomerization of membrane proteins regulates their subcellular localization and function. Here, we described a novel oligomerization mechanism affecting solute carrier family 30 member 3/zinc transporter 3 (SLC30A3/ZnT3. Oligomerization was mediated by intermolecular covalent dityrosine bonds. Using mutagenized ZnT3 expressed in PC12 cells, we identified two critical tyrosine residues necessary for dityrosine-mediated ZnT3 oligomerization. ZnT3 carrying the Y372F mutation prevented ZnT3 oligomerization, decreased ZnT3 targeting to synaptic-like microvesicles (SLMVs, and decreased resistance to zinc toxicity. Strikingly, ZnT3 harboring the Y357F mutation behaved as a "gain-of-function" mutant as it displayed increased ZnT3 oligomerization, targeting to SLMVs, and increased resistance to zinc toxicity. Single and double tyrosine ZnT3 mutants indicate that the predominant dimeric species is formed between tyrosine 357 and 372. ZnT3 tyrosine dimerization was detected under normal conditions and it was enhanced by oxidative stress. Covalent species were also detected in other SLC30A zinc transporters localized in different subcellular compartments. These results indicate that covalent tyrosine dimerization of a SLC30A family member modulates its subcellular localization and zinc transport capacity. We propose that dityrosine-dependent membrane protein oligomerization may regulate the function of diverse membrane protein in normal and disease states.

  14. Single Site Mutations in the Hetero-oligomeric Mrp Antiporter from Alkaliphilic Bacillus pseudofirmus OF4 That Affect Na+/H+ Antiport Activity, Sodium Exclusion, Individual Mrp Protein Levels, or Mrp Complex Formation*

    OpenAIRE

    Morino, Masato; Natsui, Shinsuke; Ono, Tomohiro; Swartz, Talia H.; Krulwich, Terry A.; Ito, Masahiro

    2010-01-01

    Mrp systems are widely distributed and structurally complex cation/proton antiporters. Antiport activity requires hetero-oligomeric complexes of all six or seven hydrophobic Mrp proteins (MrpA–MrpG). Here, a panel of site-directed mutants in conserved or proposed motif residues was made in the Mrp Na+(Li+)/H+ antiporter from an alkaliphilic Bacillus. The mutant operons were expressed in antiporter-deficient Escherichia coli KNabc and assessed for antiport properties, support of sodium resista...

  15. An avidin-like domain that does not bind biotin is adopted for oligomerization by the extracellular mosaic protein fibropellin.

    Science.gov (United States)

    Yanai, Itai; Yu, Yong; Zhu, Xiahui; Cantor, Charles R; Weng, Zhiping

    2005-02-01

    The protein avidin found in egg white seems optimized for binding the small vitamin biotin as a stable homotetramer. Indeed, along with its streptavidin ortholog in the bacterium Streptomyces avidinii, this protein shows the strongest known noncovalent bond of a protein with a small ligand. A third known member of the avidin family, as similar to avidin as is streptavidin, is found at the C-terminal ends of the multidomain fibropellin proteins found in sea urchin. The fibropellins form a layer known as the apical lamina that surrounds the sea urchin embryo throughout development. Based upon the structure of avidin, we deduced a structural model for the avidin-like domain of the fibropellins and found that computational modeling predicts a lack of biotin binding and the preservation of tetramerization. To test this prediction we expressed and purified the fibropellin avidin-like domain and found it indeed to be a homotetramer incapable of binding biotin. Several lines of evidence suggest that the avidin-like domain causes the entire fibropellin protein to tetramerize. We suggest that the presence of the avidin-like domain serves a structural (tetrameric form) rather than functional (biotin-binding) role and may therefore be a molecular instance of exaptation-the modification of an existing function toward a new function. Finally, based upon the oligomerization of the avidin-like domain, we propose a model for the overall structure of the apical lamina.

  16. BAX insertion, oligomerization, and outer membrane permeabilization in brain mitochondria: role of permeability transition and SH-redox regulation

    Science.gov (United States)

    Brustovetsky, Tatiana; Li, Tsyregma; Yang, Youyun; Zhang, Jiang-Ting; Antonsson, Bruno; Brustovetsky, Nickolay

    2010-01-01

    BAX cooperates with truncated BID (tBID) and Ca2+ in permeabilizing the outer mitochondrial membrane (OMM) and releasing mitochondrial apoptogenic proteins. The mechanisms of this cooperation are still unclear. Here we show that in isolated brain mitochondria, recombinant BAX readily self-integrates/oligomerizes in the OMM but produces only a minuscule release of cytochrome c, indicating that BAX insertion/oligomerization in the OMM does not always lead to massive OMM permeabilization. Ca2+ in a mitochondrial permeability transition (mPT)-dependent and recombinant tBID in an mPT-independent manner promoted BAX insertion/oligomerization in the OMM and augmented cytochrome c release. Neither tBID nor Ca2+ induced BAX oligomerization in the solution without mitochondria, suggesting that BAX oligomerization required interaction with the organelles and followed rather than preceded BAX insertion in the OMM. Recombinant Bcl-xL failed to prevent BAX insertion/oligomerization in the OMM but strongly attenuated cytochrome c release. On the other hand, a reducing agent, dithiothreitol (DTT), inhibited BAX insertion/oligomerization augmented by tBID or Ca2+ and suppressed the BAX-mediated release of cytochrome c and Smac/DIABLO but failed to inhibit Ca2+-induced swelling. Altogether, these data suggest that in brain mitochondria, BAX insertion/oligomerization can be dissociated from OMM permeabilization and that tBID and Ca2+ stimulate BAX insertion/oligomerization and BAX-mediated OMM permeabilization by different mechanisms involving mPT induction and modulation of the SH-redox state. PMID:20655869

  17. Polymer masks for structured surface and plasma etching

    Energy Technology Data Exchange (ETDEWEB)

    Vital, Alexane [Centre de Recherche sur la Matière Divisée (CRMD), 1b rue de la Férollerie, F45071 Orléans Cedex (France); Groupe de Recherches sur l’Énergétique des Milieux Ionisés (GREMI), Polytech’Orléans, 14 rue d’Issoudun, B.P. 6744, F45067 Orléans Cedex 2 (France); Vayer, Marylène, E-mail: marylene.vayer@univ-orleans.fr [Centre de Recherche sur la Matière Divisée (CRMD), 1b rue de la Férollerie, F45071 Orléans Cedex (France); Sinturel, Christophe [Centre de Recherche sur la Matière Divisée (CRMD), 1b rue de la Férollerie, F45071 Orléans Cedex (France); Tillocher, Thomas; Lefaucheux, Philippe; Dussart, Rémi [Groupe de Recherches sur l’Énergétique des Milieux Ionisés (GREMI), Polytech’Orléans, 14 rue d’Issoudun, B.P. 6744, F45067 Orléans Cedex 2 (France)

    2015-03-30

    Graphical abstract: - Highlights: • Sub-micrometric silicon structures were prepared by cryogenic plasma etching. • Polymer templates based on phase-separated films of PS/PLA were used. • Silica structured masks were prepared by filling the polymer templates. • Etching of underlying silicon through silica templates gave original structures. - Abstract: Silica and silicon structures have been prepared at the sub-micrometer length-scale, using laterally phase-separated thin films of poly(styrene) (PS) and poly(lactic acid) (PLA) homopolymer blends. The selective removal of one polymer and the filling of the released space by silica precursor solution led, after calcination, to silica structures on silicon such as arrays of bowl-shape features or pillars, layers with through or non-through cylindrical holes, which has not been observed for some of them. The control of the morphology of the initial polymer film was a key point to achieve such type of structures. Particularly relevant was the use of solvent vapor annealing (vs thermal annealing) of the initial spin-coated films that favored and stabilized laterally phase-separated morphologies. Characteristic dimension of the domains were shown to be coupled with the thickness of the film, thinner films giving smaller domain sizes. Despite a relatively high incompatibility of the two polymers, a macro-phase separation was prevented in all the studied conditions. Sub-micrometric domains were formed, and for the thinner films, nanometric domains as small as 74 nm in size can be obtained. The silica structures formed by the infiltration of the polymer templates were used as hard masks for the cryogenic etching of underlying silicon. New structured surfaces, arrays of silicon pillars which can be plain or hollow at the upper part or arrays of cylindrical holes were formed. A selectivity as high as 21 was obtained using this type of mask for 1.5 μm deep holes having a typical diameter of 200 nm.

  18. Insight into the template effect of vesicles on the laccase-catalyzed oligomerization of N-phenyl-1,4-phenylenediamine from Raman spectroscopy and cyclic voltammetry measurements

    Science.gov (United States)

    Ležaić, Aleksandra Janoševic; Luginbühl, Sandra; Bajuk-Bogdanović, Danica; Pašti, Igor; Kissner, Reinhard; Rakvin, Boris; Walde, Peter; Ćirić-Marjanović, Gordana

    2016-08-01

    We report about the first Raman spectroscopy study of a vesicle-assisted enzyme-catalyzed oligomerization reaction. The aniline dimer N-phenyl-1,4-phenylenediamine (= p-aminodiphenylamine, PADPA) was oxidized and oligomerized with Trametes versicolor laccase and dissolved O2 in the presence of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) vesicles (80-100 nm diameter) as templates. The conversion of PADPA into oligomeric products, poly(PADPA), was monitored during the reaction by in situ Raman spectroscopy. The results obtained are compared with UV/vis/NIR and EPR measurements. All three complementary methods indicate that at least some of the poly(PADPA) products, formed in the presence of AOT vesicles, resemble the conductive emeraldine salt form of polyaniline (PANI-ES). The Raman measurements also show that structural units different from those of “ordinary” PANI-ES are present too. Without vesicles PANI-ES-like products are not obtained. For the first time, the as-prepared stable poly(PADPA)-AOT vesicle suspension was used directly to coat electrodes (without product isolation) for investigating redox activities of poly(PADPA) by cyclic voltammetry (CV). CV showed that poly(PADPA) produced with vesicles is redox active not only at pH 1.1-as expected for PANI-ES-but also at pH 6.0, unlike PANI-ES and poly(PADPA) synthesized without vesicles. This extended pH range of the redox activity of poly(PADPA) is important for applications.

  19. Insight into the template effect of vesicles on the laccase-catalyzed oligomerization of N-phenyl-1,4-phenylenediamine from Raman spectroscopy and cyclic voltammetry measurements.

    Science.gov (United States)

    Ležaić, Aleksandra Janoševic; Luginbühl, Sandra; Bajuk-Bogdanović, Danica; Pašti, Igor; Kissner, Reinhard; Rakvin, Boris; Walde, Peter; Ćirić-Marjanović, Gordana

    2016-08-26

    We report about the first Raman spectroscopy study of a vesicle-assisted enzyme-catalyzed oligomerization reaction. The aniline dimer N-phenyl-1,4-phenylenediamine (= p-aminodiphenylamine, PADPA) was oxidized and oligomerized with Trametes versicolor laccase and dissolved O2 in the presence of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) vesicles (80-100 nm diameter) as templates. The conversion of PADPA into oligomeric products, poly(PADPA), was monitored during the reaction by in situ Raman spectroscopy. The results obtained are compared with UV/vis/NIR and EPR measurements. All three complementary methods indicate that at least some of the poly(PADPA) products, formed in the presence of AOT vesicles, resemble the conductive emeraldine salt form of polyaniline (PANI-ES). The Raman measurements also show that structural units different from those of "ordinary" PANI-ES are present too. Without vesicles PANI-ES-like products are not obtained. For the first time, the as-prepared stable poly(PADPA)-AOT vesicle suspension was used directly to coat electrodes (without product isolation) for investigating redox activities of poly(PADPA) by cyclic voltammetry (CV). CV showed that poly(PADPA) produced with vesicles is redox active not only at pH 1.1-as expected for PANI-ES-but also at pH 6.0, unlike PANI-ES and poly(PADPA) synthesized without vesicles. This extended pH range of the redox activity of poly(PADPA) is important for applications.

  20. Synthesis of Nonionic Oligomeric Manganese(Ⅱ) Complexes and Investigation of Their Toxicity and Τ1-Relaxation Enhancement

    Institute of Scientific and Technical Information of China (English)

    YU Kai-chao; LI Ying-xia; ZHOU Jin-lan; DING Shang-wu; YE Chao-hui

    2005-01-01

    Six nonionic oligomeric manganese(Ⅱ) complexes with oligomeric phosphate-polyglycol-EDTA ester ligands were synthesized and characterized. The longitudinal relaxivities of these complexes were measured. One of these complexes, which showed the highest relaxivity, was chosen to be used in the acute toxicity test and the Τ1-weighted imaging experiment. It has been found that compared to Gd-DTPA, this nonionic oligomeric Mn(Ⅱ) complex exhibits no acute toxicity, generates highly enhanced MRI signals and increases the intention time in the rat liver tissue.

  1. Cry1AH蛋白活性片段在溶液中的单体和寡聚体杀虫活性分析%Toxicity analysis of activated Cry1Ah of monomeric and oligomeric forms in the solution

    Institute of Scientific and Technical Information of China (English)

    周子珊; 束长龙; 梁革梅; 苍晶; 宋福平; 张杰

    2011-01-01

    [目的]分析NaCl对寡聚化的影响,以及寡聚化对Cry1Ah杀虫活性的影响.[方法]利用Superdex-75分子筛,将Cry1Ah活性片段的寡聚体和单体进行分离,通过戊二醛交联试脸分析寡聚化程度,结合生物活性测定,分析NaCl对寡聚化的影响以及寡聚化对Cry1Ah毒力的影响.[结果]在纯化的组分峰Ⅰ中寡聚体占总蛋白的63.35%.NaCl时寡聚化有促进作用,而寡聚体又以多种形式存在,并且表现出不同的生物活性.寡聚体峰Ⅰ、寡聚体峰Ⅱ、单体峰Ⅲ对棉铃虫的LC50分别为47.192、3.329、3.977μg/mL.[结论]NaCl对寡聚化有促进作用,而且,寡聚体的毒力比Cry1Ah活性片段的毒力有不同程度的降低,最多下降49.6倍.推测这种体外形成的寡聚体不同于昆虫体内形成的寡聚体,引起插膜穿孔能力下降,导致毒力不同.%[Objective] Our aim is to analyze the effect of sodium chloride on oligomerization, and the toxicity of oligomeric units. [Method] To analyze the effect of sodium chloride on oligomerization, and toxicity of oligomeric units, toxin protein was purified by chromatography with Superdex-75 to separate the oligomeric units and monomer unit. Oligomerization reaction was detected by glutaraldehyde crosslinking, and bioassay of all the oligomeric units was performed against the larvae of Helicoverpa armigera. [Result] Oligomeric units accounted for 63.35% of total Cry1Ah toxin in Peak 1. Sodium chloride could promote oligomerization. The 50% lethal concentrations (LC50) of oligomeric units (Peak 1 and Peak 2) and monomer unit (Peak 3) against H. armigera were 47.192 μg/mL, 3. 329 μg/mL and 3. 977 μg/mL, respectively. [Conclasion] Sodium chloride could promote oligomerization; the toxicity of oligomeric units declined compared with Cry1Ah polypeptide: Peak1 was 49.6 fold lower than that of activated Cry1Ah polypeptide. We deduced that the oligomerization was different in structure between vivo and in vitro reactions

  2. Small angle X-ray scattering studies of mitochondrial glutaminase C reveal extended flexible regions, and link oligomeric state with enzyme activity.

    Directory of Open Access Journals (Sweden)

    Magda Møller

    Full Text Available Glutaminase C is a key metabolic enzyme, which is unregulated in many cancer systems and believed to play a central role in the Warburg effect, whereby cancer cells undergo changes to an altered metabolic profile. A long-standing hypothesis links enzymatic activity to the protein oligomeric state, hence the study of the solution behavior in general and the oligomer state in particular of glutaminase C is important for the understanding of the mechanism of protein activation and inhibition. In this report, this is extensively investigated in correlation to enzyme concentration or phosphate level, using a high-throughput microfluidic-mixing chip for the SAXS data collection, and we confirm that the oligomeric state correlates with activity. The in-depth solution behavior analysis further reveals the structural behavior of flexible regions of the protein in the dimeric, tetrameric and octameric state and investigates the C-terminal influence on the enzyme solution behavior. Our data enable SAXS-based rigid body modeling of the full-length tetramer states, thereby presenting the first ever experimentally derived structural model of mitochondrial glutaminase C including the N- and C-termini of the enzyme.

  3. Supplementary Material for: A new mode of SAM domain mediated oligomerization observed in the CASKIN2 neuronal scaffolding protein

    KAUST Repository

    Smirnova, Ekaterina

    2016-01-01

    Abstract Background CASKIN2 is a homolog of CASKIN1, a scaffolding protein that participates in a signaling network with CASK (calcium/calmodulin-dependent serine kinase). Despite a high level of homology between CASKIN2 and CASKIN1, CASKIN2 cannot bind CASK due to the absence of a CASK Interaction Domain and consequently, may have evolved undiscovered structural and functional distinctions. Results We demonstrate that the crystal structure of the Sterile Alpha Motif (SAM) domain tandem (SAM1-SAM2) oligomer from CASKIN2 is different than CASKIN1, with the minimal repeating unit being a dimer, rather than a monomer. Analytical ultracentrifugation sedimentation velocity methods revealed differences in monomer/dimer equilibria across a range of concentrations and ionic strengths for the wild type CASKIN2 SAM tandem and a structure-directed double mutant that could not oligomerize. Further distinguishing CASKIN2 from CASKIN1, EGFP-tagged SAM tandem proteins expressed in Neuro2a cells produced punctae that were distinct both in shape and size. Conclusions This study illustrates a new way in which neuronal SAM domains can assemble into large macromolecular assemblies that might concentrate and amplify synaptic responses.

  4. Dispersion states and surface characteristics of physically blended polyhedral oligomeric silsesquioxane/polymer hybrid nanocomposites

    Science.gov (United States)

    Misra, Rahul

    Control of dispersion and segregation states of nanostructured additives is one of the biggest challenges in realizing the optimum potential of high performance hybrid polymer nanocomposites. Polyhedral oligomeric silsesquioxane (POSS) nanostructured chemicals, with their hybrid organic-inorganic nature and flexible functionalization with a variety of organic substituents, yield possibilities to control dispersion and tune compatibility in a wide range of polymer systems. The overall goal of this research is to investigate the fundamental parameters that influence the dispersion and segregation states of POSS nanostructured chemicals, and to understand chain dynamics and conformations in physically blended POSS hybrid polymer nanocomposites (HPNC's). Multiple structural and mechanical factors influencing macro to nano scale surface and bulk properties were successfully investigated and correlated. A strategy based on thermodynamic principles for selective control of POSS dispersion states in a given polymer matrix is developed and discussed. This dissertation consists of eight chapters. Chapter 1 provides a detailed introduction about the development and current research interest in POSS/polymer nanocomposites. This chapter also discusses limitations of current advanced nanoprobe techniques. Chapter 2 establishes the overall goal of this research and specific research ii objectives. Chapter 3 establishes the preferential surface migration behavior of physically dispersed, non-reactive, closed cage octaisobutyl POSS (Oib-POSS) in a non-polar polypropylene matrix. Furthermore, influence of POSS surface segregation on the surface properties, especially nano-tribomechanical behavior is also discussed. Chapter 4 expands the studies by melt blending two different types of POSS molecules, a non-reactive, closed cage Oib-POSS and an open cage trisilanolphenyl POSS (Tsp-POSS), in a nylon 6 matrix. This chapter discusses the morphology, nano-dispersion and macro- to

  5. Structure of Human Acid Sphingomyelinase Reveals the Role of the Saposin Domain in Activating Substrate Hydrolysis.

    Science.gov (United States)

    Xiong, Zi-Jian; Huang, Jingjing; Poda, Gennady; Pomès, Régis; Privé, Gilbert G

    2016-07-31

    Acid sphingomyelinase (ASM) is a lysosomal phosphodiesterase that catalyzes the hydrolysis of sphingomyelin to produce ceramide and phosphocholine. While other lysosomal sphingolipid hydrolases require a saposin activator protein for full activity, the ASM polypeptide incorporates a built-in N-terminal saposin domain and does not require an external activator protein. Here, we report the crystal structure of human ASM and describe the organization of the three main regions of the enzyme: the N-terminal saposin domain, the proline-rich connector, and the catalytic domain. The saposin domain is tightly associated along an edge of the large, bowl-shaped catalytic domain and adopts an open form that exposes a hydrophobic concave surface approximately 30Å from the catalytic center. The calculated electrostatic potential of the enzyme is electropositive at the acidic pH of the lysosome, consistent with the strict requirement for the presence of acidic lipids in target membranes. Docking studies indicate that sphingomyelin binds with the ceramide-phosphate group positioned at the binuclear zinc center and molecular dynamic simulations indicate that the intrinsic flexibility of the saposin domain is important for monomer-dimer exchange and for membrane interactions. Overall, ASM uses a combination of electrostatic and hydrophobic interactions to cause local disruptions of target bilayers in order to bring the lipid headgroup to the catalytic center in a membrane-bound reaction.

  6. Three-Dimensional Models of the Oligomeric Human Asialoglycoprotein Receptor (ASGP-R

    Directory of Open Access Journals (Sweden)

    Anna Maria Bianucci

    2010-10-01

    Full Text Available The work presented here is aimed at suggesting plausible hypotheses for functional oligomeric forms of the human asialoglycoprotein receptor (ASGP-R, by applying a combination of different computational techniques. The functional ASGP-R is a hetero-oligomer, that comprises of several subunits of two different kinds (H1 and H2, which are highly homologous. Its stoichiometry is still unknown. An articulated step-wise modeling protocol was used in order to build the receptor model in a minimal oligomeric form, necessary for it to bind multi-antennary carbohydrate ligands. The ultimate target of the study is to contribute to increasing the knowledge of interactions between the human ASGP-R and carbohydrate ligands, at the molecular level, pertinent to applications in the field of hepatic tissue engineering.

  7. Domain architecture and oligomerization properties of the paramyxovirus PIV 5 hemagglutinin-neuraminidase (HN) protein.

    Science.gov (United States)

    Yuan, Ping; Leser, George P; Demeler, Borries; Lamb, Robert A; Jardetzky, Theodore S

    2008-09-01

    The mechanism by which the paramyxovirus hemagglutinin-neuraminidase (HN) protein couples receptor binding to activation of virus entry remains to be fully understood, but the HN stalk is thought to play an important role in the process. We have characterized ectodomain constructs of the parainfluenza virus 5 HN to understand better the underlying architecture and oligomerization properties that may influence HN functions. The PIV 5 neuraminidase (NA) domain is monomeric whereas the ectodomain forms a well-defined tetramer. The HN stalk also forms tetramers and higher order oligomers with high alpha-helical content. Together, the data indicate that the globular NA domains form weak intersubunit interactions at the end of the HN stalk tetramer, while stabilizing the stalk and overall oligomeric state of the ectodomain. Electron microscopy of the HN ectodomain reveals flexible arrangements of the NA and stalk domains, which may be important for understanding how these two HN domains impact virus entry.

  8. Effect of acid density of HZSM-5 on the oligomerization of ethylene in FCC dry gas

    Institute of Scientific and Technical Information of China (English)

    Xue Ding; Shan Geng; Chunyi Li; Chaohe Yang; Guohui Wang

    2009-01-01

    The oligomerization of ethylene in FCC dry gas over HZSM-5 catalyst with different Si/Al2 ratios was studied. The effect of acid density of catalyst on the oligomerization of ethylene was discussed. By increasing the acid density of catalyst, ethylene conversion showed a linear increase, while the yields of olefins decreased when the acid density of catalyst exceeded 0.14 mmolNH3/g owing to a promotion of hydrogen transfer reaction. Through comparing the average distance between acid sites on catalyst with kinetic diameters of olefins, it was found that the dimerization of ethylene was not restrained by the sparse distribution of acid sites, while the hydrogen transfer reaction of C3 and C4 olefins was limited. On these bases, a conclusion is proposed that the dimerization of ethylene proceeded via Eley-Rideal mechanism, while the hydrogen transfer reaction of C3 and C4 olefins followed the Langmuir-Hinshelwood mechanism.

  9. Crystal Structure of Dengue Virus Type 1 Envelope Protein in the Postfusion Conformation and Its Implications for Membrane Fusion

    Energy Technology Data Exchange (ETDEWEB)

    Nayak, Vinod; Dessau, Moshe; Kucera, Kaury; Anthony, Karen; Ledizet, Michel; Modis, Yorgo; (Yale); (L2 Diagnostics)

    2009-07-31

    Dengue virus relies on a conformational change in its envelope protein, E, to fuse the viral lipid membrane with the endosomal membrane and thereby deliver the viral genome into the cytosol. We have determined the crystal structure of a soluble fragment E (sE) of dengue virus type 1 (DEN-1). The protein is in the postfusion conformation even though it was not exposed to a lipid membrane or detergent. At the domain I-domain III interface, 4 polar residues form a tight cluster that is absent in other flaviviral postfusion structures. Two of these residues, His-282 and His-317, are conserved in flaviviruses and are part of the 'pH sensor' that triggers the fusogenic conformational change in E, at the reduced pH of the endosome. In the fusion loop, Phe-108 adopts a distinct conformation, forming additional trimer contacts and filling the bowl-shaped concavity observed at the tip of the DEN-2 sE trimer.

  10. Three-Dimensional Models of the Oligomeric Human Asialoglycoprotein Receptor (ASGP-R)

    OpenAIRE

    Anna Maria Bianucci; Ilaria Massarelli; Federica Chiellini; Emo Chiellini

    2010-01-01

    The work presented here is aimed at suggesting plausible hypotheses for functional oligomeric forms of the human asialoglycoprotein receptor (ASGP-R), by applying a combination of different computational techniques. The functional ASGP-R is a hetero-oligomer, that comprises of several subunits of two different kinds (H1 and H2), which are highly homologous. Its stoichiometry is still unknown. An articulated step-wise modeling protocol was used in order to build the receptor model in a minimal...

  11. Alternative Models of Iron and Cobalt Catalysts for Ethylene Oligomerization and Polymerization

    Institute of Scientific and Technical Information of China (English)

    Katrin; Wedeking; Sherrif; Adewuyi; Maliha; Asma; Igor; Vystorop; Saliu; Amolegbe; Elena; Novikova

    2007-01-01

    1 Results Great progresses have been made in the field of transition metal-based complexes as catalytic precursors for olefin oligomerization and polymerization,in which the core subjects will remain as "know and how" to develop novel catalysts both in academic and industrial consideration.The key advantage of iron and cobalt catalyst for ethylene polymerization is to produce vinyl-type polyethylenes.Therefore following the pioneering works of bis(imino) pyridyl iron and cobalt catalyst by Brookhart[1] ...

  12. Oligomerization of DH Domain Is Essential for Dbl-Induced Transformation

    OpenAIRE

    Zhu, Kejin; Debreceni, Balazs; Bi, Feng; Zheng, Yi

    2001-01-01

    The dbl oncogene product (onco-Dbl) is the prototype member of a family of guanine nucleotide exchange factors (GEFs) for Rho GTPases. The Dbl homology (DH) domain of onco-Dbl is responsible for the GEF catalytic activity, and the DH domain, together with the immediately adjacent pleckstrin homology (PH) domain, constitutes the minimum module bearing transforming function. In the present study, we demonstrate that the onco-Dbl protein exists in oligomeric form in vitro and in cells. The oligo...

  13. Membrane Permeabilization by Oligomeric α-Synuclein: In Search of the Mechanism.

    Directory of Open Access Journals (Sweden)

    Bart D van Rooijen

    Full Text Available BACKGROUND: The question of how the aggregation of the neuronal protein α-synuclein contributes to neuronal toxicity in Parkinson's disease has been the subject of intensive research over the past decade. Recently, attention has shifted from the amyloid fibrils to soluble oligomeric intermediates in the α-synuclein aggregation process. These oligomers are hypothesized to be cytotoxic and to permeabilize cellular membranes, possibly by forming pore-like complexes in the bilayer. Although the subject of α-synuclein oligomer-membrane interactions has attracted much attention, there is only limited evidence that supports the pore formation by α-synuclein oligomers. In addition the existing data are contradictory. METHODOLOGY/PRINCIPAL FINDINGS: Here we have studied the mechanism of lipid bilayer disruption by a well-characterized α-synuclein oligomer species in detail using a number of in vitro bilayer systems and assays. Dye efflux from vesicles induced by oligomeric α-synuclein was found to be a fast all-or-none process. Individual vesicles swiftly lose their contents but overall vesicle morphology remains unaltered. A newly developed assay based on a dextran-coupled dye showed that non-equilibrium processes dominate the disruption of the vesicles. The membrane is highly permeable to solute influx directly after oligomer addition, after which membrane integrity is partly restored. The permeabilization of the membrane is possibly related to the intrinsic instability of the bilayer. Vesicles composed of negatively charged lipids, which are generally used for measuring α-synuclein-lipid interactions, were unstable to protein adsorption in general. CONCLUSIONS/SIGNIFICANCE: The dye efflux from negatively charged vesicles upon addition of α-synuclein has been hypothesized to occur through the formation of oligomeric membrane pores. However, our results show that the dye efflux characteristics are consistent with bilayer defects caused by

  14. Thermal and Chemical Stability of Two Homologous POZ/BTB Domains of KCTD Proteins Characterized by a Different Oligomeric Organization

    Directory of Open Access Journals (Sweden)

    Luciano Pirone

    2013-01-01

    Full Text Available POZ/BTB domains are widespread modules detected in a variety of different biological contexts. Here, we report a biophysical characterization of the POZ/BTB of KCTD6, a protein that is involved in the turnover of the muscle small ankyrin-1 isoform 5 and, in combination with KCTD11, in the ubiquitination and degradation of HDAC1. The analyses show that the domain is a tetramer made up by subunits with the expected α/ structure. A detailed investigation of its stability, carried out in comparison with the homologous pentameric POZ/BTB domain isolated from KCTD5, highlights a number of interesting features, which are shared by the two domains despite their different organization. Their thermal/chemical denaturation curves are characterized by a single and sharp inflection point, suggesting that the denaturation of the two domains is a cooperative two-state process. Furthermore, both domains present a significant content of secondary structure in their denatured state and a reversible denaturation process. We suggest that the ability of these domains to fold and unfold reversibly, a property that is somewhat unexpected for these oligomeric assemblies, may have important implications for their biological function. Indeed, these properties likely favor the formation of heteromeric associations that may be essential for the intricate regulation of the processes in which these proteins are involved.

  15. Interplay of histidine residues of the Alzheimer’s disease Aβ peptide governs its Zn-induced oligomerization

    Science.gov (United States)

    Istrate, Andrey N.; Kozin, Sergey A.; Zhokhov, Sergey S.; Mantsyzov, Alexey B.; Kechko, Olga I.; Pastore, Annalisa; Makarov, Alexander A.; Polshakov, Vladimir I.

    2016-02-01

    Conformational changes of Aβ peptide result in its transformation from native monomeric state to the toxic soluble dimers, oligomers and insoluble aggregates that are hallmarks of Alzheimer’s disease (AD). Interactions of zinc ions with Aβ are mediated by the N-terminal Aβ1–16 domain and appear to play a key role in AD progression. There is a range of results indicating that these interactions trigger the Aβ plaque formation. We have determined structure and functional characteristics of the metal binding domains derived from several Aβ variants and found that their zinc-induced oligomerization is governed by conformational changes in the minimal zinc binding site 6HDSGYEVHH14. The residue H6 and segment 11EVHH14, which are part of this site are crucial for formation of the two zinc-mediated interaction interfaces in Aβ. These structural determinants can be considered as promising targets for rational design of the AD-modifying drugs aimed at blocking pathological Aβ aggregation.

  16. Effects of oligomeric procyanidins on the retrogradation properties of maize starch with different amylose/amylopectin ratios.

    Science.gov (United States)

    Liu, Rui; Xu, Chen; Cong, Xu; Wu, Tao; Song, Yingshi; Zhang, Min

    2017-04-15

    The effect of oligomeric procyanidins (OPCs) on the retrogradation of maize starch with different amylose/amylopectin ratios was investigated. The apparent amylose contents in high-amylose maize (HAM), normal maize (NM), and amylopectin maize (APM) starches are 79.05%, 25.43% and 0%. Structural characterizations of retrograded maize starches in the presence of OPCs were conducted by differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and nuclear magnetic resonance (NMR). The results suggest that OPCs inhibit the retrogradation of maize starches in low concentrations (1.5-2.5%) with different inhibitory effects for HAM, NM and APM starches. It may be attributed to the variations on interaction ways and binding capabilities between different types of starches and OPCs. The in vitro enzymatic digestion result indicates HAM starch and OPCs have stronger interactions with the formation of resistant structures. These findings provide a further evidence for exploring the interactions between starches and phenolic compounds. Copyright © 2016. Published by Elsevier Ltd.

  17. Oligomeric state regulated trafficking of human platelet-activating factor acetylhydrolase type-II.

    Science.gov (United States)

    Monillas, Elizabeth S; Caplan, Jeffrey L; Thévenin, Anastasia F; Bahnson, Brian J

    2015-05-01

    The intracellular enzyme platelet-activating factor acetylhydrolase type-II (PAFAH-II) hydrolyzes platelet-activating factor and oxidatively fragmented phospholipids. PAFAH-II in its resting state is mainly cytoplasmic, and it responds to oxidative stress by becoming increasingly bound to endoplasmic reticulum and Golgi membranes. Numerous studies have indicated that this enzyme is essential for protecting cells from oxidative stress induced apoptosis. However, the regulatory mechanism of the oxidative stress response by PAFAH-II has not been fully resolved. Here, changes to the oligomeric state of human PAFAH-II were investigated as a potential regulatory mechanism toward enzyme trafficking. Native PAGE analysis in vitro and photon counting histogram within live cells showed that PAFAH-II is both monomeric and dimeric. A Gly-2-Ala site-directed mutation of PAFAH-II demonstrated that the N-terminal myristoyl group is required for homodimerization. Additionally, the distribution of oligomeric PAFAH-II is distinct within the cell; homodimers of PAFAH-II were localized to the cytoplasm while monomers were associated to the membranes of the endoplasmic reticulum and Golgi. We propose that the oligomeric state of PAFAH-II drives functional protein trafficking. PAFAH-II localization to the membrane is critical for substrate acquisition and effective oxidative stress protection. It is hypothesized that the balance between monomer and dimer serves as a regulatory mechanism of a PAFAH-II oxidative stress response.

  18. Recursive directional ligation by plasmid reconstruction allows rapid and seamless cloning of oligomeric genes.

    Science.gov (United States)

    McDaniel, Jonathan R; Mackay, J Andrew; Quiroz, Felipe García; Chilkoti, Ashutosh

    2010-04-12

    This paper reports a new strategy, recursive directional ligation by plasmid reconstruction (PRe-RDL), to rapidly clone highly repetitive polypeptides of any sequence and specified length over a large range of molecular weights. In a single cycle of PRe-RDL, two halves of a parent plasmid, each containing a copy of an oligomer, are ligated together, thereby dimerizing the oligomer and reconstituting a functional plasmid. This process is carried out recursively to assemble an oligomeric gene with the desired number of repeats. PRe-RDL has several unique features that stem from the use of type IIs restriction endonucleases: first, PRe-RDL is a seamless cloning method that leaves no extraneous nucleotides at the ligation junction. Because it uses type IIs endonucleases to ligate the two halves of the plasmid, PRe-RDL also addresses the major limitation of RDL in that it abolishes any restriction on the gene sequence that can be oligomerized. The reconstitution of a functional plasmid only upon successful ligation in PRe-RDL also addresses two other limitations of RDL: the significant background from self-ligation of the vector observed in RDL, and the decreased efficiency of ligation due to nonproductive circularization of the insert. PRe-RDL can also be used to assemble genes that encode different sequences in a predetermined order to encode block copolymers or append leader and trailer peptide sequences to the oligomerized gene.

  19. Rapid synthesis of cyclic oligomeric depsipeptides with positional, stereochemical, and macrocycle size distribution control.

    Science.gov (United States)

    Batiste, Suzanne M; Johnston, Jeffrey N

    2016-12-27

    Macrocyclic small molecules are attractive tools in the development of sensors, new materials, and therapeutics. Within early-stage drug discovery, they are increasingly sought for their potential to interact with broad surfaces of peptidic receptors rather than within their narrow folds and pockets. Cyclization of linear small molecule precursors is a straightforward strategy to constrain conformationally mobile motifs, but forging a macrocycle bond typically becomes more difficult at larger ring sizes. We report the development of a general approach to discrete collections of oligomeric macrocyclic depsipeptides using an oligomerization/macrocyclization process governed by a series of Mitsunobu reactions of hydroxy acid monomers. Ring sizes of 18, 24, 30, and 36 are formed in a single reaction from a didepsipeptide, whereas sizes of 24, 36, and 60 result from a tetradepsipeptide. The ring-size selectivity inherent to the approach can be modulated by salt additives that enhance the formation of specific ring sizes. Use of chemical synthesis to prepare the monomers suggests broad access to functionally and stereochemically diverse collections of natural product-like oligodepsipeptide macrocycles. Two cyclodepsipeptide natural products were prepared along with numerous unnatural oligomeric congeners to provide rapid access to discrete collections of complex macrocyclic small molecules from medium (18) to large (60) ring sizes.

  20. Physiological relevance of plant 2-Cys peroxiredoxin overoxidation level and oligomerization status.

    Science.gov (United States)

    Cerveau, Delphine; Ouahrani, Djelloul; Marok, Mohamed Amine; Blanchard, Laurence; Rey, Pascal

    2016-01-01

    Peroxiredoxins are ubiquitous thioredoxin-dependent peroxidases presumed to display, upon environmental constraints, a chaperone function resulting from a redox-dependent conformational switch. In this work, using biochemical and genetic approaches, we aimed to unravel the factors regulating the redox status and the conformation of the plastidial 2-Cys peroxiredoxin (2-Cys PRX) in plants. In Arabidopsis, we show that in optimal growth conditions, the overoxidation level mainly depends on the availability of thioredoxin-related electron donors, but not on sulfiredoxin, the enzyme reducing the 2-Cys PRX overoxidized form. We also observed that upon various physiological temperature, osmotic and light stress conditions, the overoxidation level and oligomerization status of 2-Cys PRX can moderately vary depending on the constraint type. Further, no major change was noticed regarding protein conformation in water-stressed Arabidopsis, barley and potato plants, whereas species-dependent up- and down-variations in overoxidation were observed. In contrast, both 2-Cys PRX overoxidation and oligomerization were strongly induced during a severe oxidative stress generated by methyl viologen. From these data, revealing that the oligomerization status of plant 2-Cys PRX does not exhibit important variation and is not tightly linked to the protein redox status upon physiologically relevant environmental constraints, the possible in planta functions of 2-Cys PRX are discussed.

  1. Changes in dynamics upon oligomerization regulate substrate binding and allostery in amino acid kinase family members.

    Directory of Open Access Journals (Sweden)

    Enrique Marcos

    2011-09-01

    Full Text Available Oligomerization is a functional requirement for many proteins. The interfacial interactions and the overall packing geometry of the individual monomers are viewed as important determinants of the thermodynamic stability and allosteric regulation of oligomers. The present study focuses on the role of the interfacial interactions and overall contact topology in the dynamic features acquired in the oligomeric state. To this aim, the collective dynamics of enzymes belonging to the amino acid kinase family both in dimeric and hexameric forms are examined by means of an elastic network model, and the softest collective motions (i.e., lowest frequency or global modes of motions favored by the overall architecture are analyzed. Notably, the lowest-frequency modes accessible to the individual subunits in the absence of multimerization are conserved to a large extent in the oligomer, suggesting that the oligomer takes advantage of the intrinsic dynamics of the individual monomers. At the same time, oligomerization stiffens the interfacial regions of the monomers and confers new cooperative modes that exploit the rigid-body translational and rotational degrees of freedom of the intact monomers. The present study sheds light on the mechanism of cooperative inhibition of hexameric N-acetyl-L-glutamate kinase by arginine and on the allosteric regulation of UMP kinases. It also highlights the significance of the particular quaternary design in selectively determining the oligomer dynamics congruent with required ligand-binding and allosteric activities.

  2. Oligomerization of Clostridium perfringens epsilon toxin is dependent upon caveolins 1 and 2.

    Directory of Open Access Journals (Sweden)

    Christine M Fennessey

    Full Text Available Evidence from multiple studies suggests that Clostridium perfringens ε-toxin is a pore-forming toxin, assembling into oligomeric complexes in the plasma membrane of sensitive cells. In a previous study, we used gene-trap mutagenesis to identify mammalian factors contributing to toxin activity, including caveolin-2 (CAV2. In this study, we demonstrate the importance of caveolin-2 and its interaction partner, caveolin-1 (CAV1, in ε-toxin-induced cytotoxicity. Using CAV2-specific shRNA in a toxin-sensitive human kidney cell line, ACHN, we confirmed that cells deficient in CAV2 exhibit increased resistance to ε-toxin. Similarly, using CAV1-specific shRNA, we demonstrate that cells deficient in CAV1 also exhibit increased resistance to the toxin. Immunoprecipitation of CAV1 and CAV2 from ε-toxin-treated ACHN cells demonstrated interaction of both CAV1 and -2 with the toxin. Furthermore, blue-native PAGE indicated that the toxin and caveolins were components of a 670 kDa protein complex. Although ε-toxin binding was only slightly perturbed in caveolin-deficient cells, oligomerization of the toxin was dramatically reduced in both CAV1- and CAV2-deficient cells. These results indicate that CAV1 and -2 potentiate ε-toxin induced cytotoxicity by promoting toxin oligomerization - an event which is requisite for pore formation and, by extension, cell death.

  3. Disruption of oligomerization induces nucleocytoplasmic shuttling of leukemia-associated rho Guanine-nucleotide exchange factor.

    Science.gov (United States)

    Grabocka, Elda; Wedegaertner, Philip B

    2007-10-01

    The rgsRhoGEFs comprise a subfamily of three guanine nucleotide exchange factors, which function in linking heterotrimeric G-proteins to the monomeric RhoGTPase. Here, we reveal the novel finding that oligomerization of leukemia-associated RhoGEF (LARG) functions to prevent nucleocytoplasmic shuttling and to retain LARG in the cytoplasm. We establish that oligomerization is mediated by a predicted coiled-coil sequence (amino acids 1507-1520) in the extreme C terminus of LARG and that substitution of isoleucines 1507/1510 with alanines disrupts homo-oligomerization and leads to nucleocytoplasmic shuttling via the CRM1 nuclear transport pathway. In addition, we demonstrate that induced dimerization of an otherwise nuclear monomeric LARG mutant promotes cytoplasmic localization. Furthermore, we establish that nuclear import of monomeric LARG is mediated by the nuclear localization sequence (29)PTDKKQK(35) in the extreme N terminus. We propose that nucleocytoplasmic shuttling provides a mechanism for spatially regulating the activity of LARG toward its cytoplasmic targets and potentially new nuclear targets.

  4. Dealuminated ZSM-5 Zeolite Catalyst for Ethylene Oligomerization to Liquid Fuels

    Institute of Scientific and Technical Information of China (English)

    Nor Aishah Saidina Amin; Didi Dwi Anggoro

    2002-01-01

    Ethylene oligomerization using ZSM-5 zeolite was investigated to study the role of Bronstedacid sites in the formation of higher hydrocarbons. The oligomerization of olefins, dependent on the acidityof ZSM-5 zeolite, is an important step in the conversion of natural gas to liquid fuels. The framework Si/Alratio reflects the number of potential acid sites and the acid strength of the ZSM-5 catalyst. ZSM-5 withthe mole ratio SiO2/Al2O3 equal to 30 was dealuminated for different periods of time according to theacidic ion-exchange method to produce ZSM-5 with various Si/Al ratios. The FT-IR analysis revealedthat the integrated framework aluminum band, non-framework aluminum band, and silanol groups areasof the ZSM-5 zeolites decreased after being dealuminated. The performance of the dealuminated zeolitewas tested for ethylene oligomerization. The results demonstrated that the dealumination of ZSM-5 ledto higher ethylene conversion, but the gasoline selectivity was reduced compared to the performance of aZSM-5 zeolite. The characterization results revealed the amount of aluminum in the zeolitic framework,the crystallinity of the ZSM-5 zeolite, and the Si/Al ratio affected the formation of Bronsted acid sites.The number of the Bronsted acid sites on the catalyst active sites is important in the olefin conversion toliquid hydrocarbons.

  5. Oligomerization of the protein tau in the Alzheimer's disease

    Science.gov (United States)

    Larini, Luca

    The Alzheimer's disease is characterized by the formation of protein aggregates both within and outside of the brain's cells, the neurons. Within the neurons, the aggregation of the microtubule associated protein tau leads to the destruction of the microtubules in the axon of the neuron. Tau is extremely flexible and is classified as an intrinsically disordered protein due to its low propensity to form secondary structure. Tau promotes tubulin assembly into microtubules, which are an essential component of the cytoskeleton of the axon. The microtubule binding region of tau consists of 4 pseudo-repeats that are critical for aggregation as well. In this study, we focus on the aggregation propensity of different segments of the microtubule binding region as well as post-translational modifications that can alter tau dynamics and structure. We have performed replica exchange molecular dynamics simulations to characterize the ensemble of conformations of the monomer and small oligomers as well as how these structures are stabilized or destabilized by mutations and post-translational modifications.

  6. Density functional theory study of the oligomerization of carboxylic acids.

    Science.gov (United States)

    Di Tommaso, Devis; Watson, Ken L

    2014-11-20

    We present a density functional theory [M06-2X/6-31+G(d,p)] study of the structures and free energies of formation of oligomers of four carboxylic acids (formic acid, acetic acid, tetrolic acid, and benzoic acid) in water, chloroform, and carbon tetrachloride. Solvation effects were treated using the SMD continuum solvation model. The low-lying energy structures of molecular complexes were located by adopting an efficient search procedure to probe the potential energy surfaces of the oligomers of carboxylic acids (CA)n (n = 2-6). The free energies of the isomers of (CA)n in solution were determined as the sum of the electronic energy, vibrational-rotational-translational gas-phase contribution, and solvation free energy. The assessment of the computational protocol adopted in this study with respect to the dimerization of acetic acid, (AA)2, and formic acid, (FA)2, located new isomers of (AA)2 and (FA)2 and gave dimerization constants in good agreement with the experimental values. The calculation of the self-association of acetic acid, tetrolic acid, and benzoic acid shows the following: (i) Classic carboxylic dimers are the most stable isomer of (CA)2 in both the gas phase and solution. (ii) Trimers of carboxylic acid are stable in apolar aprotic solvents. (iii) Molecular clusters consisting of two interacting classic carboxylic dimers (CA)4,(D+D) are the most stable type of tetramers, but their formation from the self-association of classic carboxylic dimers is highly unfavorable. (iv) For acetic acid and tetrolic acid the reactions (CA)2 + 2CA → (CA)4,(D+D) and (CA)3 + CA → (CA)4,(D+D) are exoergonic, but these aggregation pathways go through unstable clusters that could hinder the formation of tetrameric species. (v) For tetrolic acid the prenucleation species that are more likely to form in solution are dimeric and trimeric structures that have encoded structural motifs resembling the α and β solid forms of tetrolic acid. (vi) Stable tetramers of

  7. Forward Modeling of Receiver Functions to Determine Crustal Structure of the Eastern Limb in TheBushveld Complex, South Africa

    Science.gov (United States)

    Loza, E.; Ramirez, C.; Nyblade, A.; Durrheim, R. J.; Raveloson, A.

    2016-12-01

    The Bushveld Igneous Complex contains the largest layered mafic intrusion on Earth, about the size of England, and has been exploited for metals such as platinum since the 1950s. Several igneous bodies within and around the complex have been dated from 2.06 Ga, possibly representing a single massive magmatic event. The Rustenburg Layered Suite of the Bushveld Igneous Complex intruded into the Transvaal sedimentary sequence, with associated volcanic rocks of the Rooiberg Group forming the roof and part of the floor. The purpose of this study is to determine whether the Rustenburg Layered Suite is a continuous bowl-shaped formation or if it is made up of two separate dipping sheets that crop out in the western and eastern limbs. If the intrusion is connected at depth, then the Moho (crust-mantle boundary) would most likely be depressed due to the weight of the 7-8km of mafic material injected into the crust. Seismic stations were installed in the eastern and northern Bushveld in 2015 to collect teleseismic data. The use of receiver functions derived from seismic data collected since 2015 has helped determine the subsurface crustal structure of the Bushveld. Receiver functions have been used to trace the contact between the high-density mafic lower zone and the low-density Transvaal sediments. The new data gathered show the Moho boundary at about 47 km, and a 5.0 Gaussian width shows a backswing consistent with a mafic-sedimentary boundary at 8km.

  8. Liquid crystals. Oligomeric and polymeric materials for soft photonic technologies

    CERN Document Server

    Coles, M J

    2002-01-01

    The current pace of today's information technologies might lead the casual observer to believe that this is all new. However the reality is that, as with most things, this is really a long evolution of processes based on tried, tested and re-adapted techniques. This thesis represents 12 years of predominantly technology driven research and covers a whole range of characterising, evaluating and fabricating devices based on liquid crystalline systems. Firstly polymer liquid crystals are discussed with respect to the fabrication of a flexible substrate display based on standard printing techniques and this is shown to have improved display viewing properties over a standard polymer dispersed liquid crystal (PDLC) device. Following on from this work is presented that involves the production of regular grid arrays in isotropic polymers that are used as control structures in nematic liquid crystal systems. This progresses onto a now patented device that allows the production of robust ferroelectric devices based on...

  9. Pulling peptides across nanochannels: resolving peptide binding and translocation through the hetero-oligomeric channel from Nocardia farcinica.

    Science.gov (United States)

    Singh, Pratik Raj; Bárcena-Uribarri, Iván; Modi, Niraj; Kleinekathöfer, Ulrich; Benz, Roland; Winterhalter, Mathias; Mahendran, Kozhinjampara R

    2012-12-21

    We investigated translocation of cationic peptides through nanochannels derived from the Gram-positive bacterium Nocardia farcinica at the single-molecule level. The two subunits NfpA and NfpB form a hetero-oligomeric cation selective channel. On the basis of amino acid comparison we performed homology modeling and obtained a channel structurally related to MspA of Mycobacterium smegmatis. The quantitative single-molecule measurements provide an insight into transport processes of solutes through nanochannels. High-resolution ion conductance measurements in the presence of peptides of different charge and length revealed the kinetics of peptide binding. The observed asymmetry in peptide binding kinetics indicated a unidirectional channel insertion in the lipid bilayer. In the case of cationic peptides, the external voltage acts as a driving force that promotes the interaction of the peptide with the channel surface. At low voltage, the peptide just binds to the channel, whereas at higher voltage, the force is strong enough to pull the peptide across the channel. This allows distinguishing quantitatively between peptide binding and translocation through the channel.

  10. Prf immune complexes of tomato are oligomeric and contain multiple Pto-like kinases that diversify effector recognition.

    Science.gov (United States)

    Gutierrez, Jose R; Balmuth, Alexi L; Ntoukakis, Vardis; Mucyn, Tatiana S; Gimenez-Ibanez, Selena; Jones, Alexandra M E; Rathjen, John P

    2010-02-01

    Cytoplasmic recognition of pathogen virulence effectors by plant NB-LRR proteins leads to strong induction of defence responses termed effector triggered immunity (ETI). In tomato, a protein complex containing the NB-LRR protein Prf and the protein kinase Pto confers recognition of the Pseudomonas syringae effectors AvrPto and AvrPtoB. Although structurally unrelated, AvrPto and AvrPtoB interact with similar residues in the Pto catalytic cleft to activate ETI via an unknown mechanism. Here we show that the Prf complex is oligomeric, containing at least two molecules of Prf. Within the complex, Prf can associate with Pto or one of several Pto family members including Fen, Pth2, Pth3, or Pth5. The dimerization surface for Prf is the novel N-terminal domain, which also coordinates an intramolecular interaction with the remainder of the molecule, and binds Pto kinase or a family member. Thus, association of two Prf N-terminal domains brings the associated kinases into close promixity. Tomato lines containing Prf complexed with Pth proteins but not Pto possessed greater immunity against P. syringae than tomatoes lacking Prf. This demonstrates that incorporation of non-Pto kinases into the Prf complex extends the number of effector proteins that can be recognized.

  11. Unusual Emission of Polystyrene-Based Alternating Copolymers Incorporating Aminobutyl Maleimide Fluorophore-Containing Polyhedral Oligomeric Silsesquioxane Nanoparticles

    Directory of Open Access Journals (Sweden)

    Mohamed Gamal Mohamed

    2017-03-01

    Full Text Available In this study, we synthesized an unusual 2-aminobutyl maleimide isobutyl polyhedral oligomeric silsesquioxane (MIPOSS-NHBu monomer lacking conventional fluorescent groups. We then prepared poly(styrene-alt-2-aminobutyl maleimide isobutyl POSS [poly(S-alt-MIPOSS-NHBu] and poly(4-acetoxystyrene-alt-2-aminobutyl maleimide isobutyl POSS [poly(AS-alt-MIPOSS-NHBu] copolymers through facile free radical copolymerizations using azobisisobutyronitrile as the initiator and tetrahydrofuran as the solvent. A poly(4-hydroxystyrene-alt-2-aminobutyl maleimide isobutyl POSS [poly(HS-alt-MIPOSS-NHBu] copolymer was prepared through acetoxyl hydrazinolysis of poly(AS-alt-MIPOSS-NHBu. We employed 1H, 13C, and 29Si nuclear magnetic resonance spectroscopy; Fourier transform infrared spectroscopy; differential scanning calorimetry; and photoluminescence spectroscopy to investigate the structures and the thermal and optical properties of the monomers and novel POSS-containing alternating copolymers. Intramolecular hydrogen bonding between the amino and dihydrofuran-2,5-dione group and clustering of the locked C=O groups from the POSS nanoparticles in the MIPOSS-NHBu units restricted the intramolecular motion of the polymer chain, causing it to exhibit strong light emission. As a result, the MIPOSS-NHBu monomer and the poly(AS-alt-MIPOSS-NHBu copolymer both have potential applicability in the detection of metal ions with good selectivity.

  12. "Clicked" bivalent ligands containing curcumin and cholesterol as multifunctional abeta oligomerization inhibitors: design, synthesis, and biological characterization.

    Science.gov (United States)

    Lenhart, James A; Ling, Xiao; Gandhi, Ronak; Guo, Tai L; Gerk, Phillip M; Brunzell, Darlene H; Zhang, Shijun

    2010-08-26

    In our effort to develop multifunctional compounds that cotarget beta-amyloid oligomers (AbetaOs), cell membrane/lipid rafts (CM/LR), and oxidative stress, a series of bivalent multifunctional Abeta oligomerization inhibitors (BMAOIs) containing cholesterol and curcumin were designed, synthesized, and biologically characterized as potential treatments for Alzheimer's disease (AD). The in vitro assay results established that the length of spacer that links cholesterol and curcumin and the attaching position of the spacer on curcumin are important structural determinants for their biological activities. Among the BMAOIs tested, 14 with a 21-atom-spacer was identified to localize to the CM/LR of human neuroblastoma MC65 cells, to inhibit the formation of AbetaOs in MC65 cells, to protect cells from AbetaOs-induced cytotoxicity, and to retain antioxidant properties of curcumin. Furthermore, 14 was confirmed to have the potential to cross the blood-brain barrier (BBB) as demonstrated in a Caco-2 cell model. Collectively, these results strongly encourage further optimization of 14 as a new hit to develop more potent BMAOIs.

  13. Highly stable nanofluid based on polyhedral oligomeric silsesquioxane-decorated graphene oxide nanosheets and its enhanced electro-responsive behavior

    Science.gov (United States)

    Li, Yizhuo; Guan, Yanqing; Liu, Yang; Yin, Jianbo; Zhao, Xiaopeng

    2016-05-01

    Graphene oxide (GO) shows potential as an anisotropic nanofiller or a dispersed phase of electro-responsive electrorheological (ER) nanofluid due to its small size and high aspect ratio. But it is difficult to disperse GO in non-polar oil due to the hydrophilic nature of GO and thus the resulting fluid is often subject to dispersion instability and low ER effect. These disadvantages largely limit the real application of GO-based ER nanofluid. In this paper, we develop the polyhedral oligomeric silsesquioxane (POSS)-decorated GO (POSS-GO) nanosheets and demonstrate that decorating with POSS overcomes the dispersion instability of GO in silicone oil and enhances the ER effect. The morphology and structure of samples are characterized by atomic force microscopy, Fourier transform infrared spectroscopy, thermogravimetric analysis, and x-ray photoelectronic spectroscopy. The results show that the POSS-GO nanosheets are ultrathin with ∼3 nm thickness and have good compatibility with silicone oil and, as a result, the nanofluid of POSS-GO nanosheets in silicone oil shows high dispersion stability. After standing for one year at room temperature, no sedimentation occurs. Under an external electric field, the ER efficiency of the POSS-GO nanofluid is ten times as high as that of the pure GO fluid. This enhanced electro-responsive behavior is related to the fact that decorating with POSS partly reduces the GO and compresses the dielectrophoretic effect of the negatively charged pure GO fluid.

  14. Erosion effects of atomic oxygen on polyhedral oligomeric silsesquioxane-polyimide hybrid films in low earth orbit space environment.

    Science.gov (United States)

    Duo, Shuwang; Song, Mimi; Liu, Tingzhi; Hu, Changyuan; Li, Meishuan

    2013-02-01

    A novel polyimide (PI) hybrid nanocomposite containing polyhedral oligomeric silsesquioxane (POSS) had been prepared by copolymerization of trisilanolphenyl-POSS, 4,4'-oxydianiline (ODA), and pyromellitic dianhydride (PMDA). The AO resistance of these PI/POSS hybrid films was tested in the ground-based AO simulation facility. Exposed and unexposed surfaces were characterized by SEM and X-ray photoelectron spectroscopy. SEM images showed that the surface of the 20 wt% PI/POSS became much less rough than that of the pristine polyimide. Mass measurements of the samples showed that the erosion yield of the PI/POSS (20 wt.%) hybrid film was 1.2 x 10(-25) cm3/atom, and reduced to 4% of the polyimide film. The XPS data indicated that the carbon content of the near-surface region was decreased from 60.1 to 13.2 at% after AO exposure. The oxygen and silicon concentrations in the near-surface region increased to 1.96 after AO exposure. The nanometer-sized structure of POSS, with its large surface area, had led AO-irradiated samples to form a SiO2 passivation layer, which protected the underlying polymer from further AO attack. The incorporation of POSS into the polyimide could dramatically improve the AO resistance of polyimide films in low earth orbit environment.

  15. Crystal Structure of Dengue Type 1 Envelope Protein in the Postfusion Conformation and its Implication for Receptor Binding, Membrane Fusion and Antibody Recognition

    Energy Technology Data Exchange (ETDEWEB)

    Nayak, V.; Dessau, M; Kucera, K; Anthony, K; Ledizet, M; Modis, Y

    2009-01-01

    Dengue virus relies on a conformational change in its envelope protein, E, to fuse the viral lipid membrane with the endosomal membrane and thereby deliver the viral genome into the cytosol. We have determined the crystal structure of a soluble fragment E (sE) of dengue virus type 1 (DEN-1). The protein is in the postfusion conformation even though it was not exposed to a lipid membrane or detergent. At the domain I-domain III interface, 4 polar residues form a tight cluster that is absent in other flaviviral postfusion structures. Two of these residues, His-282 and His-317, are conserved in flaviviruses and are part of the pH sensor that triggers the fusogenic conformational change in E, at the reduced pH of the endosome. In the fusion loop, Phe-108 adopts a distinct conformation, forming additional trimer contacts and filling the bowl-shaped concavity observed at the tip of the DEN-2 sE trimer.

  16. Salix daphnoides: A Screening for Oligomeric and Polymeric Proanthocyanidins

    Directory of Open Access Journals (Sweden)

    Stefan Wiesneth

    2015-07-01

    Full Text Available In the present study, a qualitative analysis of proanthocyanidins (PAs from an aqueous-methanolic extract of Salix daphnoides VILL. bark is described. Procyanidin B1 (1, B2 (2, B3 (3, B4 (4, C1 (5, epicatechin-(4β→8-epicatechin-(4β→8-catechin (6 and epicatechin-(4β→8-epicatechin-(4β→8-epicatechin-(4β→8-catechin (7 have been isolated by a combination of different chromatographic separations on Sephadex® LH-20-, MCI®-, Diol-and RP-18-phases. Mass spectrometry, 1D- and 2D-NMR, circular dichroism and polarimetry were used for their structure elucidation and verification by comparison with the literature. Additionally, two fractions of very polar flavan-3-ols were compared: “regular” polymeric PAs received at the very end of the Sephadex® LH-20 chromatography showing no mobility on silica TLC and “unusual” PAs with the same RF-value but already eluting together with flavonoids in the Sephadex® LH-20 system. These “unusual” PAs were subsequently enriched by centrifugal partition chromatography (CPC. 13C-NMR, polarimetry, thiolysis, acid hydrolysis and phloroglucinol degradation were used to characterize both fractions. Differences in the composition of different flavan-3-ol units and the middle chain length were observed.

  17. Oligomerization of Indole Derivatives with Incorporation of Thiols

    Directory of Open Access Journals (Sweden)

    Jarl E.S. Wikberg

    2008-08-01

    Full Text Available Abstract: Two molecules of indole derivative, e.g. indole-5-carboxylic acid, reacted with one molecule of thiol, e.g. 1,2-ethanedithiol, in the presence of trifluoroacetic acid to yield adducts such as 3-[2-(2-amino-5-carboxyphenyl-1-(2-mercaptoethylthioethyl]-1Hindole-5-carboxylic acid. Parallel formation of dimers, such as 2,3-dihydro-1H,1'H-2,3'-biindole-5,5'-dicarboxylic acid and trimers, such as 3,3'-[2-(2-amino-5-carboxyphenyl ethane-1,1-diyl]bis(1H-indole-5-carboxylic acid of the indole derivatives was also observed. Reaction of a mixture of indole and indole-5-carboxylic acid with 2-phenylethanethiol proceeded in a regioselective way, affording 3-[2-(2-aminophenyl-1-(phenethylthioethyl]-1H-indole-5-carboxylic acid. An additional product of this reaction was 3-[2-(2-aminophenyl-1-(phenethylthioethyl]-2,3-dihydro-1H,1'H-2,3'-biindole-5'-carboxylic acid, which upon standing in DMSO-d6 solution gave 3-[2-(2-aminophenyl-1-(phenethylthioethyl]-1H,1'H-2,3'-biindole-5'-carboxylic acid. Structures of all compounds were elucidated by NMR, and a mechanism for their formation was suggested.

  18. Evidence for a Putative Impact Structure in Palm Valley, Central Australia

    Science.gov (United States)

    Hamacher, D. W.; O'Neill, C.; Buchel, A.; Britton, T. R.

    2010-07-01

    Introduction: We present evidence supporting the impact origin of a circular structure located in Palm Valley, Central Australia (24° 03' 06'' S, 132° 42' 34'' E). The ~280 m wide structure was discovered using a combination of Google Maps and a local Arrernte Aboriginal oral tradition regarding a star that fell into a waterhole called Puka in Palm Valley, Northern Territory [1][2] (see [3] for details of the discovery). Geophysical Evidence: A survey of the structure in September 2009 collected magnetic, gravity and topographic data. Geophysical modeling of the data revealed the structure has a bowl-shaped subsurface morphology, as expected for a simple impact crater. Though the structure sits within the Finke Gorge system, the models do not support an erosional origin for the structure, as no buried channels are observed. Nor does the modeling fit a volcanic origin, as the density structure at depth is consistent with fractured sandstone/sediments. Geological Evidence: One channel runs out of the crater to the south, consistent with outflow from crater-filling events, but again not with an erosional origin for the structure itself. The microstructure of rock samples collected from the site revealed the presence of planar deformation features in the quartz grains. The coincident angle of the fractures is consistent with the crystallographic fracture directions under mild-end shocks. These grains probably represent local focusing of stress as the shock wave moved through the heterogeneous grain matrix, suggesting the conditions were right for the shock pressure to locally exceed the ~7.5 GPa required to form the features, even though the bulk of the shock pressure was much less. Conclusion: Based on the level of erosion and the absence of shatter cones and meteorite fragments, we estimate the structure's age to be in the millions of years. While the presence of shocked-quartz is a direct indicator of a cosmic impact, we cannot rule out that the quartz was

  19. In vitro oligomerization and fibrillogenesis of amyloid-beta peptides.

    Science.gov (United States)

    Benseny-Cases, Núria; Klementieva, Oksana; Cladera, Josep

    2012-01-01

    frame is proposed for the interpretation of the fibril formation kinetics, intended to integrate the results from the different experimental approaches. The significance of the different aggregated species in terms of cell toxicity will be discussed. Special emphasis will be given to the influence of pH on the structural and toxic characteristics of amyloid aggregates, an aspect that may be particularly relevant in some specific physiological conditions.

  20. There's plenty of gloom at the bottom: the many challenges of accurate quantitation in size-based oligomeric separations.

    Science.gov (United States)

    Striegel, André M

    2013-11-01

    There is a variety of small-molecule species (e.g., tackifiers, plasticizers, oligosaccharides) the size-based characterization of which is of considerable scientific and industrial importance. Likewise, quantitation of the amount of oligomers in a polymer sample is crucial for the import and export of substances into the USA and European Union (EU). While the characterization of ultra-high molar mass macromolecules by size-based separation techniques is generally considered a challenge, it is this author's contention that a greater challenge is encountered when trying to perform, for quantitation purposes, separations in and of the oligomeric region. The latter thesis is expounded herein, by detailing the various obstacles encountered en route to accurate, quantitative oligomeric separations by entropically dominated techniques such as size-exclusion chromatography, hydrodynamic chromatography, and asymmetric flow field-flow fractionation, as well as by methods which are, principally, enthalpically driven such as liquid adsorption and temperature gradient interaction chromatography. These obstacles include, among others, the diminished sensitivity of static light scattering (SLS) detection at low molar masses, the non-constancy of the response of SLS and of commonly employed concentration-sensitive detectors across the oligomeric region, and the loss of oligomers through the accumulation wall membrane in asymmetric flow field-flow fractionation. The battle is not lost, however, because, with some care and given a sufficient supply of sample, the quantitation of both individual oligomeric species and of the total oligomeric region is often possible.

  1. Hsp70 oligomerization is mediated by an interaction between the interdomain linker and the substrate-binding domain.

    Directory of Open Access Journals (Sweden)

    Francesco A Aprile

    Full Text Available Oligomerization in the heat shock protein (Hsp 70 family has been extensively documented both in vitro and in vivo, although the mechanism, the identity of the specific protein regions involved and the physiological relevance of this process are still unclear. We have studied the oligomeric properties of a series of human Hsp70 variants by means of nanoelectrospray ionization mass spectrometry, optical spectroscopy and quantitative size exclusion chromatography. Our results show that Hsp70 oligomerization takes place through a specific interaction between the interdomain linker of one molecule and the substrate-binding domain of a different molecule, generating dimers and higher-order oligomers. We have found that substrate binding shifts the oligomerization equilibrium towards the accumulation of functional monomeric protein, probably by sequestering the helical lid sub-domain needed to stabilize the chaperone: substrate complex. Taken together, these findings suggest a possible role of chaperone oligomerization as a mechanism for regulating the availability of the active monomeric form of the chaperone and for the control of substrate binding and release.

  2. Hsp70 Oligomerization Is Mediated by an Interaction between the Interdomain Linker and the Substrate-Binding Domain

    Science.gov (United States)

    Aprile, Francesco A.; Dhulesia, Anne; Stengel, Florian; Roodveldt, Cintia; Benesch, Justin L. P.; Tortora, Paolo; Robinson, Carol V.; Salvatella, Xavier; Dobson, Christopher M.; Cremades, Nunilo

    2013-01-01

    Oligomerization in the heat shock protein (Hsp) 70 family has been extensively documented both in vitro and in vivo, although the mechanism, the identity of the specific protein regions involved and the physiological relevance of this process are still unclear. We have studied the oligomeric properties of a series of human Hsp70 variants by means of nanoelectrospray ionization mass spectrometry, optical spectroscopy and quantitative size exclusion chromatography. Our results show that Hsp70 oligomerization takes place through a specific interaction between the interdomain linker of one molecule and the substrate-binding domain of a different molecule, generating dimers and higher-order oligomers. We have found that substrate binding shifts the oligomerization equilibrium towards the accumulation of functional monomeric protein, probably by sequestering the helical lid sub-domain needed to stabilize the chaperone: substrate complex. Taken together, these findings suggest a possible role of chaperone oligomerization as a mechanism for regulating the availability of the active monomeric form of the chaperone and for the control of substrate binding and release. PMID:23840795

  3. Isolation and quantification of oligomeric and polymeric procyanidins in leaves and flowers of Hawthorn (Crataegus spp.).

    Science.gov (United States)

    Hellenbrand, N; Sendker, J; Lechtenberg, M; Petereit, F; Hensel, A

    2015-07-01

    Proanthocyanidins (PAs) constitute a class of polyphenols with flavan-3-ols as monomeric building blocks. These polyphenols are mostly quantified by colorimetric methods or by chromatographic determination of monomeric flavan-3-ols or low molecular oligomers as lead compounds. No reliable analytical methods are available for unambiguous identification of the homologues series of oligo- and polymeric PAs. For Hawthorn leaf and flower (Crataegi folium cum flore) from Crataegus spp. (Rosaceae) a protocol for preparative isolation of oligomeric and polymeric PAs from an acetone-water extract was developed, yielding procyanidin reference clusters with defined degree of polymerization (DP) from 2 to 10 besides a procyanidin-polymer. Identity and purity of these clusters were proven by HPLC, MS and in part NMR studies. For identification and quantification from Hawthorn an ICH-Q2 validated UHPLC method with fluorimetric detection and less than 10min runtime was developed. The method enabled quantification of procyanidin clusters with DP from 2 to 10 besides the polymer fraction. Batch analysis revealed procyanidin contents of about 20 to 45mg/g from a homologues series of oligomeric PAs and about 50% of polymer fraction. Monitoring of procyanidin distribution during seasonal growth of fresh plants of Crataegus monogyna showed more or less constant contents between 20 and 55mg/g dry weight of oligomeric procyanidins during the growing season in the different plant organs with strong accumulation in the flowers and fruits (55mg/g dry weight). From these data it can be speculated that procyanidins serve as part of the plants defense system in the reproductive organs of the plant. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. The effects of oligomerization on Saccharomyces cerevisiae Mcm4/6/7 function

    Directory of Open Access Journals (Sweden)

    Davey Megan J

    2010-09-01

    Full Text Available Abstract Background Minichromosome maintenance proteins (Mcm 2, 3, 4, 5, 6 and 7 are related by sequence and form a variety of complexes that unwind DNA, including Mcm4/6/7. A Mcm4/6/7 trimer forms one half of the Mcm2-7 hexameric ring and can be thought of as the catalytic core of Mcm2-7, the replicative helicase in eukaryotic cells. Oligomeric analysis of Mcm4/6/7 suggests that it forms a hexamer containing two Mcm4/6/7 trimers, however, under certain conditions trimeric Mcm4/6/7 has also been observed. The functional significance of the different Mcm4/6/7 oligomeric states has not been assessed. The results of such an assessment would have implications for studies of both Mcm4/6/7 and Mcm2-7. Results Here, we show that Saccharomyces cerevisiae Mcm4/6/7 reconstituted from individual subunits exists in an equilibrium of oligomeric forms in which smaller oligomers predominate in the absence of ATP. In addition, we found that ATP, which is required for Mcm4/6/7 activity, shifts the equilibrium towards larger oligomers, likely hexamers of Mcm4/6/7. ATPγS and to a lesser extent ADP also shift the equilibrium towards hexamers. Study of Mcm4/6/7 complexes containing mutations that interfere with the formation of inter-subunit ATP sites (arginine finger mutants indicates that full activity of Mcm4/6/7 requires all of its ATP sites, which are formed in a hexamer and not a trimer. In keeping with this observation, Mcm4/6/7 binds DNA as a hexamer. Conclusions The minimal functional unit of Mcm4/6/7 is a hexamer. One of the roles of ATP binding by Mcm4/6/7 may be to stabilize formation of hexamers.

  5. Isolation and identification of oligomeric procyanidins from Crataegus leaves and flowers.

    Science.gov (United States)

    Svedström, Ulla; Vuorela, Heikki; Kostiainen, Risto; Tuominen, Jari; Kokkonen, Juha; Rauha, Jussi-Pekka; Laakso, Into; Hiltunen, Raimo

    2002-08-01

    Oligomeric procyanidins were isolated from the leaves and flowers of hawthorn (Crataegus laevigata). A trimer, epicatechin-(4 beta-->8)-epicatechin-(4 beta-->6)-epicatechin, and a pentamer consisting of (-)-epicatechin units linked through C-4 beta/C-8 bonds have been isolated from hawthorn for the first time, in addition to known procyanidins including dimers B-2, B-4 and B-5, trimers C-1 and epicatechin-(4 beta-->6)-epicatechin-(4 beta-->8)-epicatechin, and tetramer D-1. A fraction containing a hexamer was also found.

  6. Thermo-mechanical characterization of a monochlorophenyl, hepta isobutyl polyhedral oligomeric silsesquioxane/polystyrene composite

    Energy Technology Data Exchange (ETDEWEB)

    Blanco, Ignazio, E-mail: iblanco@dii.unict.it; Bottino, Francesco A., E-mail: iblanco@dii.unict.it; Cicala, Gianluca, E-mail: iblanco@dii.unict.it; Cozzo, Giulia, E-mail: iblanco@dii.unict.it; Latteri, Alberta, E-mail: iblanco@dii.unict.it; Recca, Antonino, E-mail: iblanco@dii.unict.it [Department of Industrial Engineering, University of Catania, Viale A. Doria 6, 95125 Catania (Italy)

    2014-05-15

    The thermal and mechanical properties of a monochlorophenyl, hepta isobutyl Polyhedral Oligomeric Silsesquioxane/Polystyrene (ph,hib-POSS/PS) composite were studied and compared with those of pristine polymer. ph,hib-POSS/PS system was prepared by solubilization and precipitation of Polystyrene (PS) in the presence of POSS. Scanning Electron Microscopy (SEM) was performed to check the distribution of the filler in the polymer matrix. Dynamic Mechanical Analysis (DMA) was carried out to measure viscoelastic properties of solid samples. Degradations were carried out into a thermobalance and the obtained thermogravimetric (TG) and differential thermogravimetric (DTG) curves were discussed and interpreted.

  7. Esters of oligo-(glycerol carbonate-glycerol): New biobased oligomeric surfactants.

    Science.gov (United States)

    Holmiere, Sébastien; Valentin, Romain; Maréchal, Philippe; Mouloungui, Zéphirin

    2017-02-01

    Glycerol carbonate is one of the most potentially multifunction glycerol-derived compounds. Glycerol is an important by-product of the oleochemical industry. The oligomerization of glycerol carbonate, assisted by the glycerol, results in the production of polyhydroxylated oligomers rich in linear carbonate groups. The polar moieties of these oligomers (Mwesters of sorbitan polyethoxylates. The self-assembling properties of oligocarbonate esters were highlighted by their ability to stabilize inverse and multiple emulsions. The oligo-(glycerol carbonate-glycerol ether) with relatively low molecular weights showed properties of relatively high-molecular weight molecules, and constitute a viable "green" alternative to ethoxylated surfactants. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Thermo-mechanical characterization of a monochlorophenyl, hepta isobutyl polyhedral oligomeric silsesquioxane/polystyrene composite

    Science.gov (United States)

    Blanco, Ignazio; Bottino, Francesco A.; Cicala, Gianluca; Cozzo, Giulia; Latteri, Alberta; Recca, Antonino

    2014-05-01

    The thermal and mechanical properties of a monochlorophenyl, hepta isobutyl Polyhedral Oligomeric Silsesquioxane/Polystyrene (ph,hib-POSS/PS) composite were studied and compared with those of pristine polymer. ph,hib-POSS/PS system was prepared by solubilization and precipitation of Polystyrene (PS) in the presence of POSS. Scanning Electron Microscopy (SEM) was performed to check the distribution of the filler in the polymer matrix. Dynamic Mechanical Analysis (DMA) was carried out to measure viscoelastic properties of solid samples. Degradations were carried out into a thermobalance and the obtained thermogravimetric (TG) and differential thermogravimetric (DTG) curves were discussed and interpreted.

  9. Polyhedral oligomeric silsesquioxanes/carbon nanotube/carbon fiber multiscale composite: Influence of a novel hierarchical reinforcement on the interfacial properties

    Science.gov (United States)

    Zhang, R. L.; Wang, C. G.; Liu, L.; Cui, H. Z.; Gao, B.

    2015-10-01

    A novel hierarchical reinforcing carbon fiber through co-grafting carbon nanotube (CNTs) and polyhedral oligomeric silsesquioxanes (POSS) was prepared in this paper. The structure and surface characteristics of the grafted carbon fiber were investigated by Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), thermogravimetry (TG) and scanning electron microscope (SEM), respectively. The surface energy and the functional groups of the carbon fiber surface were increased obviously after modification. The ILSS results showed that there was a remarkable improvement in the interfacial properties of the new hybrid CF-CNTs-POSS composites. The investigation can prove an effective way to increase the interfacial adhesion and improve the mechanical performance of the fiber/resin composites on the desired application.

  10. A complementary electrochromic device based on polyaniline tethered polyhedral oligomeric silsesquioxane and poly(3,4-ethylenedioxythiophene)/poly(4-styrene sulfonic acid)

    Energy Technology Data Exchange (ETDEWEB)

    Xiong, Shanxin [Temasek Laboratories, Nanyang Technological University, 50 Nanyang Drive, Singapore 637553 (Singapore); Ma, Jan; Lu, Xuehong [Temasek Laboratories, Nanyang Technological University, 50 Nanyang Drive, Singapore 637553 (Singapore); School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798 (Singapore)

    2009-12-15

    A high-contrast complementary electrochromic device based on polyaniline (PANI) tethered polyhedral oligomeric silsesquioxane (POSS) (POSS-PANI) and poly(3,4-ethylenedioxythiophene)/poly(4-styrene sulfonic acid) (PEDOT:PSS) is assembled. The electrochromic properties, cyclic voltammetry behavior and coloration efficiency of the device are studied. Due to the loosely packed structure, POSS-PANI gives rise to a significantly higher electrochromic contrast, coloration efficiency and faster switching speed than PANI. Despite its high contrast, the combination of POSS-PANI with PEDOT:PSS still shows synergy in terms of contrast enhancement, which can be attributed to the additional driving force for the diffusion of dopants into PEDOT:PSS provided by the dedoping of POSS-PANI. (author)

  11. Small Angle X-Ray Scattering Studies of Mitochondrial Glutaminase C Reveal Extended Flexible Regions, and Link Oligomeric State with Enzyme Activity

    DEFF Research Database (Denmark)

    Møller, M.; Nielsen, Søren Skou; Ramachandran, Siddharth;

    2013-01-01

    Glutaminase C is a key metabolic enzyme, which is unregulated in many cancer systems and believed to play a central role in the Warburg effect, whereby cancer cells undergo changes to an altered metabolic profile. A long-standing hypothesis links enzymatic activity to the protein oligomeric state......, hence the study of the solution behavior in general and the oligomer state in particular of glutaminase C is important for the understanding of the mechanism of protein activation and inhibition. In this report, this is extensively investigated in correlation to enzyme concentration or phosphate level...... state and investigates the C-terminal influence on the enzyme solution behavior. Our data enable SAXS-based rigid body modeling of the full-length tetramer states, thereby presenting the first ever experimentally derived structural model of mitochondrial glutaminase C including the N- and C...

  12. A Rapid One-Pot Synthesis of Novel High-Purity Methacrylic Phosphonic Acid (PA-Based Polyhedral Oligomeric Silsesquioxane (POSS Frameworks via Thiol-Ene Click Reaction

    Directory of Open Access Journals (Sweden)

    K. Karuppasamy

    2017-05-01

    Full Text Available Herein, we demonstrate a facile methodology to synthesis a novel methacrylic phosphonic acid (PA-functionalized polyhedral oligomeric silsesquioxanes (POSSs via thiol-ene click reaction using octamercapto thiol-POSS and ethylene glycol methacrylate phosphate (EGMP monomer. The presence of phosphonic acid moieties and POSS-cage structure in POSS-S-PA was confirmed by Fourier transform infrared (FT-IR and nuclear magnetic resonance (1H, 29Si and 31P-NMR analyses. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF mass spectrum of POSS-S-PA acquired in a dithranol matrix, which has specifically designed for intractable polymeric materials. The observed characterization results signposted that novel organo-inorganic hybrid POSS-S-PA would be an efficacious material for fuel cells as a proton exchange membrane and high-temperature applications due to its thermal stability of 380 °C.

  13. Biocomputational analysis of evolutionary relationship between toll-like receptor and nucleotide-binding oligomerization domain-like receptors genes

    Directory of Open Access Journals (Sweden)

    Rabia Bhardwaj

    2016-11-01

    Full Text Available Aim: The active domains (TIR and NACHT of the pattern recognition receptors (PRRs: Toll-like receptors [TLRs] and nucleotide-binding oligomerization domain [NOD]-like receptors [NLR], respectively are the major hotspots of evolution as natural selection has crafted their final structure by substitution of residues over time. This paper addresses the evolutionary perspectives of the TLR and NLR genes with respect to the active domains in terms of their chronological fruition, functional diversification, and species-specific stipulation. Materials and Methods: A total of 48 full-length cds (and corresponding peptide of the domains were selected as representatives of each type of PRRs, belonging to divergent animal species, for the biocomputational analyses. The secondary and tertiary structure of the taurine TIR and NACHT domains was predicted to compare the relatedness among the domains under study. Results: Multiple sequence alignment and phylogenetic tree results indicated that these host-specific PRRs formed entirely different clusters, with active domains of NLRs (NACHT evolved earlier as compared to the active domains of TLRs (TIR. Each type of TLR or NLR shows comparatively less variation among the animal species due to the specificity of action against the type of microbes. Conclusion: It can be concluded from the study that there has been no positive selection acting on the domains associated with disease resistance which is a fitness trait indicating the extent of purifying pressure on the domains. Gene duplication could be a possible reason of genesis of similar kinds of TLRs (virus or bacteria specific.

  14. Biocomputational analysis of evolutionary relationship between toll-like receptor and nucleotide-binding oligomerization domain-like receptors genes

    Science.gov (United States)

    Bhardwaj, Rabia; Mukhopadhyay, Chandra Shekhar; Deka, Dipak; Verma, Ramneek; Dubey, P. P.; Arora, J. S.

    2016-01-01

    Aim: The active domains (TIR and NACHT) of the pattern recognition receptors (PRRs: Toll-like receptors [TLRs] and nucleotide-binding oligomerization domain [NOD]-like receptors [NLR], respectively) are the major hotspots of evolution as natural selection has crafted their final structure by substitution of residues over time. This paper addresses the evolutionary perspectives of the TLR and NLR genes with respect to the active domains in terms of their chronological fruition, functional diversification, and species-specific stipulation. Materials and Methods: A total of 48 full-length cds (and corresponding peptide) of the domains were selected as representatives of each type of PRRs, belonging to divergent animal species, for the biocomputational analyses. The secondary and tertiary structure of the taurine TIR and NACHT domains was predicted to compare the relatedness among the domains under study. Results: Multiple sequence alignment and phylogenetic tree results indicated that these host-specific PRRs formed entirely different clusters, with active domains of NLRs (NACHT) evolved earlier as compared to the active domains of TLRs (TIR). Each type of TLR or NLR shows comparatively less variation among the animal species due to the specificity of action against the type of microbes. Conclusion: It can be concluded from the study that there has been no positive selection acting on the domains associated with disease resistance which is a fitness trait indicating the extent of purifying pressure on the domains. Gene duplication could be a possible reason of genesis of similar kinds of TLRs (virus or bacteria specific). PMID:27956772

  15. Zinc ion conducting polymer electrolytes based on oligomeric polyether/PVDF-HFP blends

    Science.gov (United States)

    Ye, Hui; Xu, Jun John

    Here we report novel zinc ion conducting polymer electrolytes based on oligomeric polyether/PVDF-HFP blends with or without the incorporation of a small amount of organic carbonates. Their thermal properties, ionic conductivity and electrochemical properties are characterized and the effect of different Zn salts and incorporation of a small amount of organic carbonates are investigated. These polymer electrolyte membranes exhibit essentially no or very low volatility, high thermal stability, high ionic conductivity, wide electrochemical stability window, acceptable interfacial resistance with zinc, and the capability for reversible Zn plating/stripping. Particularly promising are electrolyte systems based on the combination of low lattice energy zinc imide salt and a special co-solvent of oligomeric poly(ethylene glycol) dimethyl ether (PEGDME) mixed with a small amount of ethylene carbonate (EC), dimensionally stabilized with PVDF-HFP. Such novel polymer electrolyte membranes could lead to the development of new kinds of electrochemical energy storage devices based on zinc electrochemistry, including solid-state, thin-film rechargeable zinc/air cells envisaged.

  16. Zinc ion conducting polymer electrolytes based on oligomeric polyether/PVDF-HFP blends

    Energy Technology Data Exchange (ETDEWEB)

    Ye, Hui; Xu, Jun John [Department of Materials Science and Engineering, Rutgers, the State University of New Jersey, Piscataway, NJ 08854 (United States)

    2007-03-20

    Here we report novel zinc ion conducting polymer electrolytes based on oligomeric polyether/PVDF-HFP blends with or without the incorporation of a small amount of organic carbonates. Their thermal properties, ionic conductivity and electrochemical properties are characterized and the effect of different Zn salts and incorporation of a small amount of organic carbonates are investigated. These polymer electrolyte membranes exhibit essentially no or very low volatility, high thermal stability, high ionic conductivity, wide electrochemical stability window, acceptable interfacial resistance with zinc, and the capability for reversible Zn plating/stripping. Particularly promising are electrolyte systems based on the combination of low lattice energy zinc imide salt and a special co-solvent of oligomeric poly(ethylene glycol) dimethyl ether (PEGDME) mixed with a small amount of ethylene carbonate (EC), dimensionally stabilized with PVDF-HFP. Such novel polymer electrolyte membranes could lead to the development of new kinds of electrochemical energy storage devices based on zinc electrochemistry, including solid-state, thin-film rechargeable zinc/air cells envisaged. (author)

  17. Oligomerization of SCFTIR1 Is Essential for Aux/IAA Degradation and Auxin Signaling in Arabidopsis.

    Science.gov (United States)

    Dezfulian, Mohammad H; Jalili, Espanta; Roberto, Don Karl A; Moss, Britney L; Khoo, Kerry; Nemhauser, Jennifer L; Crosby, William L

    2016-09-01

    The phytohormone auxin is a key regulator of plant growth and development. Molecular studies in Arabidopsis have shown that auxin perception and signaling is mediated via TIR1/AFB-Aux/IAA co-receptors that assemble as part of the SCFTIR1/AFB E3 ubiquitin-ligase complex and direct the auxin-regulated degradation of Aux/IAA transcriptional repressors. Despite the importance of auxin signaling, little is known about the functional regulation of the TIR1/AFB receptor family. Here we show that TIR1 can oligomerize in planta via a set of spatially clustered amino acid residues. While none of the residues identified reside in the interaction interface of the TIR1-Aux/IAA degron, they nonetheless regulate the binding of TIR1 to Aux/IAA substrate proteins and their subsequent degradation in vivo as an essential aspect of auxin signaling. We propose oligomerization of TIR1 as a novel regulatory mechanism in the regulation of auxin-mediated plant patterning and development.

  18. Glycation of Wild-Type Apomyoglobin Induces Formation of Highly Cytotoxic Oligomeric Species.

    Science.gov (United States)

    Iannuzzi, Clara; Carafa, Vincenzo; Altucci, Lucia; Irace, Gaetano; Borriello, Margherita; Vinciguerra, Roberto; Sirangelo, Ivana

    2015-11-01

    Protein glycation is a non-enzymatic, irreversible modification of protein amino groups by reactive carbonyl species leading to the formation of advanced glycation end products (AGEs). Several proteins implicated in neurodegenerative diseases have been found to be glycated in vivo and the extent of glycation is related to the pathologies of the patients. Although it is now accepted that there is a direct correlation between AGEs formation and the development of neurodegenerative diseases related to protein misfolding and amyloid aggregation, several questions still remain unanswered: whether glycation is the triggering event or just an additional factor acting on the aggregation pathway. We have recently shown that glycation of the amyloidogenic W7FW14F apomyoglobin mutant significantly accelerates the amyloid fibrils formation providing evidence that glycation actively participates to the process. In the present study, to test if glycation can be considered also a triggering factor in amyloidosis, we evaluated the ability of different glycation agents to induce amyloid aggregation in the soluble wild-type apomyoglobin. Our results show that glycation covalently modifies apomyoglobin and induces conformational changes that lead to the formation of oligomeric species that are not implicated in amyloid aggregation. Thus, AGEs formation does not trigger amyloid aggregation in the wild-type apomyoglobin but only induce the formation of soluble oligomeric species able to affect cell viability. The molecular bases of cell toxicity induced by AGEs formed upon glycation of wild-type apomyoglobin have been also investigated.

  19. Protein Cross-Linking and Oligomerization through Dityrosine Formation upon Exposure to Ozone.

    Science.gov (United States)

    Kampf, Christopher J; Liu, Fobang; Reinmuth-Selzle, Kathrin; Berkemeier, Thomas; Meusel, Hannah; Shiraiwa, Manabu; Pöschl, Ulrich

    2015-09-15

    Air pollution is a potential driver for the increasing prevalence of allergic disease, and post-translational modification by air pollutants can enhance the allergenic potential of proteins. Here, the kinetics and mechanism of protein oligomerization upon ozone (O3) exposure were studied in coated-wall flow tube experiments at environmentally relevant O3 concentrations, relative humidities and protein phase states (amorphous solid, semisolid, and liquid). We observed the formation of protein dimers, trimers, and higher oligomers, and attribute the cross-linking to the formation of covalent intermolecular dityrosine species. The oligomerization proceeds fast on the surface of protein films. In the bulk material, reaction rates are limited by diffusion depending on phase state and humidity. From the experimental data, we derive a chemical mechanism and rate equations for a kinetic multilayer model of surface and bulk reaction enabling the prediction of oligomer formation. Increasing levels of tropospheric O3 in the Anthropocene may promote the formation of protein oligomers with enhanced allergenicity and may thus contribute to the increasing prevalence of allergies.

  20. Quercetin Inhibits Inflammasome Activation by Interfering with ASC Oligomerization and Prevents Interleukin-1 Mediated Mouse Vasculitis

    Science.gov (United States)

    Domiciano, Talita P.; Wakita, Daiko; Jones, Heather D.; Crother, Timothy R.; Verri, Waldiceu A.; Arditi, Moshe; Shimada, Kenichi

    2017-01-01

    Interleukin-1β (IL-1β) is a highly inflammatory cytokine that significantly contributes to both acute and chronic inflammatory diseases. The secretion of IL-1β requires a unique protease, caspase-1, which is activated by various protein platforms called inflammasomes. Data suggests a key role for mitochondrial reactive oxygen species for inflammasome activation. Flavonoids constitute a group of naturally occurring polyphenolic molecules with many biological activities, including antioxidant effects. In this study, we investigated the effect of three flavonoids, quercetin (QUC), naringenin, and silymarim on inflammasome activation. We found that QUC inhibits IL-1β secretion by both the NLRP3 and AIM2 inflammasome in a dose dependent manner, but not the NLRC4 inflammasome. QUC inhibition of the inflammasome was still observed in Atg16l1 knockout macrophages, indicating that QUC’s effect was autophagy independent. Since QUC inhibited both NLRP3 and AIM2 inflammasomes but not NLRC4, we assessed ASC speck formation. QUC reduced ASC speck formation and ASC oligomerization compared with controls. Additionally, QUC inhibited IL-1β in Cryopyrin-Associated Periodic Syndromes (CAPS) macrophages, where NLRP3 inflammasome is constitutively activated. In conclusion, QUC inhibits both the NLRP3 and AIM2 inflammasome by preventing ASC oligomerization and may be a potential therapeutic candidate for Kawasaki disease vasculitis and other IL-1 mediated inflammatory diseases. PMID:28148962

  1. Integration and oligomerization of Bax protein in lipid bilayers characterized by single molecule fluorescence study.

    Science.gov (United States)

    Luo, Lu; Yang, Jun; Liu, Dongxiang

    2014-11-14

    Bax is a pro-apoptotic Bcl-2 family protein. The activated Bax translocates to mitochondria, where it forms pore and permeabilizes the mitochondrial outer membrane. This process requires the BH3-only activator protein (i.e. tBid) and can be inhibited by anti-apoptotic Bcl-2 family proteins such as Bcl-xL. Here by using single molecule fluorescence techniques, we studied the integration and oligomerization of Bax in lipid bilayers. Our study revealed that Bax can bind to lipid membrane spontaneously in the absence of tBid. The Bax pore formation undergoes at least two steps: pre-pore formation and membrane insertion. The activated Bax triggered by tBid or BH3 domain peptide integrates on bilayers and tends to form tetramers, which are termed as pre-pore. Subsequent insertion of the pre-pore into membrane is highly dependent on the composition of cardiolipin in lipid bilayers. Bcl-xL can translocate Bax from membrane to solution and inhibit the pore formation. The study of Bax integration and oligomerization at the single molecule level provides new evidences that may help elucidate the pore formation of Bax and its regulatory mechanism in apoptosis. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Quercetin Inhibits Inflammasome Activation by Interfering with ASC Oligomerization and Prevents Interleukin-1 Mediated Mouse Vasculitis.

    Science.gov (United States)

    Domiciano, Talita P; Wakita, Daiko; Jones, Heather D; Crother, Timothy R; Verri, Waldiceu A; Arditi, Moshe; Shimada, Kenichi

    2017-02-02

    Interleukin-1β (IL-1β) is a highly inflammatory cytokine that significantly contributes to both acute and chronic inflammatory diseases. The secretion of IL-1β requires a unique protease, caspase-1, which is activated by various protein platforms called inflammasomes. Data suggests a key role for mitochondrial reactive oxygen species for inflammasome activation. Flavonoids constitute a group of naturally occurring polyphenolic molecules with many biological activities, including antioxidant effects. In this study, we investigated the effect of three flavonoids, quercetin (QUC), naringenin, and silymarim on inflammasome activation. We found that QUC inhibits IL-1β secretion by both the NLRP3 and AIM2 inflammasome in a dose dependent manner, but not the NLRC4 inflammasome. QUC inhibition of the inflammasome was still observed in Atg16l1 knockout macrophages, indicating that QUC's effect was autophagy independent. Since QUC inhibited both NLRP3 and AIM2 inflammasomes but not NLRC4, we assessed ASC speck formation. QUC reduced ASC speck formation and ASC oligomerization compared with controls. Additionally, QUC inhibited IL-1β in Cryopyrin-Associated Periodic Syndromes (CAPS) macrophages, where NLRP3 inflammasome is constitutively activated. In conclusion, QUC inhibits both the NLRP3 and AIM2 inflammasome by preventing ASC oligomerization and may be a potential therapeutic candidate for Kawasaki disease vasculitis and other IL-1 mediated inflammatory diseases.

  3. The oligomeric Rep protein of Mungbean yellow mosaic India virus (MYMIV) is a likely replicative helicase.

    Science.gov (United States)

    Choudhury, Nirupam Roy; Malik, Punjab Singh; Singh, Dharmendra Kumar; Islam, Mohammad Nurul; Kaliappan, Kosalai; Mukherjee, Sunil Kumar

    2006-01-01

    Geminiviruses replicate by rolling circle mode of replication (RCR) and the viral Rep protein initiates RCR by the site-specific nicking at a conserved nonamer (TAATATT downward arrow AC) sequence. The mechanism of subsequent steps of the replication process, e.g. helicase activity to drive fork-elongation, etc. has largely remained obscure. Here we show that Rep of a geminivirus, namely, Mungbean yellow mosaic India virus (MYMIV), acts as a replicative helicase. The Rep-helicase, requiring > or =6 nt space for its efficient activity, translocates in the 3'-->5' direction, and the presence of forked junction in the substrate does not influence the activity to any great extent. Rep forms a large oligomeric complex and the helicase activity is dependent on the oligomeric conformation ( approximately 24mer). The role of Rep as a replicative helicase has been demonstrated through ex vivo studies in Saccharomyces cerevisiae and in planta analyses in Nicotiana tabacum. We also establish that such helicase activity is not confined to the MYMIV system alone, but is also true with at least two other begomoviruses, viz., Mungbean yellow mosaic virus (MYMV) and Indian cassava mosaic virus (ICMV).

  4. Lipid-II Independent Antimicrobial Mechanism of Nisin Depends On Its Crowding And Degree Of Oligomerization

    Science.gov (United States)

    Prince, Ashutosh; Sandhu, Padmani; Kumar, Pankaj; Dash, Eva; Sharma, Shingarika; Arakha, Manoranjan; Jha, Suman; Akhter, Yusuf; Saleem, Mohammed

    2016-11-01

    Nisin inhibits bacterial growth by generating pores in cell membrane and interrupting cell-wall biosynthesis through specific lipid II interaction. However, the role of the hinge region and C-terminus residues of the peptide in antibacterial action of nisin is largely unknown. Here, using molecular dynamics simulations and experimental approach, we report that at high concentration regimes of nisin, interaction with phospholipids may equally deform the bacterial cell membranes even under significantly varying amounts of lipid-II. Membrane thinning, destabilization and decrease in lipid density depend on the degree of oligomerization of nisin. Growth kinetics of Bacillus subtilis and Escherichia coli interestingly show recovery by extended lag phase under low concentrations of nisin treatment while high concentrations of nisin caused decrease in cell viability as recorded by striking reduction in membrane potential and surface area. The significant changes in the dipole potential and fluorescence anisotropy were observed in negatively charged membranes in the absence of lipid-II with increasing concentration of nisin. The identical correlation of cell viability, membrane potential dissipation and morphology with the concentration regime of nisin, in both Bacillus subtilis (lipid II rich) and Escherichia coli (lipid II impoverished), hints at a non-specific physical mechanism where degree of membrane deformation depends on degree of crowding and oligomerization of nisin.

  5. Evolutionary trace-based peptides identify a novel asymmetric interaction that mediates oligomerization in nuclear receptors.

    Science.gov (United States)

    Gu, Peili; Morgan, Daniel H; Sattar, Minawar; Xu, Xueping; Wagner, Ryan; Raviscioni, Michele; Lichtarge, Olivier; Cooney, Austin J

    2005-09-01

    Germ cell nuclear factor (GCNF) is an orphan nuclear receptor that plays important roles in development and reproduction, by repressing the expression of essential genes such as Oct4, GDF9, and BMP15, through binding to DR0 elements. Surprisingly, whereas recombinant GCNF binds to DR0 sequences as a homodimer, endogenous GCNF does not exist as a homodimer but rather as part of a large complex termed the transiently retinoid-induced factor (TRIF). Here, we use evolutionary trace (ET) analysis to design mutations and peptides that probe the molecular basis for the formation of this unusual complex. We find that GCNF homodimerization and TRIF complex formation are DNA-dependent, and ET suggests that dimerization involves key functional sites on both helix 3 and helix 11, which are located on opposing surfaces of the ligand binding domain. Targeted mutations in either helix of GCNF disrupt the formation of both the homodimer and the endogenous TRIF complex. Moreover, peptide mimetics of both of these ET-determined sites inhibit dimerization and TRIF complex formation. This suggests that a novel helix 3-helix 11 heterotypic interaction mediates GCNF interaction and would facilitate oligomerization. Indeed, it was determined that the endogenous TRIF complex is composed of a GCNF oligomer. These findings shed light on an evolutionarily selected mechanism that reveals the unusual DNA-binding, dimerization, and oligomerization properties of GCNF.

  6. Apolipoprotein E, especially apolipoprotein E4, increases the oligomerization of amyloid β peptide.

    Science.gov (United States)

    Hashimoto, Tadafumi; Serrano-Pozo, Alberto; Hori, Yukiko; Adams, Kenneth W; Takeda, Shuko; Banerji, Adrian Olaf; Mitani, Akinori; Joyner, Daniel; Thyssen, Diana H; Bacskai, Brian J; Frosch, Matthew P; Spires-Jones, Tara L; Finn, Mary Beth; Holtzman, David M; Hyman, Bradley T

    2012-10-24

    Alzheimer's disease (AD) is the most common progressive neurodegenerative disorder causing dementia. Massive deposition of amyloid β peptide (Aβ) as senile plaques in the brain is the pathological hallmark of AD, but oligomeric, soluble forms of Aβ have been implicated as the synaptotoxic component. The apolipoprotein E ε 4 (apoE ε4) allele is known to be a genetic risk factor for developing AD. However, it is still unknown how apoE impacts the process of Aβ oligomerization. Here, we found that the level of Aβ oligomers in APOE ε4/ε4 AD patient brains is 2.7 times higher than those in APOE ε3/ε3 AD patient brains, matched for total plaque burden, suggesting that apoE4 impacts the metabolism of Aβ oligomers. To test this hypothesis, we examined the effect of apoE on Aβ oligomer formation. Using both synthetic Aβ and a split-luciferase method for monitoring Aβ oligomers, we observed that apoE increased the level of Aβ oligomers in an isoform-dependent manner (E2 apoE4, increases Aβ oligomers in the brain. Higher levels of Aβ oligomers in the brains of APOE ε4/ε4 carriers compared with APOE ε3/ε3 carriers may increase the loss of dendritic spines and accelerate memory impairments, leading to earlier cognitive decline in AD.

  7. Phosphate and HEPES buffers potently affect the fibrillation and oligomerization mechanism of Alzheimer's Aβ peptide.

    Science.gov (United States)

    Garvey, Megan; Tepper, Katharina; Haupt, Caroline; Knüpfer, Uwe; Klement, Karolin; Meinhardt, Jessica; Horn, Uwe; Balbach, Jochen; Fändrich, Marcus

    2011-06-10

    The oligomerization of Aβ peptide into amyloid fibrils is a hallmark of Alzheimer's disease. Due to its biological relevance, phosphate is the most commonly used buffer system for studying the formation of Aβ and other amyloid fibrils. Investigation into the characteristics and formation of amyloid fibrils frequently relies upon material formed in vitro, predominantly in phosphate buffers. Herein, we examine the effects on the fibrillation and oligomerization mechanism of Aβ peptide that occur due solely to the influence of phosphate buffer. We reveal that significant differences in amyloid fibrillation are observed due to fibrillation being initiated in phosphate or HEPES buffer (at physiological pH and temperature). Except for the differing buffer ions, all experimental parameters were kept constant. Fibril formation was assessed using fluorescently monitored kinetic studies, microscopy, X-ray fiber diffraction and infrared and nuclear magnetic resonance spectroscopies. Based on this set up, we herein reveal profound effects on the mechanism and speed of Aβ fibrillation. The three histidine residues at positions 6, 13 and 14 of Aβ(1-40) are instrumental in these mechanistic changes. We conclude that buffer plays a more significant role in fibril formation than has been generally acknowledged.

  8. Characterization of oligomeric procyanidins and identification of quercetin glucuronide from lotus ( Nelumbo nucifera Gaertn.) seedpod.

    Science.gov (United States)

    Xiao, Jun-Song; Xie, Bi-Jun; Cao, Yan-Ping; Wu, Hua; Sun, Zhi-Da; Xiao, Di

    2012-03-21

    Procyanidins are a class of polyphenols in the plant kingdom. Lotus ( Nelumbo nucifera Gaertn.) seedpods, the inedible part of lotus and a byproduct during the production of lotus seeds, were found to be a new source rich in procyanidins. Detailed information about oligomeric procyanidins in lotus seedpods remains unknown. In this study, lotus seedpods were extracted using 60% aqueous methanol and characterized with phloroglucinolysis and liquid chromatography (mass spectrometry with an electrospray ionization source). The results indicate that the oligomeric and polymeric fraction had a mean degree of polymerization of 3.2 and 15.4, respectively, and consisted of (+)-catechin (m/z 289), gallocatechin or epigallocatechin (m/z 305), quercetin glycoside (m/z 463), quercetin glucuronide (m/z 477), procyanidin dimers (m/z 577.1), proanthocyanidin dimer gallate (m/z 593.3), prodelphinidin dimers (m/z 609.1), procyanidin trimers (m/z 865.1), etc. Quercetin glucuronide was further purified using flash chromatography and identified as quercetin-3-O-β-glucuronide by determining its exact mass using ion-trap time-of-flight mass spectrometry and ¹H and ¹³C nuclear magnetic resonance, ¹H-detected heteronuclear single-quantum coherence, and ¹H-detected heteronuclear multiple-bond correlation analyses.

  9. Dealuminated ZSM—5 Zeolite Catalyst for Ethylene Oligomerization to Liquid Fuels

    Institute of Scientific and Technical Information of China (English)

    NorAishahSaidinaAmin; DidiDwiAnggoro

    2002-01-01

    Ethylene oligomerization using ZSM-5 zeolite was investigated to study the role of Broensted acid sites in the formation of higher hydrocarbons,The oligomeriztion of olefins,dependent on the acidity of ZSM-5 zeolite ,is an important step in the conversion of natural gas to liquied fuels,The framework Si/Al ratio reflects the number of potential acid sites and the acid strength of the ZSM-5 catalyst,ZSM-5 with the mole ratio SiO2/Al2O3 equal to 30 was dealuminated for different periods of time according to the acidic ion-exchange method to produce ZSM-5 with various Si/Al ratios,The FT-IR analysis revealed that the integrated framework aluminum band,non-framework aluminum band,and silanol groups areas of the ZSM-5 zeolites decreased after being dealuminated,The performanc of the dealuminated zeolite was tested for ethylene oligomerization.The results demonstrated that the dealumination of ZSM-5 led to higher ethylene conversion,but the gasoline selectivity was reduced compared to the performance of a ZSM-5 zeolite ,The characterization results revealed the amount of aluminum in the zeolitic framework,the crystallinity of the ZSM-5 zeolite,and the Si/Al ration affected the formation of Broensted acid sites,The number of the Broensted acid sites on the catalyst active sites is important in the olefin conversion to liquied hydrocarbons.

  10. Oligomerization of Glycine and Alanine Catalyzed by Iron Oxides: Implications for Prebiotic Chemistry

    Science.gov (United States)

    Shanker, Uma; Bhushan, Brij; Bhattacharjee, G.; Kamaluddin

    2012-02-01

    Iron oxide minerals are probable constituents of the sediments present in geothermal regions of the primitive earth. They might have adsorbed different organic monomers (amino acids, nucleotides etc.) and catalyzed polymerization processes leading to the formation of the first living cell. In the present work we tested the catalytic activity of three forms of iron oxides (Goethite, Akaganeite and Hematite) in the intermolecular condensation of each of the amino acids glycine and L-alanine. The effect of zinc oxide and titanium dioxide on the oligomerization has also been studied. Oligomerization studies were performed for 35 days at three different temperatures 50, 90 and 120°C without applying drying/wetting cycling. The products formed were characterized by HPLC and ESI-MS techniques. All three forms of iron oxides catalyzed peptide bond formation (23.2% of gly2 and 10.65% of ala2). The reaction was monitored every 7 days. Formation of peptides was observed to start after 7 days at 50°C. Maximum yield of peptides was found after 35 days at 90°C. Reaction at 120°C favors formation of diketopiperazine derivatives. It is also important to note that after 35 days of reaction, goethite produced dimer and trimer with the highest yield among the oxides tested. We suggest that the activity of goethite could probably be due to its high surface area and surface acidity.

  11. Development of a fast atom bombardment tandem mass spectrometric screening method for alkyl-ended oligomeric biguanides

    Science.gov (United States)

    Monaghan, J. J.; Morden, W. E.

    1992-12-01

    The FAB-MS and FAB-MS---MS behaviour of a series of "alkyl-ended" oligomeric biguanides has been studied. MS---MS product ion scans showed that fragmentation of these species occurred by predictable cleavages of the biguanide chains. Results are presented from a number of MS---MS precursor ion and neutral loss scans studied in an attempt to develop a screening method for biguanides in complex mixtures. The most effective of these scans is shown to be the neutral loss of the N-alkylguanidine molecule. Use of this scan is demonstrated for a mixture of oligomeric biguanides.

  12. Catalysis of the Oligomerization of O-Phospho-Serine, Aspartic Acid, or Glutamic Acid by Cationic Micelles

    Science.gov (United States)

    Boehler, Christof; Hill, Aubrey R., Jr.; Orgel, Leslie E.

    1996-01-01

    Treatment of relatively concentrated aqueous solutions of O-phospho-serine (50 mM), aspartic acid (100 mM) or glutamic acid (100 mM) with carbonyldiimidazole leads to the formation of an activated intermediate that oligomerizes efficiently. When the concentration of amino acid is reduced tenfold, few long oligomers can be detected. Positively-charged cetyltrimethyl ammonium bromide micelles concentrate the negatively-charged activated intermediates of the amino acids at their surfaces and catalyze efficient oligomerization even from dilute solutions.

  13. Influence of point mutations on the stability, dimerization and oligomerization of human cystatin C and its L68Q variant

    Directory of Open Access Journals (Sweden)

    Aneta eSzymanska

    2012-07-01

    Full Text Available Human cystatin C (hCC is a small but very intriguing protein. Produced by all nucleated cells is found in almost all tissues and body fluids where, at physiological conditions, plays a role of a very potent inhibitor of cysteine proteases. Biologically active hCC is a monomeric protein but during cellular trafficking it forms dimers, transiently loosing its inhibitory activity. In vitro, dimerization of cystatin C was observed for the mature protein during crystallization trials, revealing that the mechanism of this process is based on the three dimensional swapping of the protein domains. In our work we have focused on the impact of two proposed hot spots in cystatin C structure on its conformational stability. Encouraged by promising results of the theoretical calculations, we designed and produced several hCC hinge region point mutation variants that display a variety of conformational stability and propensity for dimerization and aggregation. A similar approach, i.e. rational mutagenesis, has been also applied to study the amyloidogenic L68Q variant to determine the contribution of hydrophobic interactions and steric effect on the stability of monomeric cystatin C. In this overview we would like to summarize the results of our studies. The impact of a particular mutation on the properties of the studied proteins will be presented in the context of their thermal and mechanical stability, in vitro dimerization tendency as well as the outcome of crystallization. Better understanding of the mechanism and, especially, factors affecting conformational stability of cystatin C and access to stable monomeric and dimeric versions of the protein opens new perspectives in explaining the role of dimers and the domain swapping process in hCC oligomerization, as well as designing potential inhibitors of this process.

  14. Punching Holes in Membranes: How Oligomeric Pore-Forming Proteins and Lipids Cooperate to Form Aqueous Channels in Membranes

    Science.gov (United States)

    Fradin, Cécile; Satsoura, Dmitri; Andrews, David W.

    Many important biological processes are carried out by a small number of proteins working together as a team to accomplish a specific task. Cooperation between the different proteins is often accomplished through the formation of a supramolecular complex, comprised of either identical or different subunits. Although the formation of protein assemblies is a favored mechanism throughout the cell, it becomes especially important in lipid membranes, as evidenced by the numerous cellular events that are either triggered by or result in the formation of protein complexes in membranes. However, due to the difficulties associated with the study of membrane proteins, the formation of oligomers in lipid membranes is perhaps one of the least understood cellular processes. In this chapter we focus our attention on a subset of membrane complexes — namely, those formed by proteins that are able to pass from a water-soluble to a transmembrane form in order to create a water-filled channel through the lipid membrane. These pore-forming proteins (PFPs) are found in many organisms throughout different kingdoms of life, from bacteria to human. They are often involved in cell death mechanisms through their capacity to break membrane permeability barriers, which can lead to dissipation of the membrane potential as well as introduction or leakage of enzymatic proteins. In fact, a large subset of the PFPs are toxins, and referred to in the literature as pore-forming toxins (PFTs). The association of several monomers into an oligomer is almost always an important aspect of the modus operandi of these proteins. Oligomerization can be useful in several ways: it results in structures large enough to delineate nanometer-size water-filled channels in lipid bilayers, it ensures the presence of large hydrophobic surfaces that can support insertion in the membrane, and it permits cooperative formation and insertion mechanisms.

  15. Cobalt bis(dicarbollides)(1-) covalently attached to the calyx[4]arene platform: the first combination of organic bowl-shaped matrices and inorganic metallaborane cluster anions

    NARCIS (Netherlands)

    Grüner, Bohumír; Mikulasek, Libor; Baca, Jirí; Cisarova, Ivana; Böhmer, Volker; Danila, Crenguta; Reinoso-Garcia, Marta M.; Verboom, Willem; Reinhoudt, David N.; Casnati, Alessandro; Ungaro, Rocco

    2005-01-01

    Various calix[4]arene and resorc[4]arene ionic compounds substituted by cobalt bis(dicarbollide) anions (1) have been prepared for the first time. From tBu-calix[4]arene (A) the complete series of mono-, di-, tri- and tetrasubstituted derivatives bearing one to four cluster anions on the lower rim (

  16. Thiophene-fused bowl-shaped polycyclic aromatics with a dibenzo[a,g]corannulene core for organic field-effect transistors.

    Science.gov (United States)

    Lu, Ru-Qiang; Zhou, Yi-Nyu; Yan, Xiao-Yun; Shi, Ke; Zheng, Yu-Qing; Luo, Ming; Wang, Xin-Chang; Pei, Jian; Xia, Haiping; Zoppi, Laura; Baldridge, Kim K; Siegel, Jay S; Cao, Xiao-Yu

    2015-01-31

    For the first time, electron-rich thiophene units were fused into the skeleton of corannulene to extend π-surfaces and tune arrangement in single crystals. Two isomeric butterfly-like thiophene-fused dibenzo[a,g]corannulenes (3 and 5) were synthesized. Isomer 3 showed p-type transport properties, with a hole mobility of 0.06 cm(2) V(-1) s(-1).

  17. Styrene oligomerization as a molecular probe reaction for zeolite acidity: a UV-Vis spectroscopy and DFT study

    NARCIS (Netherlands)

    Buurmans, I.L.C.; Pidko, E.A.; de Groot, J.M.; Stavitski, I.; van Santen, R.A.; Weckhuysen, B.M.

    2013-01-01

    A series of H-ZSM-5 crystallites with different framework Si/Al ratios was studied by analyzing the kinetics and reaction mechanism of the oligomerization of 4-fluorostyrene as molecular probe reaction for Brønsted acidity. The formation of carbocationic species was followed by UV-Vis spectroscopy.

  18. The upstream conserved regions (UCRs) mediate homo- and hetero-oligomerization of type 4 cyclic nucleotide phosphodiesterases (PDE4s).

    Science.gov (United States)

    Xie, Moses; Blackman, Brigitte; Scheitrum, Colleen; Mika, Delphine; Blanchard, Elise; Lei, Tao; Conti, Marco; Richter, Wito

    2014-05-01

    PDE4s (type 4 cyclic nucleotide phosphodiesterases) are divided into long and short forms by the presence or absence of conserved N-terminal domains termed UCRs (upstream conserved regions). We have shown previously that PDE4D2, a short variant, is a monomer, whereas PDE4D3, a long variant, is a dimer. In the present study, we have determined the apparent molecular masses of various long and short PDE4 variants by size-exclusion chromatography and sucrose density-gradient centrifugation. Our results indicate that dimerization is a conserved property of all long PDE4 forms, whereas short forms are monomers. Dimerization is mediated by the UCR domains. Given their high sequence conservation, the UCR domains mediate not only homo-oligomerization, but also hetero-oligomerization of distinct PDE4 long forms as detected by co-immunoprecipitation assays and FRET microscopy. Endogenous PDE4 hetero-oligomers are, however, low in abundance compared with homo-dimers, revealing the presence of mechanisms that predispose PDE4s towards homo-oligomerization. Oligomerization is a prerequisite for the regulatory properties of the PDE4 long forms, such as their PKA (protein kinase A)-dependent activation, but is not necessary for PDE4 protein-protein interactions. As a result, individual PDE4 protomers may independently mediate protein-protein interactions, providing a mechanism whereby PDE4s contribute to the assembly of macromolecular signalling complexes.

  19. A Small-Molecule Inhibitor of Bax and Bak Oligomerization Prevents Genotoxic Cell Death and Promotes Neuroprotection.

    Science.gov (United States)

    Niu, Xin; Brahmbhatt, Hetal; Mergenthaler, Philipp; Zhang, Zhi; Sang, Jing; Daude, Michael; Ehlert, Fabian G R; Diederich, Wibke E; Wong, Eve; Zhu, Weijia; Pogmore, Justin; Nandy, Jyoti P; Satyanarayana, Maragani; Jimmidi, Ravi K; Arya, Prabhat; Leber, Brian; Lin, Jialing; Culmsee, Carsten; Yi, Jing; Andrews, David W

    2017-04-20

    Aberrant apoptosis can lead to acute or chronic degenerative diseases. Mitochondrial outer membrane permeabilization (MOMP) triggered by the oligomerization of the Bcl-2 family proteins Bax/Bak is an irreversible step leading to execution of apoptosis. Here, we describe the discovery of small-molecule inhibitors of Bax/Bak oligomerization that prevent MOMP. We demonstrate that these molecules disrupt multiple, but not all, interactions between Bax dimer interfaces thereby interfering with the formation of higher-order oligomers in the MOM, but not recruitment of Bax to the MOM. Small-molecule inhibition of Bax/Bak oligomerization allowed cells to evade apoptotic stimuli and rescued neurons from death after excitotoxicity, demonstrating that oligomerization of Bax is essential for MOMP. Our discovery of small-molecule Bax/Bak inhibitors provides novel tools for the investigation of the mechanisms leading to MOMP and will ultimately facilitate development of compounds inhibiting Bax/Bak in acute and chronic degenerative diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Orally administrated cinnamon extract reduces β-amyloid oligomerization and corrects cognitive impairment in Alzheimer's disease animal models.

    Directory of Open Access Journals (Sweden)

    Anat Frydman-Marom

    Full Text Available An increasing body of evidence indicates that accumulation of soluble oligomeric assemblies of β-amyloid polypeptide (Aβ play a key role in Alzheimer's disease (AD pathology. Specifically, 56 kDa oligomeric species were shown to be correlated with impaired cognitive function in AD model mice. Several reports have documented the inhibition of Aβ plaque formation by compounds from natural sources. Yet, evidence for the ability of common edible elements to modulate Aβ oligomerization remains an unmet challenge. Here we identify a natural substance, based on cinnamon extract (CEppt, which markedly inhibits the formation of toxic Aβ oligomers and prevents the toxicity of Aβ on neuronal PC12 cells. When administered to an AD fly model, CEppt rectified their reduced longevity, fully recovered their locomotion defects and totally abolished tetrameric species of Aβ in their brain. Furthermore, oral administration of CEppt to an aggressive AD transgenic mice model led to marked decrease in 56 kDa Aβ oligomers, reduction of plaques and improvement in cognitive behavior. Our results present a novel prophylactic approach for inhibition of toxic oligomeric Aβ species formation in AD through the utilization of a compound that is currently in use in human diet.

  1. Recognition of coxiella burnetii by toll-like receptors and nucleotide-binding oligomerization domain-like receptors

    NARCIS (Netherlands)

    Ammerdorffer, Anne; Schoffelen, Teske; Gresnigt, Mark S.; Oosting, Marije; Brok, Den Martijn H.; Abdollahi-Roodsaz, Shahla; Kanneganti, Thirumala Devi; Jong, De Dirk J.; Deuren, Van Marcel; Roest, Hendrik Jan; Rebel, Johanna M.J.; Netea, Mihai G.; Joosten, Leo A.B.; Sprong, Tom

    2015-01-01

    Background. Infection with Coxiella burnetii can lead to acute and chronic Q fever. Toll-like receptor 1 (TLR1), TLR2, TLR4, TLR6, nucleotide-binding oligomerization domain receptor 1 (NOD1), NOD2, and the mitogen-activated protein kinases are central in the innate immune response against

  2. Recognition of Coxiella burnetii by Toll-like Receptors and Nucleotide-Binding Oligomerization Domain-like Receptors

    NARCIS (Netherlands)

    Ammerdorffer, A.; Schoffelen, T.; Gresnigt, M.S.; Oosting, M.; Brok, M.H.M.G.M. den; Abdollahi-Roodsaz, S.; Kanneganti, T.D.; Jong, D.J. de; Deuren, M. van; Roest, H.J.; Rebel, J.M.; Netea, M.G.; Joosten, L.A.B.; Sprong, T.

    2015-01-01

    BACKGROUND: Infection with Coxiella burnetii can lead to acute and chronic Q fever. Toll-like receptor 1 (TLR1), TLR2, TLR4, TLR6, nucleotide-binding oligomerization domain receptor 1 (NOD1), NOD2, and the mitogen-activated protein kinases are central in the innate immune response against

  3. Interference with RUNX1/ETO Leukemogenic Function by Cell-Penetrating Peptides Targeting the NHR2 Oligomerization Domain

    Directory of Open Access Journals (Sweden)

    Yvonne Bartel

    2013-01-01

    Full Text Available The leukemia-associated fusion protein RUNX1/ETO is generated by the chromosomal translocation t(8;21 which appears in about 12% of all de novo acute myeloid leukemias (AMLs. Essential for the oncogenic potential of RUNX1/ETO is the oligomerization of the chimeric fusion protein through the nervy homology region 2 (NHR2 within ETO. In previous studies, we have shown that the intracellular expression of peptides containing the NHR2 domain inhibits RUNX1/ETO oligomerization, thereby preventing cell proliferation and inducing differentiation of RUNX1/ETO transformed cells. Here, we show that introduction of a recombinant TAT-NHR2 fusion polypeptide into the RUNX1/ETO growth-dependent myeloid cell line Kasumi-1 results in decreased cell proliferation and increased numbers of apoptotic cells. This effect was highly specific and mediated by binding the TAT-NHR2 peptide to ETO sequences, as TAT-polypeptides containing the oligomerization domain of BCR did not affect cell proliferation or apoptosis in Kasumi-1 cells. Thus, the selective interference with NHR2-mediated oligomerization by peptides represents a challenging but promising strategy for the inhibition of the leukemogenic potential of RUNX1/ETO in t(8;21-positive leukemia.

  4. Interference with RUNX1/ETO Leukemogenic Function by Cell-Penetrating Peptides Targeting the NHR2 Oligomerization Domain

    Science.gov (United States)

    Bartel, Yvonne; Grez, Manuel; Wichmann, Christian

    2013-01-01

    The leukemia-associated fusion protein RUNX1/ETO is generated by the chromosomal translocation t(8;21) which appears in about 12% of all de novo acute myeloid leukemias (AMLs). Essential for the oncogenic potential of RUNX1/ETO is the oligomerization of the chimeric fusion protein through the nervy homology region 2 (NHR2) within ETO. In previous studies, we have shown that the intracellular expression of peptides containing the NHR2 domain inhibits RUNX1/ETO oligomerization, thereby preventing cell proliferation and inducing differentiation of RUNX1/ETO transformed cells. Here, we show that introduction of a recombinant TAT-NHR2 fusion polypeptide into the RUNX1/ETO growth-dependent myeloid cell line Kasumi-1 results in decreased cell proliferation and increased numbers of apoptotic cells. This effect was highly specific and mediated by binding the TAT-NHR2 peptide to ETO sequences, as TAT-polypeptides containing the oligomerization domain of BCR did not affect cell proliferation or apoptosis in Kasumi-1 cells. Thus, the selective interference with NHR2-mediated oligomerization by peptides represents a challenging but promising strategy for the inhibition of the leukemogenic potential of RUNX1/ETO in t(8;21)-positive leukemia. PMID:23865046

  5. The Solvent Effect on the Chemoselectivity of Palladium-catalyzed Oligomerization of 3, 3-Dimethyl-1-butyne

    Institute of Scientific and Technical Information of China (English)

    Jin Sheng CHENG; Jin Heng LI; Huan Feng JIANG; Xiao Yue OUYANG

    2003-01-01

    The chemoselectivities of PdC12 and CuC12-catalyzed oligomerization of 3, 3-dimethyl-butyne: 1, 3, 5-tri-tert-butylbenzene, 2, 2, 7, 7-tetramethyl-3, 6-dichloro-3, 5-octadiene and 2, 2, 7,7- tetramethyl-3,5-octadiyne were obtained, respectively, by regulating the polarity of the solvent.

  6. A rapid, semi-quantitative assay to screen for modulators of alpha-synuclein oligomerization ex vivo

    Directory of Open Access Journals (Sweden)

    Marion eDelenclos

    2016-01-01

    Full Text Available Alpha synuclein (αsyn aggregates are associated with the pathogenesis of Parkinson’s disease and others related disorders. Although modulation of αsyn aggregation is an attractive therapeutic target, new powerful methodologies are desperately needed to facilitate in vivo screening of novel therapeutics. Here we describe an in vivo rodent model with the unique ability to rapidly track αsyn-αsyn interactions and thus oligomerization using a bioluminescent protein complementation strategy that monitors spatial and temporal αsyn oligomerization ex vivo. We find that αsyn forms oligomers in vivo as early as 1 week after stereotactic AAV injection into rat substantia nigra. Strikingly, although abundant αsyn expression is also detected in striatum at one week, no αsyn oligomers are detected at this time point. By 4 weeks, oligomerization of αsyn is detected in both striatum and substantia nigra homogenates. Moreover, in a proof-of-principle experiment, the effect of a previously described Hsp90 inhibitor known to prevent αsyn oligomer formation, demonstrates the utility of this rapid and sensitive animal model to monitor αsyn oligomerization status in the rat brain.

  7. Structure of the Nemrut caldera (Eastern Anatolia, Turkey) and associated hydrothermal fluid circulation

    Science.gov (United States)

    Ulusoy, İnan; Labazuy, Philippe; Aydar, Erkan; Ersoy, Orkun; Çubukçu, Evren

    2008-07-01

    Plio-Quaternary volcanism played an important role in the present physical state of Eastern Anatolia. Mount Nemrut, situated to the west of Lake Van is one of the main volcanic centers in the region, with a spectacular summit caldera 8.5 × 7 km in diameter. The most recent eruptions of the volcano were in 1441, 1597 and 1692. Nemrut Lake covers the western half of the caldera; it is a deep, half-bowl-shaped lake with a maximum depth of 176 m. Numerous eruption centers are exposed within the caldera as a consequence of magma-water interaction. Current activity of Nemrut caldera is revealed as hot springs, fumaroles and a small, hot lake. Self-potential and bathymetric surveys carried out in the caldera were used to characterize the structure of the caldera and the associated hydrothermal fluid circulation. In addition, analyses based on digital elevation models and satellite imagery were used to improve our knowledge about the structure of the caldera. According to SP results, the flanks of the volcano represent "the hydrogeologic zone", whereas the intra-caldera region is an "active hydrothermal area" where the fluid circulation is controlled by structural discontinuities. There is also a northern fissure zone which exhibits hydrothermal signatures. Nemrut caldera collapsed piecemeal, with three main blocks. Stress controlling the collapse mechanism seems to be highly affected by the regional neotectonic regime. In addition to the historical activity, current hydrothermal and hydrogeologic conditions in the caldera, in which there is a large lake and shallow water table, increase the risk of the quiescent volcano.

  8. Oligomerization of Vibrio cholerae Hemolysin Induces CXCR3 Upregulation and Activation of B-1a Cell

    Institute of Scientific and Technical Information of China (English)

    Gayatri Mukherjee; Kalyan K Banerjee; Tapas Biswas

    2008-01-01

    The hemolysin oligomer promotes the proliferation of B-1a cells and the expression of CD25, which is indicative of cell activation, on B-1a cells. The upregulation of CD86 induced by the oligomer showed its selective bias for the B7-2 member of B7 family while the monomer failed to induce these effects. The oligomer induced the expression of CXCR3, associated with B cell activation, while the monomer induced the expression of CXCL4, a powerful angiostatic chemokine. In conclusion, we found that B-1a cells responded to the apoptogenic monomer by expressing CXCL4, whereas oligomerization of the immunogen induced CXCR3 to shift the response towards activation. Cellular & Molecular Immunology. 2008;5(3):231-234.

  9. Efficient light-emitting devices based on platinum-complexes-anchored polyhedral oligomeric silsesquioxane materials

    KAUST Repository

    Yang, Xiaohui

    2010-08-24

    The synthesis, photophysical, and electrochemical characterization of macromolecules, consisting of an emissive platinum complex and carbazole moieties covalently attached to a polyhedral oligomeric silsesquioxane (POSS) core, is reported. Organic light-emitting devices based on these POSS materials exhibit a peak external quantum efficiency of ca. 8%, which is significantly higher than that of the analogous devices with a physical blend of the platinum complexes and a polymer matrix, and they represent noticeable improvement in the device efficiency of solution-processable phosphorescent excimer devices. Furthermore, the ratio of monomer and excimer/aggregate electroluminescent emission intensity, as well as the device efficiency, increases as the platinum complex moiety presence on the POSS macromolecules decreases. © 2010 American Chemical Society.

  10. Entropic estimate of cooperative binding of substrate on a single oligomeric enzyme: An index of cooperativity

    CERN Document Server

    Banerjee, Kinshuk; Gangopadhyay, Gautam

    2012-01-01

    Here we have systematically studied the cooperative binding of substrate molecules on the active sites of a single oligomeric enzyme in a chemiostatic condition. The average number of bound substrate and the net velocity of the enzyme catalyzed reaction are studied by the formulation of stochastic master equation for the cooperative binding classified here as spatial and temporal. We have estimated the entropy production for the cooperative binding schemes based on single trajectory analysis using a kinetic Monte Carlo technique. It is found that the total as well as the medium entropy production show the same generic diagnostic signature for detecting the cooperativity, usually characterized in terms of the net velocity of the reaction. This feature is also found to be valid for the total entropy production rate at the nonequilibrium steady state. We have introduced an index of cooperativity, C, defined in terms of the ratio of the surprisals or equivalently, the stochastic system entropy associated with the...

  11. Energetic Calculations to Decipher pH-Dependent Oligomerization and Domain Swapping of Proteins.

    Science.gov (United States)

    Shingate, Prashant; Warwicker, Jim; Sowdhamini, Ramanathan

    2015-01-01

    Domain swapping mechanism is a specialised mode of oligomerization of proteins in which part of a protein is exchanged in a non-covalent manner between constituent subunits. This mechanism is highly affected by several physiological conditions. Here, we present a detailed analysis ofthe effect of pH on different regions of the domain swapped oligomer by considering examples which are known to be sensitive to pH in transiting from monomeric to domain-swapped dimeric form. The energetic calculations were performed using a specialized method which considers changes in pH and subsequent changes in the interactions between subunits. This analysis provides definitive hints about the pH-dependence switch from monomer to domain-swapped oligomer and the steps that may be involved in the swapping mechanism.

  12. The Role of Nucleotide-Binding Oligomerization Domain-Like Receptors in Pulmonary Infection.

    Science.gov (United States)

    Wiese, Kristin M; Coates, Bria M; Ridge, Karen M

    2017-08-01

    Pneumonia is caused by both viral and bacterial pathogens and is responsible for a significant health burden in the Unites States. The innate immune system is the human body's first line of defense against these pathogens. The recognition of invading pathogens via pattern recognition receptors leads to proinflammatory cytokine and chemokine production, followed by recruitment and activation of effector immune cells. The nonspecific inflammatory nature of the innate immune response can result in immunopathology that is detrimental to the host. In this review, we focus on one class of pattern recognition receptors, the nucleotide-binding oligomerization domain (NOD)-like receptors, specifically NOD1 and NOD2, and their role in host defense against viral and bacterial pathogens of the lung, including influenza, respiratory syncytial virus, Streptococcus pneumoniae, Chlamydophila pneumoniae, and Staphylococcus aureus. It is hoped that improved understanding of NOD1 and NOD2 activity in pneumonia will facilitate the development of novel therapies and promote improved patient outcomes.

  13. Building blocks of the apoptotic pore: how Bax and Bak are activated and oligomerize during apoptosis

    Science.gov (United States)

    Westphal, D; Kluck, R M; Dewson, G

    2014-01-01

    The central role of the Bcl-2 family in regulating apoptotic cell death was first identified in the 1980s. Since then, significant in-roads have been made in identifying the multiple members of this family, characterizing their form and function and understanding how their interactions determine whether a cell lives or dies. In this review we focus on the recent progress made in characterizing the proapoptotic Bcl-2 family members, Bax and Bak. This progress has resolved longstanding controversies, but has also challenged established theories in the apoptosis field. We will discuss different models of how these two proteins become activated and different ‘modes' by which they are inhibited by other Bcl-2 family members. We will also discuss novel conformation changes leading to Bak and Bax oligomerization and speculate how these oligomers might permeabilize the mitochondrial outer membrane. PMID:24162660

  14. Dissecting functions of the conserved oligomeric Golgi tethering complex using a cell-free assay.

    Science.gov (United States)

    Cottam, Nathanael P; Wilson, Katherine M; Ng, Bobby G; Körner, Christian; Freeze, Hudson H; Ungar, Daniel

    2014-01-01

    Vesicle transport sorts proteins between compartments and is thereby responsible for generating the non-uniform protein distribution along the eukaryotic secretory and endocytic pathways. The mechanistic details of specific vesicle targeting are not yet well characterized at the molecular level. We have developed a cell-free assay that reconstitutes vesicle targeting utilizing the recycling of resident enzymes within the Golgi apparatus. The assay has physiological properties, and could be used to show that the two lobes of the conserved oligomeric Golgi tethering complex play antagonistic roles in trans-Golgi vesicle targeting. Moreover, we can show that the assay is sensitive to several different congenital defects that disrupt Golgi function and therefore cause glycosylation disorders. Consequently, this assay will allow mechanistic insight into the targeting step of vesicle transport at the Golgi, and could also be useful for characterizing some novel cases of congenital glycosylation disorders.

  15. Energetic Calculations to Decipher pH-Dependent Oligomerization and Domain Swapping of Proteins.

    Directory of Open Access Journals (Sweden)

    Prashant Shingate

    Full Text Available Domain swapping mechanism is a specialised mode of oligomerization of proteins in which part of a protein is exchanged in a non-covalent manner between constituent subunits. This mechanism is highly affected by several physiological conditions. Here, we present a detailed analysis ofthe effect of pH on different regions of the domain swapped oligomer by considering examples which are known to be sensitive to pH in transiting from monomeric to domain-swapped dimeric form. The energetic calculations were performed using a specialized method which considers changes in pH and subsequent changes in the interactions between subunits. This analysis provides definitive hints about the pH-dependence switch from monomer to domain-swapped oligomer and the steps that may be involved in the swapping mechanism.

  16. Inhibition of listeriolysin O oligomerization by lutein prevents Listeria monocytogenes infection.

    Science.gov (United States)

    Liu, Bowen; Teng, Zihao; Wang, Jianfeng; Lu, Gejin; Deng, Xuming; Li, Li

    2017-01-01

    The foodborne pathogenic bacterial species Listeria monocytogenes (L. monocytogenes) has caused incalculable damages to public health, and its successful infection requires various virulence factors, including Listeriolysin O (LLO). By forming pores in phagosomal membranes and even in some organelles, LLO plays an indispensable role in the ability of L. monocytogenes to escape from host immune attacks. Because of its critical role, LLO offers an appropriate therapeutic target against L. monocytogenes infection. Here, lutein, a natural small molecule existing widely in fruits and vegetables, is demonstrated as an effective inhibitor of LLO that works by blocking its oligomerization during invasion without showing significant bacteriostatic activity. Further assays applying lutein in cell culture models of invasion and in animal models showed that lutein could effectively inhibit L. monocytogenes infection. Overall, our results indicate that lutein may represent a promising and novel therapeutic agent against L. monocytogenes infection. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Radiation-induced pink nickel oligomeric clusters in water. Pulse radiolysis study.

    Science.gov (United States)

    Hioul, Mohamed Larbi; Lin, Mingzhang; Belloni, Jacqueline; Keghouche, Nassira; Marignier, Jean-Louis

    2014-10-09

    γ-rays and pulse radiolysis of aqueous solutions of Ni(2+) ions in the presence of polyacrylate (PA(-)) and 2-propanol leads to the formation of metastable species absorbing at 540 nm that are ascribed to "pink" oligomeric clusters of a few nickel atoms only. The molar absorption coefficient is evaluated as ε540 nm = 3300 ± 300 L mol(-1) cm(-1) per Ni(0) atom. The successive steps from the reduction of Ni(2+) into Ni(+) ions to the formation of the pink clusters at 540 nm under conditions of complexation by PA(-) are investigated by pulse radiolysis. The yield of the formation of pink clusters increases markedly with the irradiation dose rate, demonstrating the occurrence of the disproportionation of the [Ni(+), PA(-)] complex after a single electron pulse. The reduction and nucleation mechanisms, including rate constants, in competition with the back oxidation by protons, particularly at low dose rate, are discussed.

  18. Understanding Controls on Wetting at Fluorinated Polyhedral Oligomeric Silsesquioxane/Polymer Surfaces.

    Science.gov (United States)

    Ye, Yi; Tian, Ming; Zhang, Chen; Du, Zhongjie; Mi, Jianguo

    2016-01-12

    Fluorinated polyhedral oligomeric silsesquioxane (F-POSS) nanoparticles have been widely used to enhance the hydrophobicity or oleophobicity of polymer films via constructing the specific micro/nanoscale roughness. In this work, we study the oleophobicity of pure and F-POSS-decorated poly(vinylidene fluoride) (PVDF) and poly(methyl methacrylate) (PMMA) films using a dynamic density functional theory approach. The role of nanoparticle size and coverage and the chemical features of F-POSS and the polymer film in the wetting behavior of diiodomethane droplets has been integrated to the remaining ratio of surface potential to quantitatively characterize the corner effect. It is shown that, on the basis of universal force field parameters, the theoretically predicted contact angles are in general agreement with the available experimental data.

  19. Oligomeric α-synuclein and β-amyloid variants as potential biomarkers for Parkinson's and Alzheimer's diseases.

    Science.gov (United States)

    Williams, Stephanie M; Schulz, Philip; Sierks, Michael R

    2016-01-01

    Oligomeric forms of α-synuclein and β-amyloid are toxic protein variants that are thought to contribute to the onset and progression of Parkinson's disease (PD) and Alzheimer's disease (AD), respectively. The detection of toxic variants in human cerebrospinal fluid (CSF) and blood has great promise for facilitating early and accurate diagnoses of these devastating diseases. Two hurdles that have impeded the use of these protein variants as biomarkers are the availability of reagents that can bind the different variants and a sensitive assay to detect their very low concentrations. We previously isolated antibody-based reagents that selectively bind two different oligomeric variants of α-synuclein and two of β-amyloid, and developed a phage-based capture enzyme-linked immunosorbent assay (ELISA) with subfemtomolar sensitivity to quantify their presence. Here, we used these reagents to show that these oligomeric α-synuclein variants are preferentially present in PD brain tissue, CSF and serum, and that the oligomeric β-amyloid variants are preferentially present in AD brain tissue, CSF, and serum. Some AD samples also had α-synuclein pathology and some PD samples also had β-amyloid pathology, and, very intriguingly, these PD cases also had a history of dementia. Detection of different oligomeric α-synuclein and β-amyloid species is an effective method for identifying tissue, CSF and sera from PD and AD samples, respectively, and samples that also contained early stages of other protein pathologies, indicating their potential value as blood-based biomarkers for neurodegenerative diseases.

  20. The protective effects and underlying mechanism of an anti-oligomeric Aβ42 single-chain variable fragment antibody.

    Science.gov (United States)

    Zhang, Yuan; Chen, Xu; Liu, Jinyu; Zhang, Yingjiu

    2015-12-01

    Oligomeric Aβ42 aggregates have been identified as one of the major neurotoxic components of Alzheimer's disease (AD). Immunotherapy targeted against these Aβ42 aggregates has been proposed as an appropriate therapeutic approach for the treatment of AD. Here, we report an anti-oligomeric Aβ42 single-chain variable fragment (scFv) antibody, named MO6, obtained from the human antibody library of a healthy donor. ScFv MO6 specifically recognized and bound to the oligomeric Aβ42 (Aβ42 oligomers and immature protofibrils; 18-37 kDa), and reduced their levels mainly by blocking their formation, although scFv MO6 also induced disaggregation of Aβ42 aggregates. More importantly, scFv MO6 ameliorated or attenuated Aβ42-induced cytotoxicity and increased cell viability by up to 33%. Furthermore, scFv MO6 efficiently passed through an in vitro blood-brain barrier (BBB) model with a delivery efficiency of 66% after 60 min post-administration. ScFv MO6 is a monovalent antibody with an affinity constant (KD) of 5.2×10(-6) M for Aβ42 oligomers. Molecular docking simulations of Aβ42 to scFv MO6 revealed that the approach and specific binding of scFv MO6 to oligomeric Aβ42 aggregates was achieved by conformational recognition and directed induction, which resulted in a more dynamic adaptation of Aβ42 to scFv MO6, occurring mainly in the N-terminal (3-4), middle (12-19) and C-terminal (34-42) regions of Aβ42. This binding mode of scFv MO6 to Aβ42 explains its protective effects against oligomeric Aβ42. Our findings may be applied for the design of a smaller antibody specific for Aβ42 oligermers.

  1. Aqueous SOA formation from radical oligomerization of methyl vinyl ketone (MVK) and methacrolein (MACR)

    Science.gov (United States)

    Renard, P.; Siekmann, F.; Ravier, S.; Temime-Roussel, B.; Clément, J.; Ervens, B.; Monod, A.

    2013-12-01

    It is now accepted that one of the important pathways of secondary organic aerosol (SOA) formation occurs through aqueous phase chemistry in the atmosphere. However, the chemical mechanisms leading to macromolecules are still not well understood. It was recently shown that oligomer production by OH radical oxidation in the aerosol aqueous phase from α-dicarbonyl precursors, such as methylglyoxal and glyoxal, is irreversible and fast. We have investigated the aqueous phase photooxidation of MACR and MVK, which are biogenic organic compounds derived from isoprene. Aqueous phase photooxidation of MVK and MACR was investigated in a photoreactor using photolysis of H2O2 as OH radical source. Electrospray high resolution mass spectrometry analysis of the solutions brought clear evidence for the formation of oligomer systems having a mass range of up to 1800 Da within less than 15 minutes of reaction. Highest oligomer formation rates were obtained under conditions of low dissolved oxygen, highest temperature (T = 298 K) and highest precursor initial concentrations ([MVK]0 = 20 mM). A radical mechanism of oligomerization is proposed to explain the formation of the high molecular weight products. Furthermore, we quantified the total amount of carbon present in oligomers. Kinetic parameters of the proposed oligomerization mechanism are constrained by means of a box model that is able to reproduce the temporal evolution of intermediates and products as observed in the laboratory experiments. Additional model simulations for atmospherically-relevant conditions will be presented that show the extent to which these radical processes contribute to SOA formation in the atmospheric multiphase system as compared to other aqueous phase as well as traditional SOA sources. MVK time profile (as measured by UV Spectroscopy) and mass spectra (obtained using UPLC-ESI-MS for the retention time range 0-5 min in the positive mode) at 5, 10 and 50 min of reaction (MVK 20 mM, 25° C, under

  2. Targeting Bax interaction sites reveals that only homo-oligomerization sites are essential for its activation

    Science.gov (United States)

    Peng, R; Tong, J-S; Li, H; Yue, B; Zou, F; Yu, J; Zhang, L

    2013-01-01

    Bax is a proapoptotic Bcl-2 family member that has a central role in the initiation of mitochondria-dependent apoptosis. However, the mechanism of Bax activation during apoptosis remains unsettled. It is believed that the activation of Bax is mediated by either dissociation from prosurvival Bcl-2 family members, or direct association with BH3-only members. Several interaction sites on Bax that mediate its interactions with other Bcl-2 family members, as well as its proapoptotic activity, have been identified in previous studies by other groups. To rigorously investigate the functional role of these interaction sites, we knocked in their respective mutants using HCT116 colon cancer cells, in which apoptosis induced by several stimuli is strictly Bax-dependent. Bax-mediated apoptosis was intact upon knock-in (KI) of K21E and D33A, which were shown to block the interaction of Bax with BH3-only activators. Apoptosis was partially reduced by KI of D68R, which impairs the interaction of Bax with prosurvival members, and S184V, a constitutively mitochondria-targeting mutant. In contrast, apoptosis was largely suppressed by KI of L70A/D71A, which blocks homo-oligomerization of Bax and its binding to prosurvival Bcl-2 family proteins. Collectively, our results suggest that the activation of endogenous Bax in HCT116 cells is dependent on its homo-oligomerization sites, but not those previously shown to interact with BH3-only activators or prosurvival proteins only. We therefore postulate that critical interaction sites yet to be identified, or mechanisms other than protein-protein interactions, need to be pursued to delineate the mechanism of Bax activation during apoptosis. PMID:23392123

  3. Defining the oligomerization state of γ-synuclein in solution and in cells.

    Science.gov (United States)

    Golebiewska, Urszula; Zurawsky, Cassandra; Scarlata, Suzanne

    2014-01-21

    γ-Synuclein is expressed at high levels in neuronal cells and in multiple invasive cancers. Like its family member α-synuclein, γ-synuclein is thought to be natively unfolded but does not readily form fibrils. The function of γ-synuclein is unknown, but we have found that it interacts strongly with the enzyme phospholipase Cβ (PLCβ), altering its interaction with G proteins. As a first step in determining its role, we have characterized its oligomerization using fluorescence homotransfer, photon-counting histogram analysis, and native gel electrophoresis. We found that when its expressed in Escherichia coli and purified, γ-synuclein appears monomeric on chromatographs under denaturing conditions, but under native conditions, it appears as oligomers of varying sizes. We followed the monomer-to-tetramer association by labeling the protein with fluorescein and following the concentration-dependent loss in fluorescence anisotropy resulting from fluorescence homotransfer. We also performed photon-counting histogram analysis at increasing concentrations of fluorescein-labeled γ-synuclein and found concentration-dependent oligomerization. Addition of PLCβ2, a strong γ-synuclein binding partner whose cellular expression is correlated with γ-synuclein, results in disruption of γ-synuclein oligomers. Similarly, its binding to lipid membranes promotes the monomer form. When we exogenously express γ-synuclein or microinject purified protein into cells, the protein appears monomeric. Our studies show that even though purified γ-synuclein form oligomers, when binding partners are present, as in cells, it dissociates to a monomer to bind these partners, which in turn may modify protein function and integrity.

  4. The R439C mutation in LMNA causes lamin oligomerization and susceptibility to oxidative stress

    Science.gov (United States)

    Verstraeten, Valerie LRM; Caputo, Sandrine; Van Steensel, Maurice AM; Duband-Goulet, Isabelle; Zinn-Justin, Sophie; Kamps, Miriam; Kuijpers, Helma JH; Östlund, Cecilia; Worman, Howard J; Briedé, Jacob J; Le Dour, Caroline; Marcelis, Carlo LM; Van Geel, Michel; Steijlen, Peter M; Van Den Wijngaard, Arthur; Ramaekers, Frans CS; Broers, Jos LV

    2009-01-01

    Abstract Dunnigan-type familial partial lipodystrophy (FPLD) is a laminopathy characterized by an aberrant fat distribution and a metabolic syndrome for which oxidative stress has recently been suggested as one of the disease-causing mechanisms. In a family affected with FPLD, we identified a heterozygous missense mutation c.1315C>T in the LMNA gene leading to the p.R439C substitution. Cultured patient fibroblasts do not show any prelamin A accumulation and reveal honeycomb-like lamin A/C formations in a significant percentage of nuclei. The mutation affects a region in the C-terminal globular domain of lamins A and C, different from the FPLD-related hot spot. Here, the introduction of an extra cysteine allows for the formation of disulphide-mediated lamin A/C oligomers. This oligomerization affects the interaction properties of the C-terminal domain with DNA as shown by gel retardation assays and causes a DNA-interaction pattern that is distinct from the classical R482W FPLD mutant. Particularly, whereas the R482W mutation decreases the binding efficiency of the C-terminal domain to DNA, the R439C mutation increases it. Electron spin resonance spectroscopy studies show significantly higher levels of reactive oxygen species (ROS) upon induction of oxidative stress in R439C patient fibroblasts compared to healthy controls. This increased sensitivity to oxidative stress seems independent of the oligomerization and enhanced DNA binding typical for R439C, as both the R439C and R482W mutants show a similar and significant increase in ROS upon induction of oxidative stress by H2O2. PMID:19220582

  5. Genetic analysis and serum level of cartilage oligomeric matrix protein in patients with pseudoachondroplasia

    Institute of Scientific and Technical Information of China (English)

    LIU Feng-xia; LI Zhi-ling; WEI Zhen-ji; MENG yan; REN Cui-ai; ZHANG Xu-de; YU Meng-xue; HUANG Shang-zhi

    2010-01-01

    Background Pseudoachondroplasia (PSACH) is an autosomal-dominant osteochondrodysplasia due to mutations in the gene encoding cartilage oligomeric matrix protein (COMP).Clinical diagnosis of PSACH is based primarily on family history, physical examination, and radiographic evaluation.There is evidence that decreased serum COMP concentration may serve as a diagnostic marker in PSACH.Here, we investigated the role of this gene and the serum COMP concentration in Chinese patients with PSACH.Methods A family with three patients and a sporadic case were recruited.Genomic and phenotypic data were recorded.The diagnosis of PSACH was made on the base of clinical evaluation.The genomic DNA was extracted from peripheral blood leukocytes.The 8-19 exons and flanking intron-exon boundary sequences of COMP were amplified by polymerase chain reaction (PCR) and screened for mutation by direct DNA sequencing.Serum COMP concentrations of 4 patients and age-compatible control group of 20 unrelated healthy subjects were analyzed on the basis of an ELISA Kit for human cartilage oligomeric matrix protein.Results A deletion (c.1447-1455del) was identified in exon 13 in the sporadic case.The mean serum COMP concentrations of four patients (3.12±2.28) were significantly lower than those of control group (10.86±2.21, P <0.05).There was no overlap in the distribution of serum COMP concentration between PSACH patients and controls.Conclusions Mutations in COMP gene are responsible for the PSACH.Serum COMP concentration may be suggested as an additional diagnostic marker to aid clinical findings in suspected cases of PSACH.

  6. Following activation of the amyloid cascade, apolipoprotein E4 drives the in vivo oligomerization of amyloid-β resulting in neurodegeneration.

    Science.gov (United States)

    Belinson, Haim; Kariv-Inbal, Zehavit; Kayed, Rakez; Masliah, Eliezer; Michaelson, Daniel M

    2010-01-01

    According to the amyloid hypothesis, the accumulation of oligomerized amyloid-β (Aβ) is a primary event in the pathogenesis of Alzheimer's disease (AD). The trigger of the amyloid cascade and of Aβ oligomerization in sporadic AD, the most prevalent form of the disease, remains elusive. Here, we examined the hypothesis that apolipoprotein E4 (ApoE4), the most prevalent genetic risk factor for AD, triggers the accumulation of intraneuronal oligomerized Aβ following activation of the amyloid cascade. We investigated the intracellular organelles that are targeted by these processes and govern their pathological consequences. This revealed that activation of the amyloid cascade in vivo by inhibition of the Aβ degrading enzyme neprilysin specifically results in accumulation of Aβ and oligomerized Aβ and of ApoE4 in the CA1 neurons of ApoE4 mice. This was accompanied by lysosomal and mitochondrial pathology and the co-localization of Aβ, oligomerized Aβ, and ApoE4 with enlarged lysosomes and of Aβ and oligomerized Aβ with mitochondria. The time course of the lysosomal effects paralleled that of the loss of CA1 neurons, whereas the mitochondrial effects reached an earlier plateau. These findings suggest that ApoE4 potentiates the pathological effects of Aβ and the amyloid cascade by triggering the oligomerization of Aβ, which in turn, impairs intraneuronal mitochondria and lysosomes and drives neurodegeneration.

  7. Effects of aerobic exercise intervention on serum cartilage oligomeric matrix protein levels and lymphocyte dna damage in obese elderly females

    Science.gov (United States)

    Cho, Su Youn; Roh, Hee Tae

    2016-01-01

    [Purpose] The aim of the reported research was to investigate the effects of regular aerobic exercise on cartilage oligomeric matrix protein and oxidative DNA damage in obese, elderly females. [Subjects and Methods] Sixteen class I obese, elderly females, according to World Health Organization criteria, were randomly and equally assigned to a control group (n=8) or an exercise group (n=8). The exercise group participated in exercise sessions of 60 minutes per day, 3 days per week, for a period of 8 weeks. [Results] After aerobic exercise intervention, weight, body mass index, body fat, waist circumference, and DNA damage (Tail moment) were significantly decreased, compared with baseline values. In contrast, serum cartilage oligomeric matrix protein levels were not significantly different among any groups or time-points. [Conclusion] Regular aerobic exercise may be effective for reducing obesity-induced high DNA damage levels in obese females, without causing the deformation or degradation of lower extremity articular cartilage. PMID:27390441

  8. Characterization of oligomerization of a peptide from the ebola virus glycoprotein by small-angle neutron scattering

    Energy Technology Data Exchange (ETDEWEB)

    Egorov, V. V., E-mail: vlaegur@omrb.pnpi.spb.ru [National Research Center “Kurchatov Institute”, Konstantinov Petersburg Nuclear Physics Institute (Russian Federation); Gorshkov, A. N. [Ministry of Health of the Russian Federation, Research Institute of Influenza (Russian Federation); Murugova, T. N. [Joint Institute for Nuclear Research (Russian Federation); Vasin, A. V. [Ministry of Health of the Russian Federation, Research Institute of Influenza (Russian Federation); Lebedev, D. V.; Isaev-Ivanov, V. V. [National Research Center “Kurchatov Institute”, Konstantinov Petersburg Nuclear Physics Institute (Russian Federation); Kiselev, O. I. [Ministry of Health of the Russian Federation, Research Institute of Influenza (Russian Federation)

    2016-01-15

    Transmission electron microscopy (TEM) and small-angle neutron scattering (SANS) studies showed that model peptides QNALVCGLRQ (G33) and QNALVCGLRG (G31) corresponding to region 551–560 of the GP protein of the Sudan Ebola virus are prone to oligomerization in solution. Both peptides can form amyloid-like fibrills. The G33 peptide forms fibrils within one day of incubation, whereas the fibrillogenesis of the G31 peptide is observed only after incubation for several months. The possible role of the observed processes in the pathogenesis and the possibility of applying a combination of the TEM and SANS techniques to search for new compounds that are able to influence the protein oligomerization are discussed.

  9. Bax monomers form dimer units in the membrane that further self-assemble into multiple oligomeric species

    Science.gov (United States)

    Subburaj, Yamunadevi; Cosentino, Katia; Axmann, Markus; Pedrueza-Villalmanzo, Esteban; Hermann, Eduard; Bleicken, Stephanie; Spatz, Joachim; García-Sáez, Ana J.

    2015-08-01

    Bax is a key regulator of apoptosis that mediates the release of cytochrome c to the cytosol via oligomerization in the outer mitochondrial membrane before pore formation. However, the molecular mechanism of Bax assembly and regulation by other Bcl-2 members remains obscure. Here, by analysing the stoichiometry of Bax oligomers at the single-molecule level, we find that Bax binds to the membrane in a monomeric state and then self-assembles in Bax does not exist in a unique oligomeric state, but as several different species based on dimer units. Moreover, we show that cBid activates Bax without affecting its assembly, while Bcl-xL induces the dissociation of Bax oligomers. On the basis of our experimental data and theoretical modelling, we propose a new mechanism for the molecular pathway of Bax assembly to form the apoptotic pore.

  10. Characterization of oligomerization of a peptide from the ebola virus glycoprotein by small-angle neutron scattering

    Science.gov (United States)

    Egorov, V. V.; Gorshkov, A. N.; Murugova, T. N.; Vasin, A. V.; Lebedev, D. V.; Isaev-Ivanov, V. V.; Kiselev, O. I.

    2016-01-01

    Transmission electron microscopy (TEM) and small-angle neutron scattering (SANS) studies showed that model peptides QNALVCGLRQ (G33) and QNALVCGLRG (G31) corresponding to region 551-560 of the GP protein of the Sudan Ebola virus are prone to oligomerization in solution. Both peptides can form amyloid-like fibrills. The G33 peptide forms fibrils within one day of incubation, whereas the fibrillogenesis of the G31 peptide is observed only after incubation for several months. The possible role of the observed processes in the pathogenesis and the possibility of applying a combination of the TEM and SANS techniques to search for new compounds that are able to influence the protein oligomerization are discussed.

  11. Increased serum cartilage oligomeric matrix protein levels and decreased patellar bone mineral density in patients with chondromalacia patellae.

    OpenAIRE

    2002-01-01

    BACKGROUND: Chondromalacia patellae is a potentially disabling disorder characterised by features of patellar cartilage degradation. OBJECTIVE: To evaluate markers of cartilage and bone turnover in patients with chondromalacia patellae. METHODS: 18 patients with chondromalacia patellae were studied. Serum cartilage oligomeric matrix protein (s-COMP) and bone sialoprotein (s-BSP) levels were measured by enzyme linked immunosorbent assay (ELISA) and compared with those of age and sex matched he...

  12. Atmospheric protein chemistry influenced by anthropogenic air pollutants: nitration and oligomerization upon exposure to ozone and nitrogen dioxide.

    Science.gov (United States)

    Liu, Fobang; Lakey, Pascale S J; Berkemeier, Thomas; Tong, Haijie; Kunert, Anna Theresa; Meusel, Hannah; Cheng, Yafang; Su, Hang; Fröhlich-Nowoisky, Janine; Lai, Senchao; Weller, Michael G; Shiraiwa, Manabu; Pöschl, Ulrich; Kampf, Christopher J

    2017-08-24

    The allergenic potential of airborne proteins may be enhanced via post-translational modification induced by air pollutants like ozone (O3) and nitrogen dioxide (NO2). The molecular mechanisms and kinetics of the chemical modifications that enhance the allergenicity of proteins, however, are still not fully understood. Here, protein tyrosine nitration and oligomerization upon simultaneous exposure of O3 and NO2 were studied in coated-wall flow-tube and bulk solution experiments under varying atmospherically relevant conditions (5-200 ppb O3, 5-200 ppb NO2, 45-96% RH), using bovine serum albumin as a model protein. Generally, more tyrosine residues were found to react via the nitration pathway than via the oligomerization pathway. Depending on reaction conditions, oligomer mass fractions and nitration degrees were in the ranges of 2.5-25% and 0.5-7%, respectively. The experimental results were well reproduced by the kinetic multilayer model of aerosol surface and bulk chemistry (KM-SUB). The extent of nitration and oligomerization strongly depends on relative humidity (RH) due to moisture-induced phase transition of proteins, highlighting the importance of cloud processing conditions for accelerated protein chemistry. Dimeric and nitrated species were major products in the liquid phase, while protein oligomerization was observed to a greater extent for the solid and semi-solid phase states of proteins. Our results show that the rate of both processes was sensitive towards ambient ozone concentration, but rather insensitive towards different NO2 levels. An increase of tropospheric ozone concentrations in the Anthropocene may thus promote pro-allergic protein modifications and contribute to the observed increase of allergies over the past decades.

  13. Screening of drugs inhibiting in vitro oligomerization of Cu/Zn-superoxide dismutase with a mutation causing amyotrophic lateral sclerosis

    OpenAIRE

    Itsuki Anzai; Keisuke Toichi; Eiichi Tokuda; Atsushi Mukaiyama; Shuji Akiyama; Yoshiaki Furukawa

    2016-01-01

    Dominant mutations in Cu/Zn-superoxide dismutase (SOD1) gene have been shown to cause a familial form of amyotrophic lateral sclerosis (SOD1-ALS). A major pathological hallmark of this disease is abnormal accumulation of mutant SOD1 oligomers in the affected spinal motor neurons. While no effective therapeutics for SOD1-ALS is currently available, SOD1 oligomerization will be a good target for developing cures of this disease. Recently, we have reproduced the formation of SOD1 oligomers ab...

  14. Both Ca2+ and Zn2+ are essential for S100A12 protein oligomerization and function

    Directory of Open Access Journals (Sweden)

    Shekhtman Alexander

    2009-04-01

    Full Text Available Abstract Background Human S100A12 is a member of the S100 family of EF-hand calcium-modulated proteins that are associated with many diseases including cancer, chronic inflammation and neurological disorders. S100A12 is an important factor in host/parasite defenses and in the inflammatory response. Like several other S100 proteins, it binds zinc and copper in addition to calcium. Mechanisms of zinc regulation have been proposed for a number of S100 proteins e.g. S100B, S100A2, S100A7, S100A8/9. The interaction of S100 proteins with their targets is strongly dependent on cellular microenvironment. Results The aim of the study was to explore the factors that influence S100A12 oligomerization and target interaction. A comprehensive series of biochemical and biophysical experiments indicated that changes in the concentration of calcium and zinc led to changes in the oligomeric state of S100A12. Surface plasmon resonance confirmed that the presence of both calcium and zinc is essential for the interaction of S100A12 with one of its extracellular targets, RAGE – the Receptor for Advanced Glycation End products. By using a single-molecule approach we have shown that the presence of zinc in tissue culture medium favors both the oligomerization of exogenous S100A12 protein and its interaction with targets on the cell surface. Conclusion We have shown that oligomerization and target recognition by S100A12 is regulated by both zinc and calcium. Our present work highlighted the potential role of calcium-binding S100 proteins in zinc metabolism and, in particular, the role of S100A12 in the cross talk between zinc and calcium in cell signaling.

  15. Increased serum cartilage oligomeric matrix protein levels and decreased patellar bone mineral density in patients with chondromalacia patellae.

    OpenAIRE

    Murphy, E; Fitzgerald, O; Saxne, Tore; Bresnihan, B

    2002-01-01

    BACKGROUND: Chondromalacia patellae is a potentially disabling disorder characterised by features of patellar cartilage degradation. OBJECTIVE: To evaluate markers of cartilage and bone turnover in patients with chondromalacia patellae. METHODS: 18 patients with chondromalacia patellae were studied. Serum cartilage oligomeric matrix protein (s-COMP) and bone sialoprotein (s-BSP) levels were measured by enzyme linked immunosorbent assay (ELISA) and compared with those of age and sex matched he...

  16. Synthesis and Catalytic Performances of a Novel Zn-MOF Catalyst Bearing Nickel Chelating Diimine Carboxylate Ligands for Ethylene Oligomerization

    Directory of Open Access Journals (Sweden)

    Suyan Liu

    2015-01-01

    Full Text Available A novel Zn-MOF[Zn3(OH2L2] was synthesized from dicarboxylate ligands with diimine groups (1,4-bis(4-CO2HC6H4-2,3-dimethyl-1,4-diazabutadiene. The physicochemical properties of the material were characterized by a series of technologies including XRD, SEM, and ICP. In order to adapt to the ethylene oligomerization process, a catalyst [Zn3OH2L1Ni2] (denoted as Cat.A possessing active Ni2+ centers was prepared by a postsynthetic treatment method using dichloride nickel as a nickel source in this work. For comparison, α-diimine ligands with/without dicarboxylic acid groups reacted with dichloride nickel to obtain homogenous Cat.B and Cat.C, respectively. The effects of reaction parameters, including n(Al/n(Ni, temperature, and pressure on the oligomerization activities and oligomers distribution were investigated. The results demonstrated that all of catalysts used with diethylaluminum chloride were active for the ethylene oligomerization. Among them, Cat.A and Cat.B showed higher catalytic activities and higher selectivities to low-carbon α-olefins at atmospheric pressure. The Cat.A exhibited the optimal catalytic activity [6.7 × 105 g/(mol·Ni·h·atm] for C4 (91.8% under the conditions of Al/Ni = 1500, P = 1.0 atm, T = 20°C. In addition, Cat.A and Cat.B presented large amount of ethylene polymer, while Cat.C had a higher catalytic activity of ethylene oligomerization at high pressure.

  17. Oligomeric adiponectin forms and their complexes in the blood of healthy donors and patients with type 2 diabetes mellitus.

    Science.gov (United States)

    Kogan, Alexander E; Filatov, Vladimir L; Kolosova, Olga V; Katrukha, Ivan A; Mironova, Ekaterina V; Zhuravleva, Natalya S; Nagibin, Oleg A; Kara, Andrei N; Bereznikova, Anastasiya V; Katrukha, Alexey G

    2013-01-01

    Adiponectin (Adn) is a protein that circulates in the blood in several oligomeric forms, namely low-, medium-, and high-molecular-weight forms. Adn may serve as a risk factor for type 2 diabetes mellitus (T2DM). The aims of this work were (1) to produce monoclonal antibodies (MAbs) specific to different Adn oligomeric forms, (2) to design immunoassays suitable for measuring the Adn forms present in human blood, and (3) to investigate the changes in Adn forms that occur in patients with T2DM. Gel filtration, fluoroimmunoassays, and Western blotting were utilized as major techniques in this study. MAbs recognizing various oligomeric forms of Adn were obtained. Complexes between Adn and complement component C1q and between the low molecular weight form of Adn and albumin were described in human blood. A decrease in the total Adn and Adn-albumin complex levels in the blood of patients with T2DM and no difference in the levels of the Adn-C1q complex in comparison with healthy volunteers were demonstrated. An Adn94-Adn63 fluoroimmunoassay was selected as the technique that most accurately measured the mass ratio of Adn oligomers in blood samples, and an Adn214-Adn27 assay that measured the low-molecular-weight form of Adn only.

  18. Microwave dielectric study of an oligomeric electrolyte gelator by time domain reflectometry.

    Science.gov (United States)

    Kundu, Shyamal Kumar; Yagihara, Shin; Yoshida, Masaru; Shibayama, Mitsuhiro

    2009-07-30

    The dynamics of water molecules in aqueous solutions of an oligomeric electrolyte gelator, poly[pyridinium-1,4-diyliminocarbonyl-1,4-phenylene-methylene chloride] (1-Cl) was characterized by microwave dielectric measurements using the time domain reflectometry method. The dielectric dispersion and absorption curves related to the orientational motion of water molecules were described by the Cole-Cole equation. Discontinuities were observed in the concentration dependence of the dielectric relaxation strength, Deltaepsilonh, as well as in the Cole-Cole parameter, betah. These discontinuities were observed between the samples with concentrations of 6 and 7 g/L 1-Cl/water, which correspond to a change in the transparency. Such a discontinuity corresponds to the observation of the critical concentration of gelation. The interaction between water and 1-Cl molecules was discussed from the tauh-betah diagram. As 1-Cl carries an amide group, it could be expected that 1-Cl may interact hydrophilically with water, but the present result suggests that 1-Cl interact hydrophobically with water.

  19. Oligomeric synthesis and density functional theory of leucoemeraldine base form of polyaniline

    Science.gov (United States)

    Ullah, Rizwan; Ullah, Habib; Shah, Anwar-ul-Haq Ali; Bilal, Salma; Ali, Khurshid

    2017-01-01

    Oligomeric synthesis of phenyl-end-capped oligoaniline (4PANI LB) has been carried out through a weak oxidizing agent, CuCl2, using chemical oxidative polymerization protocol. The sample was characterized by mass spectrometry, UV-vis, IR, and CHN elemental analysis. The experimental results are counterchecked with the aid of Quantum mechanical calculations such as density functional theory (DFT). DFT at B3LYP/6-31 G (d) level of theory was used for the geometric and electronic properties simulations which also confirm the existence of 4PANI LB. Excellent correlation is observed between the experiment and theory, particularly in the UV-vis spectra which conclude the formation of tetramer (fully reduced form) 4PANI LB (C24H20.06N4.07). Electronic properties such as Ionization Potential (I.P), Electron Affinities (E.A), the coefficient of highest occupied molecular orbital (HOMO), the coefficient of lowest unoccupied molecular orbital (LUMO) of 4PANI LB were evaluated at the above-mentioned level of theory.

  20. Determination of GLUT1 Oligomerization Parameters using Bioluminescent Förster Resonance Energy Transfer.

    Science.gov (United States)

    Looyenga, Brendan; VanOpstall, Calvin; Lee, Zion; Bell, Jed; Lodge, Evans; Wrobel, Katherine; Arnoys, Eric; Louters, Larry

    2016-06-30

    The facilitated glucose transporter GLUT1 (SLC2A1) is an important mediator of glucose homeostasis in humans. Though it is found in most cell types to some extent, the level of GLUT1 expression across different cell types can vary dramatically. Prior studies in erythrocytes-which express particularly high levels of GLUT1-have suggested that GLUT1 is able to form tetrameric complexes with enhanced transport activity. Whether dynamic aggregation of GLUT1 also occurs in cell types with more modest expression of GLUT1, however, is unclear. To address this question, we developed a genetically encoded bioluminescent Förster resonance energy transfer (BRET) assay using the luminescent donor Nanoluciferase and fluorescent acceptor mCherry. By tethering these proteins to the N-terminus of GLUT1 and performing saturation BRET analysis, we were able to demonstrate the formation of multimeric complexes in live cells. Parallel use of flow cytometry and immunoblotting further enabled us to estimate the density of GLUT1 proteins required for spontaneous oligomerization. These data provide new insights into the physiological relevance of GLUT1 multimerization as well as a new variant of BRET assay that is useful for measuring the interactions among other cell membrane proteins in live cells.

  1. Role of redox environment on the oligomerization of higher molecular weight adiponectin

    Directory of Open Access Journals (Sweden)

    Nuñez Martha

    2011-05-01

    Full Text Available Abstract Background Adiponectin is an adipocyte-secreted hormone with insulin-sensitizing and anti-inflammatory actions. The assembly of trimeric, hexameric, and higher molecular weight (HMW species of adiponectin is a topic of significant interest because physiological actions of adiponectin are oligomer-specific. In addition, adiponectin assembly is an example of oxidative oligomerization of multi-subunit protein complexes in endoplasmic reticulum (ER. Results We previously reported that trimers assemble into HMW adiponectin via intermediates stabilized by disulfide bonds, and complete oxidation of available cysteines locks adiponectin in hexameric conformation. In this study, we examined the effects of redox environment on the rate of oligomer formation and the distribution of oligomers. Reassembly of adiponectin under oxidizing conditions accelerated disulfide bonding but favored formation of hexamers over the HMW species. Increased ratios of HMW to hexameric adiponectin could be achieved rapidly under oxidizing conditions by promoting disulfide rearrangement. Conclusions Based upon these observations, we propose oxidative assembly of multi-subunit adiponectin complexes in a defined and stable redox environment is favored under oxidizing conditions coupled with high rates of disulfide rearrangement.

  2. An integrative approach to the study of filamentous oligomeric assemblies, with application to RecA.

    Science.gov (United States)

    Boyer, Benjamin; Ezelin, Johann; Poulain, Pierre; Saladin, Adrien; Zacharias, Martin; Robert, Charles H; Prévost, Chantal

    2015-01-01

    Oligomeric macromolecules in the cell self-organize into a wide variety of geometrical motifs such as helices, rings or linear filaments. The recombinase proteins involved in homologous recombination present many such assembly motifs. Here, we examine in particular the polymorphic characteristics of RecA, the most studied member of the recombinase family, using an integrative approach that relates local modes of monomer/monomer association to the global architecture of their screw-type organization. In our approach, local modes of association are sampled via docking or Monte Carlo simulations. This enables shedding new light on fiber morphologies that may be adopted by the RecA protein. Two distinct RecA helical morphologies, the so-called "extended" and "compressed" forms, are known to play a role in homologous recombination. We investigate the variability within each form in terms of helical parameters and steric accessibility. We also address possible helical discontinuities in RecA filaments due to multiple monomer-monomer association modes. By relating local interface organization to global filament morphology, the strategies developed here to study RecA self-assembly are particularly well suited to other DNA-binding proteins and to filamentous protein assemblies in general.

  3. Oligomeric procyanidins of French maritime pine bark extract (Pycnogenol) effectively inhibit alpha-glucosidase.

    Science.gov (United States)

    Schäfer, Angelika; Högger, Petra

    2007-07-01

    The standardized maritime pine bark extract (Pycnogenol) was reported to exert clinical anti-diabetic effects after peroral intake. However, an increased insulin secretion was not observed after administration of the extract to patients. Our aim was to elucidate whether the described clinical effects of Pycnogenol are related to inhibition of alpha-glucosidase. Therefore, we analyzed the inhibitory activity of Pycnogenol, green tea extract and acarbose towards alpha-glucosidase. Furthermore, we explored different fractions of Pycnogenol containing compounds of diverse molecular masses from polyphenolic monomers, dimers and higher oligomers to uncover which components exhibited the most pronounced inhibitory activity. We found that Pycnogenol exhibited the most potent inhibition (IC(50) about 5 microg/mL) on alpha-glucosidase compared to green tea extract (IC(50) about 20 microg/mL) and acarbose (IC(50) about 1mg/mL). The inhibitory action of Pycnogenol was stronger in extract fractions containing higher procyanidin oligomers. The results obtained assign a novel, local effect to oligomeric procyanidins and contribute to the explanation of glucose-lowering effects of Pycnogenol observed in clinical trials with diabetic patients.

  4. Oligomerization of 10,16-Dihydroxyhexadecanoic Acid and Methyl 10,16-Dihydroxyhexadecanoate Catalyzed by Lipases

    Directory of Open Access Journals (Sweden)

    Daniel Arrieta-Baez

    2013-08-01

    Full Text Available The main monomer of tomato cuticle, 10,16-dihydroxyhexadecanoic acid (10,16-DHPA and its methyl ester derivative (methyl-10,16-dihydroxyhexadecanote; methyl-10,16-DHHD, were used to study their oligomerization reactions catalyzed by five lipases: Candida antarctica lipase B (CAL-B, Rhizomucor miehei lipase (RM, Thermomyces lanuginosus lipase (TL, Pseudomonas cepacia lipase (PCL and porcine pancreatic lipase (PPL. For 10,16-DHPA, optimum yields were obtained at 60 °C using toluene and 2-methyl-2-butanol (2M2B as solvent, while for methyl-10,16-DHHD the bests yields were obtained in toluene and acetonitrile. Both reactions leaded to linear polyesters according to the NMR and FT-IR analysis, and there was no data indicating the presence of branched polymers. Using optimized conditions, poly(10,16-DHPA and poly(methyl-10,16-DHHD with Mw = 814 and Mn = 1,206 Da, and Mw = 982 and Mn = 860 Da, respectively, were formed according to their MALDI-TOF MS and ESI-MS data. The self-assembly of the polyesters obtained were analyzed by AFM.

  5. Strong Screening Effect of Polyhedral Oligomeric Silsesquioxanes (POSS Nanoparticles on Hydrogen Bonded Polymer Blends

    Directory of Open Access Journals (Sweden)

    Chin-Wei Chiou

    2014-03-01

    Full Text Available In this study we used anionic living polymerization to prepare two different homopolymers: a poly(methyl methacrylate (PMMA and a PMMA derivative presenting polyhedral oligomeric silsesquioxane (PMA-POSS units as its side chains. We then employed differential scanning calorimetry (DSC, Fourier transform infrared (FTIR spectroscopy, and wide-angle X-ray diffraction (WAXD to investigate the miscibility and specific interactions of PMMA and PMA-POSS with three hydrogen bonding donor compounds: poly(vinyl phenol (PVPh, phenolic resin, and bisphenol A (BPA. DSC revealed that all of the PVPh/PMMA, phenolic/PMMA, and BPA/PMMA blends exhibited a single glass transition temperature, characteristic of miscible systems; FTIR spectroscopic analyses revealed that such miscibility resulted from hydrogen bonding interactions between the C=O groups of PMMA and the OH groups of these three hydrogen bonding donor compounds. In contrast, all of the PVPh/PMA-POSS, phenolic/PMA-POSS, and BPA/PMA-POSS blends were immiscible: DSC revealed two glass transition temperatures arising from strong screening effects (FTIR spectroscopy and high degrees of aggregation (WAXD of the POSS nanoparticles. We propose that the value of the intramolecular screening effect (γ should be very close to 1 for all PMA-POSS blend systems when POSS nanoparticles appear as the side chains of PMMA, such that the OH groups of the hydrogen bonding donor compounds cannot interact with the C=O groups of PMA-POSS.

  6. The role of Vif oligomerization and RNA chaperone activity in HIV-1 replication.

    Science.gov (United States)

    Batisse, Julien; Guerrero, Santiago; Bernacchi, Serena; Sleiman, Dona; Gabus, Caroline; Darlix, Jean-Luc; Marquet, Roland; Tisné, Carine; Paillart, Jean-Christophe

    2012-11-01

    The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells that involve most natural HIV-1 target cells. Vif counteracts the packaging of two cellular cytidine deaminases named APOBEC3G (A3G) and A3F by diverse mechanisms including the recruitment of an E3 ubiquitin ligase complex and the proteasomal degradation of A3G/A3F, the inhibition of A3G mRNA translation or by a direct competition mechanism. In addition, Vif appears to be an active partner of the late steps of viral replication by participating in virus assembly and Gag processing, thus regulating the final stage of virion formation notably genomic RNA dimerization and by inhibiting the initiation of reverse transcription. Vif is a small pleiotropic protein with multiple domains, and recent studies highlighted the importance of Vif conformation and flexibility in counteracting A3G and in binding RNA. In this review, we will focus on the oligomerization and RNA chaperone properties of Vif and show that the intrinsic disordered nature of some Vif domains could play an important role in virus assembly and replication. Experimental evidence demonstrating the RNA chaperone activity of Vif will be presented.

  7. Serum cartilage oligomeric matrix protein: is there a repeated bout effect?

    Directory of Open Access Journals (Sweden)

    Michael Behringer

    2014-10-01

    Full Text Available The primary aim of the present study was to investigate if there is a repeated bout effect for cartilage tissue, evident in the marker serum cartilage oligomeric matrix protein (sCOMP. Ten healthy male subjects (26.4±3.14 years performed two high impact interventions (100 drop jumps with a 30 second interval carried out at a 3 week interval. After each intervention, sCOMP and muscle soreness were assessed on 8 and 6 occasions respectively. Muscle soreness was determined via a visual analog scale with a maximum pain score of 10. sComp levels did not show a blunted response after the second bout (Bout 1: 12.2±3.3 U/L−1; Bout 2: 13.1±4.0 U/L−1; P>0.05. Remarkably, sCOMP increased from baseline levels by 16% after bout 1 and 15% after bout 2. Muscle soreness was blunted following the second intervention (Bout 1: 5.0±1.8; Bout 2: 1.6±0.8. Unlike the known repeated bout effect for muscle damage markers, sCOMP levels do not show a blunted response after two similar loading interventions. This information on biomarker behavior is essential to clinicians attempting to use this marker as an indicator of cartilage damage associated with the development or progression of osteoarthritis.

  8. Anticancer effects of oligomeric proanthocyanidins on human colorectal cancer cell line, SNU-C4

    Institute of Scientific and Technical Information of China (English)

    Youn-Jung Kim; Hae-Jeong Park; Seo-Hyun Yoon; Mi-Ja Kim; Kang-Hyun Leem; Joo-Ho Chung; Hye-Kyung Kim

    2005-01-01

    AIM: Oligomeric proanthocyanidins (OPC), natural polyphenolic compounds found in plants, are known to have antioxidant and anti-cancer effects. We investigated whether the anti-cancer effects of the OPC are induced by apoptosis on human colorectal cancer cell line, SNU-C4.METHODS: Colorectal cancer cell line, SNU-C4 was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. The cytotoxic effect of OPC was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenylt-etrazolium bromide (MTT) assay. To find out the apoptotic cell death, 4, 6-diamidino-2-phenylindole (DAPI) staining,terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, reverse transcriptionpolymerase chain reaction (RT-PCR), and caspase-3 enzyme assay were performed.RESULTS: In this study, cytotoxic effect of OPC on SNUC4 cells appeared in a dose-dependent manner. OPC treatment (100 μg/mL) revealed typical morphological apoptotic features. Additionally OPC treatment (100 μg/mL)increased level of BAX and CASPASE-3, and decreased level of BCL-2 mRNA expression. Caspase-3 enzyme activity was also significantly increased by treatment of OPC (100 μg/mL) compared with control.CONCLUSION: These data indicate that OPC caused cell death by apoptosis through caspase pathways on human colorectal cancer cell line, SNU-C4.

  9. Coatings of molecularly imprinted polymers based on polyhedral oligomeric silsesquioxane for open tubular capillary electrochromatography.

    Science.gov (United States)

    Zhao, Qing-Li; Zhou, Jin; Zhang, Li-Shun; Huang, Yan-Ping; Liu, Zhao-Sheng

    2016-05-15

    Polyhedral oligomeric silsesquioxane (POSS) was successfully applied, for the first time, to prepare imprinted monolithic coating for capillary electrochromatography. The imprinted monolithic coating was synthesized with a mixture of PSS-(1-Propylmethacrylate)-heptaisobutyl substituted (MA 0702), S-amlodipine (template), methacrylic acid (functional monomer), and 2-methacrylamidopropyl methacrylate (crosslinker), in a porogenic mixture of toluene-isooctane. The influence of synthesis parameters on the imprinting effect and separation performance, including the amount of MA 0702, the ratio of template to monomer, and the ratio of monomer to crosslinker, was investigated. The greatest resolution for enantiomers separation on the imprinted monolithic column prepared with MA 0702 was up to 22.3, about 2 times higher than that prepared in absence of the POSS. Column efficiency on the POSS-based MIP coatings was beyond 30,000 plate m(-1). The comparisons between MIP coating synthesized with the POSS and without the POSS were made in terms of selectivity, column efficiency, and resolution. POSS-based MIP capillaries with naproxen or zopiclone was also prepared and separation of enantiomers can be achieved.

  10. Non-oxidative dehydro-oligomerization of methane to higher molecular weight hydrocarbons at low temperatures

    Institute of Scientific and Technical Information of China (English)

    王林胜; 徐奕德; 陶龙骧

    1997-01-01

    The non-oxidative dehydro-oligomerization of methane to higher molecular weight hydrocarbons such as aroma tics and C2 hydrocarbons in a low temperature range of 773-973 K with Mo/HZSM-5,Mo-Zr/HZSM-5 and Mo-W/HZSM-5 catalysts is studied.The means for enhancing the activity and stability of the Mo-containing catalysts under the reaction conditions is reported.Quite a stable methane conversion rate of over 10% with a high selectivity to the higher hydrocarbons has been obtained at a temperature of 973 K.Pure methane conversions of about 5.2% and 2.0% have been obtained at 923 and 873 K,respectively.In addition,accompanied by the C2-C3 mixture,tht- methane reaction can be initiated even at a lower temperature and the conversion rate of methane is enhanced by the presence of tne initiator of C2-C3 hydrocarbons.Compared with methane oxidative coupling to ethylene,the novel way for methane transformation is significant and reasonable for its lower reaction temperatures and high selectivity to the desired prod

  11. An integrative approach to the study of filamentous oligomeric assemblies, with application to RecA.

    Directory of Open Access Journals (Sweden)

    Benjamin Boyer

    Full Text Available Oligomeric macromolecules in the cell self-organize into a wide variety of geometrical motifs such as helices, rings or linear filaments. The recombinase proteins involved in homologous recombination present many such assembly motifs. Here, we examine in particular the polymorphic characteristics of RecA, the most studied member of the recombinase family, using an integrative approach that relates local modes of monomer/monomer association to the global architecture of their screw-type organization. In our approach, local modes of association are sampled via docking or Monte Carlo simulations. This enables shedding new light on fiber morphologies that may be adopted by the RecA protein. Two distinct RecA helical morphologies, the so-called "extended" and "compressed" forms, are known to play a role in homologous recombination. We investigate the variability within each form in terms of helical parameters and steric accessibility. We also address possible helical discontinuities in RecA filaments due to multiple monomer-monomer association modes. By relating local interface organization to global filament morphology, the strategies developed here to study RecA self-assembly are particularly well suited to other DNA-binding proteins and to filamentous protein assemblies in general.

  12. Small-molecule inhibition of STOML3 oligomerization reverses pathological mechanical hypersensitivity.

    Science.gov (United States)

    Wetzel, Christiane; Pifferi, Simone; Picci, Cristina; Gök, Caglar; Hoffmann, Diana; Bali, Kiran K; Lampe, André; Lapatsina, Liudmila; Fleischer, Raluca; Smith, Ewan St John; Bégay, Valérie; Moroni, Mirko; Estebanez, Luc; Kühnemund, Johannes; Walcher, Jan; Specker, Edgar; Neuenschwander, Martin; von Kries, Jens Peter; Haucke, Volker; Kuner, Rohini; Poulet, James F A; Schmoranzer, Jan; Poole, Kate; Lewin, Gary R

    2017-02-01

    The skin is equipped with specialized mechanoreceptors that allow the perception of the slightest brush. Indeed, some mechanoreceptors can detect even nanometer-scale movements. Movement is transformed into electrical signals via the gating of mechanically activated ion channels at sensory endings in the skin. The sensitivity of Piezo mechanically gated ion channels is controlled by stomatin-like protein-3 (STOML3), which is required for normal mechanoreceptor function. Here we identify small-molecule inhibitors of STOML3 oligomerization that reversibly reduce the sensitivity of mechanically gated currents in sensory neurons and silence mechanoreceptors in vivo. STOML3 inhibitors in the skin also reversibly attenuate fine touch perception in normal mice. Under pathophysiological conditions following nerve injury or diabetic neuropathy, the slightest touch can produce pain, and here STOML3 inhibitors can reverse mechanical hypersensitivity. Thus, small molecules applied locally to the skin can be used to modulate touch and may represent peripherally available drugs to treat tactile-driven pain following neuropathy.

  13. Cartilage Oligomeric Matrix Protein Angiopoeitin-1 Provides Benefits During Nerve Regeneration In Vivo and In Vitro.

    Science.gov (United States)

    Qiu, Longhai; He, Bo; Hu, Jun; Zhu, Zhaowei; Liu, Xiaolin; Zhu, Jiakai

    2015-12-01

    Our group pioneered the study of nerve regeneration in China and has successfully developed human "acellular nerve grafts (ACNGs)". However, our clinical studies revealed that the effects of ACNGs for long and large nerve defects are far from satisfactory. To improve the efficacy of ACNGs, we combined Cartilage oligomeric matrix protein angiopoietin-1 (COMP-Ang1) with ACNGs in rat sciatic nerve injury models and observed the outcomes via angiographic, morphological, and functional analyses. Co-cultures of endothelial cells (ECs) and dorsal root ganglion neurons (DRGs) were also used to characterize the relationship between neovascularization and nerve regeneration. The results showed significant improvements in early neovascularization, nerve regeneration, and functional outcomes in vivo in the ACNG + COMP-Ang1 group. In vitro, neurite length, and density as well as the expression levels of neurofilament 68 (NF68) and phosphorylated-Tie-2 (p-Tie-2) significantly increased when ECs were co-cultured with DRGs using COMP-Ang1. p-Tie-2 expression dramatically decreased after treatment with a Tie-2 kinase inhibitor (S157701), which consequently decreased the level of NF68. COMP-Ang1 can be concluded to promote early neovascularization followed by brisk nerve regeneration, and the mechanism of this regeneration may involve the modulation of the p-Tie-2 and Tie-2 receptors on ECs. These findings demonstrate that ACNGs can be modified using COMP-Ang1 to improve their efficacy in repairing peripheral nerve defects in clinical trials.

  14. Molecular cloning and functional characterization of duck nucleotide-binding oligomerization domain 1 (NOD1).

    Science.gov (United States)

    Li, Huilin; Jin, Hui; Li, Yaqian; Liu, Dejian; Foda, Mohamed Frahat; Jiang, Yunbo; Luo, Rui

    2017-09-01

    Nucleotide-binding oligomerization domain 1 (NOD1) is an imperative cytoplasmic pattern recognition receptor (PRR) and considered as a key member of the NOD-like receptor (NLR) family which plays a critical role in innate immunity through sensing microbial components derived from bacterial peptidoglycan. In the current study, the full-length of duck NOD1 (duNOD1) cDNA from duck embryo fibroblasts (DEFs) was cloned. Multiple sequence alignment and phylogenetic analysis demonstrated that duNOD1 exhibited a strong evolutionary relationship with chicken and rock pigeon NOD1. Tissue-specific expression analysis showed that duNOD1 was widely distributed in various organs, with the highest expression observed in the liver. Furthermore, duNOD1 overexpression induced NF-κB activation in DEFs and the CARD domain is crucial for duNOD1-mediated NF-κB activation. In addition, silencing the duNOD1 decreased the activity of NF-κB in DEFs stimulated by iE-DAP. Overexpression of duNOD1 significantly increased the expression of TNF-α, IL-6, and RANTES in DEFs. These findings highlight the crucial role of duNOD1 as an intracellular sensor in duck innate immune system. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Microscale fish bowls: a new class of latex particles with hollow interiors and engineered porous structures in their surfaces.

    Science.gov (United States)

    Jeong, Unyong; Im, Sang Hyuk; Camargo, Pedro H C; Kim, Jung Hyun; Xia, Younan

    2007-10-23

    Microscale fish bowls, hollow particles with engineered holes in their surfaces, were prepared using two different methods. In the first method, commercial latex beads suspended in water were swollen with a good solvent of the polymer, followed by freezing with liquid nitrogen and evaporation of the solvent below 0 degrees C. While one big hole was generated when the amount of solvent used for the swelling was relatively low, small holes could be produced in the outer surface of each bowl by increasing the degree of swelling. The porosity and pore structure show a similar dependence on the degree of swelling for both amorphous and semicrystalline polymers even though they are supposed to exhibit different phase behaviors during the freezing and solvent evaporation processes. In the second method, a polymer emulsion in water was prepared and then frozen with liquid nitrogen, followed by solvent evaporation below 0 degrees C. The porosity and pore structure could be controlled by adjusting the concentration of the polymer solution used to prepare the emulsion. As for encapsulation, the bowl-shaped particles could be transformed back into solid beads via thermal annealing at a temperature near the glass transition temperature of the polymer or by adding a good solvent of the polymer to the colloidal suspension. In a proof-of-concept experiment, microscale fish bowls were fabricated from poly(caprolactone), quickly loaded with a fluorescent dye, and sealed through thermal annealing. The encapsulated dye could then be slowly released in a phosphate buffered saline, suggesting their potential use as a new class of microscale capsules for drug delivery.

  16. Low molar excess of 4-oxo-2-nonenal and 4-hydroxy-2-nonenal promote oligomerization of alpha-synuclein through different pathways.

    Science.gov (United States)

    Almandoz-Gil, Leire; Welander, Hedvig; Ihse, Elisabet; Khoonsari, Payam Emami; Musunuri, Sravani; Lendel, Christofer; Sigvardson, Jessica; Karlsson, Mikael; Ingelsson, Martin; Kultima, Kim; Bergström, Joakim

    2017-09-01

    Aggregated alpha-synuclein is the main component of Lewy bodies, intraneuronal inclusions found in brains with Parkinson's disease and dementia with Lewy bodies. A body of evidence implicates oxidative stress in the pathogenesis of these diseases. For example, a large excess (30:1, aldehyde:protein) of the lipid peroxidation end products 4-oxo-2-nonenal (ONE) or 4-hydroxy-2-nonenal (HNE) can induce alpha-synuclein oligomer formation. The objective of the study was to investigate the effect of these reactive aldehydes on alpha-synuclein at a lower molar excess (3:1) at both physiological (7.4) and acidic (5.4) pH. As observed by size-exclusion chromatography, ONE rapidly induced the formation of alpha-synuclein oligomers at both pH values, but the effect was less pronounced under the acidic condition. In contrast, only a small proportion of alpha-synuclein oligomers were formed with low excess HNE-treatment at physiological pH and no oligomers at all under the acidic condition. With prolonged incubation times (up to 96h), more alpha-synuclein was oligomerized at physiological pH for both ONE and HNE. As determined by Western blot, ONE-oligomers were more SDS-stable and to a higher-degree cross-linked as compared to the HNE-induced oligomers. However, as shown by their greater sensitivity to proteinase K treatment, ONE-oligomers, exhibited a less compact structure than HNE-oligomers. As indicated by mass spectrometry, ONE modified most Lys residues, whereas HNE primarily modified the His50 residue and fewer Lys residues, albeit to a higher degree than ONE. Taken together, our data show that the aldehydes ONE and HNE can modify alpha-synuclein and induce oligomerization, even at low molar excess, but to a higher degree at physiological pH and seemingly through different pathways. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  17. A method for the separation of hybrids of chromatographically identical oligomeric proteins. Use of 3,4,5,6-tetrahydrophthaloyl groups as a reversible "chromatographic handle".

    Science.gov (United States)

    Gibbons, I; Schachman, H K

    1976-01-13

    Hybridization experiments with variants of an oligomeric protein often provide important information regarding subunit structure, function, and interactions. In some systems, however, the variants are so similar electrophoretically and chromatographically that purification of individual hybrids is not feasible. Therefore a method was developed for preparing hybrids by using 3,4,5,6-tetrahydrophthalic anhydride as a reversible acylating agent for protein amino groups. The technique involved acylating about 30% of the amino groups at pH 8 to give a derivative with a markedly altered net charge, formation of the hybrid set with unmodified and modified species, separation of the individual components by ion-exchange chromatography, and finally removal of the tetrahydrophthaloyl groups from the desired hybrid by incubation for about 1 day at pH 6 and room temperature. Experiments with model compounds and two enzymes showed that the anhydride was sepcific for amino groups. The extent of modification of proteins was measured by the spectral change at 250 nm, the loss of free amino groups, and the change in electrophoretic mobility of the polypeptide chains in polyacrylamide gels containing 8 M urea. Deacylation of modified, inactive aldolase and the catalytic subunit of aspartate transcarbamylase led to the restoration of the enzyme activity and electrophoretic mobility of the unmodified proteins. Both intra- and inter-subunit hybrids of aspartate transcarbamylase were prepared and isolated by using the tetrahydrophthaloyl groups as a reversible "chromatographic handle". Prior to deacylation the inter-subunit hybrid containing one acylated and one native catalytic subunit (and negative regulatory sub-units) exhibited no homotropic cooperativity and after deacylation the characteristic allosteric properties of the enzyme were regained. Similarly the ligand-promoted conformational changes associated with the allosteric transition were resotred upon deacylation of the intra

  18. Investigation of monomeric and oligomeric wine stilbenoids in red wines by ultra-high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry.

    Science.gov (United States)

    Moss, Ryan; Mao, Qinyong; Taylor, Dennis; Saucier, Cédric

    2013-08-30

    Stilbenoids are secondary plant metabolites responsible for the protection of multiple plant species including grape vine from bacterial and fungal infection. Red wine has been shown to be a major source of these compounds in the human diet, where they display an array of health benefits. Providing a more complete profile of the stilbenoids present in red wine, this study detects 41 stilbenoid compounds, 23 of which have never before been detected in red wine. Red wine extracts were scanned using an ultra-high-performance liquid chromatograph coupled to a hybrid quadrupole time-of-flight mass analyzer. Multiple targeted MS/MS precursor ion scan experiments were performed using electrospray ionization operated in negative mode. Precursor ion masses were scanned for the monomeric and oligomeric stilbenoids, as well as modifications such as O-glycosylation, methoxylation and oxidation products of these compounds. Accurate mass precursor and characteristic product ions afforded partial structural elucidation and assignment of these compounds. A total of 41 (both known and novel) stilbenoids were detected in extracted red wine. In addition to the well-known monomeric stilbenes, several resveratrol-resveratrol homodimers (m/z 453.1344), resveratrol-piceatannol heterodimers (m/z 469.1293) and piceatannol-piceatannol homodimers (m/z 485.1236) were detected. Modified dimers of resveratrol including O-glycosylated (m/z 615.1872), methoxylated (m/z 485.1606) and oxidized (m/z 471.1449) dimers were also detected. Multiple trimers of resveratrol (m/z 679.1978) were detected for the first time in red wine, as well as some known and some novel stilbenoid tetramers (m/z 905.2604). In summary, 41 stilbenoids were detected in red wine, 23 for the first time. Both monomeric and oligomeric stilbenoids were partially identified and assigned by their accurate mass precursor ions and characteristic stilbenoid fragmentation patterns. Knowledge gained from these experiments contributes to

  19. Ligand-dependent conformations and dynamics of the serotonin 5-HT(2A receptor determine its activation and membrane-driven oligomerization properties.

    Directory of Open Access Journals (Sweden)

    Jufang Shan

    Full Text Available From computational simulations of a serotonin 2A receptor (5-HT(2AR model complexed with pharmacologically and structurally diverse ligands we identify different conformational states and dynamics adopted by the receptor bound to the full agonist 5-HT, the partial agonist LSD, and the inverse agonist Ketanserin. The results from the unbiased all-atom molecular dynamics (MD simulations show that the three ligands affect differently the known GPCR activation elements including the toggle switch at W6.48, the changes in the ionic lock between E6.30 and R3.50 of the DRY motif in TM3, and the dynamics of the NPxxY motif in TM7. The computational results uncover a sequence of steps connecting these experimentally-identified elements of GPCR activation. The differences among the properties of the receptor molecule interacting with the ligands correlate with their distinct pharmacological properties. Combining these results with quantitative analysis of membrane deformation obtained with our new method (Mondal et al, Biophysical Journal 2011, we show that distinct conformational rearrangements produced by the three ligands also elicit different responses in the surrounding membrane. The differential reorganization of the receptor environment is reflected in (i-the involvement of cholesterol in the activation of the 5-HT(2AR, and (ii-different extents and patterns of membrane deformations. These findings are discussed in the context of their likely functional consequences and a predicted mechanism of ligand-specific GPCR oligomerization.

  20. A High Diversity in Chitinolytic and Chitosanolytic Species and Enzymes and Their Oligomeric Products Exist in Soil with a History of Chitin and Chitosan Exposure.

    Science.gov (United States)

    Nampally, Malathi; Rajulu, M B Govinda; Gillet, Dominique; Suryanarayanan, T S; Moerschbacher, Bruno B

    2015-01-01

    Chitin is one of the most abundant biomolecules on earth, and its partially de-N-acetylated counterpart, chitosan, is one of the most promising biotechnological resources due to its diversity in structure and function. Recently, chitin and chitosan modifying enzymes (CCMEs) have gained increasing interest as tools to engineer chitosans with specific functions and reliable performance in biotechnological and biomedical applications. In a search for novel CCME, we isolated chitinolytic and chitosanolytic microorganisms from soils with more than ten-years history of chitin and chitosan exposure and screened them for chitinase and chitosanase isoenzymes as well as for their patterns of oligomeric products by incubating their secretomes with chitosan polymers. Of the 60 bacterial strains isolated, only eight were chitinolytic and/or chitosanolytic, while 20 out of 25 fungal isolates were chitinolytic and/or chitosanolytic. The bacterial isolates produced rather similar patterns of chitinolytic and chitosanolytic enzymes, while the fungal isolates produced a much broader range of different isoenzymes. Furthermore, diverse mixtures of oligosaccharides were formed when chitosan polymers were incubated with the secretomes of select fungal species. Our study indicates that soils with a history of chitin and chitosan exposure are a good source of novel CCME for chitosan bioengineering.

  1. In vitro recombination catalyzed by bacterial class 1 integron integrase IntI1 involves cooperative binding and specific oligomeric intermediates.

    Directory of Open Access Journals (Sweden)

    Véronique Dubois

    Full Text Available Gene transfer via bacterial integrons is a major pathway for facilitating the spread of antibiotic resistance genes across bacteria. Recently the mechanism underlying the recombination catalyzed by class 1 integron recombinase (IntI1 between attC and attI1 was highlighted demonstrating the involvement of a single-stranded intermediary on the attC site. However, the process allowing the generation of this single-stranded substrate has not been determined, nor have the active IntI1*DNA complexes been identified. Using the in vitro strand transfer assay and a crosslink strategy we previously described we demonstrated that the single-stranded attC sequences could be generated in the absence of other bacterial proteins in addition to IntI. This suggests a possible role for this protein in stabilizing and/or generating this structure. The mechanism of folding of the active IntI*DNA complexes was further analyzed and we show here that it involves a cooperative binding of the protein to each recombination site and the emergence of different oligomeric species specific for each DNA substrate. These findings provide further insight into the recombination reaction catalyzed by IntI1.

  2. Ubiquitin regulates caspase recruitment domain-mediated signaling by nucleotide-binding oligomerization domain-containing proteins NOD1 and NOD2.

    Science.gov (United States)

    Ver Heul, Aaron M; Fowler, C Andrew; Ramaswamy, S; Piper, Robert C

    2013-03-08

    NOD1 and NOD2 (nucleotide-binding oligomerization domain-containing proteins) are intracellular pattern recognition receptors that activate inflammation and autophagy. These pathways rely on the caspase recruitment domains (CARDs) within the receptors, which serve as protein interaction platforms that coordinately regulate immune signaling. We show that NOD1 CARD binds ubiquitin (Ub), in addition to directly binding its downstream targets receptor-interacting protein kinase 2 (RIP2) and autophagy-related protein 16-1 (ATG16L1). NMR spectroscopy and structure-guided mutagenesis identified a small hydrophobic surface of NOD1 CARD that binds Ub. In vitro, Ub competes with RIP2 for association with NOD1 CARD. In vivo, we found that the ligand-stimulated activity of NOD1 with a mutant CARD lacking Ub binding but retaining ATG16L1 and RIP2 binding is increased relative to wild-type NOD1. Likewise, point mutations in the tandem NOD2 CARDs at positions analogous to the surface residues defining the Ub interface on NOD1 resulted in loss of Ub binding and increased ligand-stimulated NOD2 signaling. These data suggest that Ub binding provides a negative feedback loop upon NOD-dependent activation of RIP2.

  3. The role of surface electrostatics on the stability, function and regulation of human cystathionine β-synthase, a complex multidomain and oligomeric protein.

    Science.gov (United States)

    Pey, Angel L; Majtan, Tomas; Kraus, Jan P

    2014-09-01

    Human cystathionine β-synthase (hCBS) is a key enzyme of sulfur amino acid metabolism, controlling the commitment of homocysteine to the transsulfuration pathway and antioxidant defense. Mutations in hCBS cause inherited homocystinuria (HCU), a rare inborn error of metabolism characterized by accumulation of toxic homocysteine in blood and urine. hCBS is a complex multidomain and oligomeric protein whose activity and stability are independently regulated by the binding of S-adenosyl-methionine (SAM) to two different types of sites at its C-terminal regulatory domain. Here we study the role of surface electrostatics on the complex regulation and stability of hCBS using biophysical and biochemical procedures. We show that the kinetic stability of the catalytic and regulatory domains is significantly affected by the modulation of surface electrostatics through noticeable structural and energetic changes along their denaturation pathways. We also show that surface electrostatics strongly affect SAM binding properties to those sites responsible for either enzyme activation or kinetic stabilization. Our results provide new insight into the regulation of hCBS activity and stability in vivo with implications for understanding HCU as a conformational disease. We also lend experimental support to the role of electrostatic interactions in the recently proposed binding modes of SAM leading to hCBS activation and kinetic stabilization.

  4. New nanoplatforms based on UCNPs linking with polyhedral oligomeric silsesquioxane (POSS) for multimodal bioimaging

    Science.gov (United States)

    Ge, Xiaoqian; Dong, Liang; Sun, Lining; Song, Zhengmei; Wei, Ruoyan; Shi, Liyi; Chen, Haige

    2015-04-01

    A new and facile method was used to transfer upconversion luminescent nanoparticles from hydrophobic to hydrophilic using polyhedral oligomeric silsesquioxane (POSS) linking on the surface of upconversion nanoparticles. In comparison with the unmodified upconversion nanoparticles, the POSS modified upconversion nanoplatforms [POSS-UCNPs(Er), POSS-UCNPs(Tm)] displayed good monodispersion in water and exhibited good water-solubility, while their particle size did not change substantially. Due to the low cytotoxicity and good biocompatibility as determined by methyl thiazolyl tetrazolium (MTT) assay and histology and hematology analysis, the POSS modified upconversion nanoplatforms were successfully applied to upconversion luminescence imaging of living cells in vitro and nude mouse in vivo (upon excitation at 980 nm). In addition, the doped Gd3+ ion endows the POSS-UCNPs with effective T1 signal enhancement and the POSS-UCNPs were successfully applied to in vivo magnetic resonance imaging (MRI) for a Kunming mouse, which makes them potential MRI positive-contrast agents. More importantly, the corner organic groups of POSS can be easily modified, resulting in kinds of POSS-UCNPs with many potential applications. Therefore, the method and results may provide more exciting opportunities for multimodal bioimaging and multifunctional applications.A new and facile method was used to transfer upconversion luminescent nanoparticles from hydrophobic to hydrophilic using polyhedral oligomeric silsesquioxane (POSS) linking on the surface of upconversion nanoparticles. In comparison with the unmodified upconversion nanoparticles, the POSS modified upconversion nanoplatforms [POSS-UCNPs(Er), POSS-UCNPs(Tm)] displayed good monodispersion in water and exhibited good water-solubility, while their particle size did not change substantially. Due to the low cytotoxicity and good biocompatibility as determined by methyl thiazolyl tetrazolium (MTT) assay and histology and hematology

  5. Quantitative FRET imaging of leptin receptor oligomerization kinetics in single cells.

    Science.gov (United States)

    Biener, Eva; Charlier, Madia; Ramanujan, V Krishnan; Daniel, Nathalie; Eisenberg, Avital; Bjørbaek, Christian; Herman, Brian; Gertler, Arieh; Djiane, Jean

    2005-12-01

    Leptin, an adipocyte-secreted hormone, signals through activation of its membrane-embedded receptor (LEPR). To study the leptin-induced events occurring in short (LEPRa) and long (LEPRb) LEPRs in the cell membrane, by FRET (fluorescence resonance energy transfer) methodology, the respective receptors, tagged at their C-terminal with CFP (cyan fluorescent protein) or YFP (yellow fluorescent protein), were prepared. The constructs encoding mLEPRa (mouse LEPRa)-YFP and mLEPRa-CFP, mLEPRb-YFP and mLEPRb-CFP were tested for biological activity in transiently transfected CHO cells (Chinese-hamster ovary cells) and HEK-293T cells (human embryonic kidney 293 T cells) for activation of STAT3 (signal transduction and activators of transcription 3)-mediated LUC (luciferase) activity and binding of radiolabelled leptin. All four constructs were biologically active and were as potent as their untagged counterparts. The localization pattern of the fused protein appeared to be confined almost entirely to the cell membrane. The leptin-dependent interaction between various types of receptors in fixed cells were studied by measuring FRET, using fluorescence lifetime imaging microscopy and acceptor photobleaching methods. Both methods yielded similar results, indicating that (1) leptin receptors expressed in the cell membrane exist mostly as preformed LEPRa/LEPRa or LEPRb/LEPRb homo-oligomers but not as LEPRb/LEPRa hetero-oligomers; (2) the appearance of transient leptin-induced FRET in cells transfected with LEPRb/LEPRb reflects both a conformational change that leads to closer interaction in the cytosolic part and a higher FRET signal, as well as de novo homo-oligomerization; (3) in LEPRa/LEPRa, exposure to leptin does not lead to any increase in FRET signalling as the proximity of CFP and YFP fluorophores in space already gives maximal FRET efficiency of the preoligomerized receptors.

  6. Marginal integrity of restorations produced with a model composite based on polyhedral oligomeric silsesquioxane (POSS

    Directory of Open Access Journals (Sweden)

    Luciano Ribeiro CORREA NETTO

    2015-10-01

    Full Text Available Marginal integrity is one of the most crucial aspects involved in the clinical longevity of resin composite restorations.Objective To analyze the marginal integrity of restorations produced with a model composite based on polyhedral oligomeric silsesquioxane (POSS.Material and Methods A base composite (B was produced with an organic matrix with UDMA/TEGDMA and 70 wt.% of barium borosilicate glass particles. To produce the model composite, 25 wt.% of UDMA were replaced by POSS (P25. The composites P90 and TPH3 (TP3 were used as positive and negative controls, respectively. Marginal integrity (%MI was analyzed in bonded class I cavities. The volumetric polymerization shrinkage (%VS and the polymerization shrinkage stress (Pss - MPa were also evaluated.Results The values for %MI were as follows: P90 (100% = TP3 (98.3% = B (96.9% > P25 (93.2%, (p<0.05. The %VS ranged from 1.4% (P90 to 4.9% (P25, while Pss ranged from 2.3 MPa (P90 to 3.9 MPa (B. For both properties, the composite P25 presented the worst results (4.9% and 3.6 MPa. Linear regression analysis showed a strong positive correlation between %VS and Pss (r=0.97, whereas the correlation between Pss and %MI was found to be moderate (r=0.76.Conclusions The addition of 25 wt.% of POSS in methacrylate organic matrix did not improve the marginal integrity of class I restorations. Filtek P90 showed lower polymerization shrinkage and shrinkage stress when compared to the experimental and commercial methacrylate composite.

  7. Isolated Toll-like receptor transmembrane domains are capable of oligomerization.

    Directory of Open Access Journals (Sweden)

    James I Godfroy

    Full Text Available Toll-like receptors (TLRs act as the first line of defense against bacterial and viral pathogens by initiating critical defense signals upon dimer activation. The contribution of the transmembrane domain in the dimerization and signaling process has heretofore been overlooked in favor of the extracellular and intracellular domains. As mounting evidence suggests that the transmembrane domain is a critical region in several protein families, we hypothesized that this was also the case for Toll-like receptors. Using a combined biochemical and biophysical approach, we investigated the ability of isolated Toll-like receptor transmembrane domains to interact independently of extracellular domain dimerization. Our results showed that the transmembrane domains had a preference for the native dimer partners in bacterial membranes for the entire receptor family. All TLR transmembrane domains showed strong homotypic interaction potential. The TLR2 transmembrane domain demonstrated strong heterotypic interactions in bacterial membranes with its known interaction partners, TLR1 and TLR6, as well as with a proposed interaction partner, TLR10, but not with TLR4, TLR5, or unrelated transmembrane receptors providing evidence for the specificity of TLR2 transmembrane domain interactions. Peptides for the transmembrane domains of TLR1, TLR2, and TLR6 were synthesized to further study this subfamily of receptors. These peptides validated the heterotypic interactions seen in bacterial membranes and demonstrated that the TLR2 transmembrane domain had moderately strong interactions with both TLR1 and TLR6. Combined, these results suggest a role for the transmembrane domain in Toll-like receptor oligomerization and as such, may be a novel target for further investigation of new therapeutic treatments of Toll-like receptor mediated diseases.

  8. Binding and Oligomerization of Modified and Native Bt Toxins in Resistant and Susceptible Pink Bollworm.

    Directory of Open Access Journals (Sweden)

    Josue Ocelotl

    Full Text Available Insecticidal proteins from Bacillus thuringiensis (Bt are used extensively in sprays and transgenic crops for pest control, but their efficacy is reduced when pests evolve resistance. Better understanding of the mode of action of Bt toxins and the mechanisms of insect resistance is needed to enhance the durability of these important alternatives to conventional insecticides. Mode of action models agree that binding of Bt toxins to midgut proteins such as cadherin is essential for toxicity, but some details remain unresolved, such as the role of toxin oligomers. In this study, we evaluated how Bt toxin Cry1Ac and its genetically engineered counterpart Cry1AcMod interact with brush border membrane vesicles (BBMV from resistant and susceptible larvae of Pectinophora gossypiella (pink bollworm, a global pest of cotton. Compared with Cry1Ac, Cry1AcMod lacks 56 amino acids at the amino-terminus including helix α-1; previous work showed that Cry1AcMod formed oligomers in vitro without cadherin and killed P. gossypiella larvae harboring cadherin mutations linked with >1000-fold resistance to Cry1Ac. Here we found that resistance to Cry1Ac was associated with reduced oligomer formation and insertion. In contrast, Cry1AcMod formed oligomers in BBMV from resistant larvae. These results confirm the role of cadherin in oligomerization of Cry1Ac in susceptible larvae and imply that forming oligomers without cadherin promotes toxicity of Cry1AcMod against resistant P. gossypiella larvae that have cadherin mutations.

  9. Rapid mutation of endogenous zebrafish genes using zinc finger nucleases made by Oligomerized Pool ENgineering (OPEN.

    Directory of Open Access Journals (Sweden)

    Jonathan E Foley

    Full Text Available BACKGROUND: Customized zinc finger nucleases (ZFNs form the basis of a broadly applicable tool for highly efficient genome modification. ZFNs are artificial restriction endonucleases consisting of a non-specific nuclease domain fused to a zinc finger array which can be engineered to recognize specific DNA sequences of interest. Recent proof-of-principle experiments have shown that targeted knockout mutations can be efficiently generated in endogenous zebrafish genes via non-homologous end-joining-mediated repair of ZFN-induced DNA double-stranded breaks. The Zinc Finger Consortium, a group of academic laboratories committed to the development of engineered zinc finger technology, recently described the first rapid, highly effective, and publicly available method for engineering zinc finger arrays. The Consortium has previously used this new method (known as OPEN for Oligomerized Pool ENgineering to generate high quality ZFN pairs that function in human and plant cells. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that OPEN can also be used to generate ZFNs that function efficiently in zebrafish. Using OPEN, we successfully engineered ZFN pairs for five endogenous zebrafish genes: tfr2, dopamine transporter, telomerase, hif1aa, and gridlock. Each of these ZFN pairs induces targeted insertions and deletions with high efficiency at its endogenous gene target in somatic zebrafish cells. In addition, these mutations are transmitted through the germline with sufficiently high frequency such that only a small number of fish need to be screened to identify founders. Finally, in silico analysis demonstrates that one or more potential OPEN ZFN sites can be found within the first three coding exons of more than 25,000 different endogenous zebrafish gene transcripts. CONCLUSIONS AND SIGNIFICANCE: In summary, our study nearly triples the total number of endogenous zebrafish genes successfully modified using ZFNs (from three to eight and suggests that OPEN

  10. Composite polymer electrolyte containing ionic liquid and functionalized polyhedral oligomeric silsesquioxanes for anhydrous PEM applications.

    Science.gov (United States)

    Subianto, Surya; Mistry, Mayur K; Choudhury, Namita Roy; Dutta, Naba K; Knott, Robert

    2009-06-01

    A new type of supported liquid membrane was made by combining an ionic liquid (IL) with a Nafion membrane reinforced with multifunctional polyhedral oligomeric silsesquioxanes (POSSs) using a layer-by-layer strategy for anhydrous proton-exchange membrane (PEM) application. The POSS was functionalized by direct sulfonation, and the sulfonated POSS (S-POSS) was incorporated into Nafion 117 membranes by the infiltration method. The resultant hybrid membrane shows strong ionic interaction between the Nafion matrix and the multifunctional POSS, resulting in increased glass transition temperature and thermal stability at very low loadings of S-POSS (1%). The presence of S-POSS has also improved the proton conductivity especially at low humidities, where it shows a marked increase due to its confinement in the ionic domains and promotes water uptake by capillary condensation. In order to achieve anhydrous conductivity, the IL 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (BMI-BTSI) was incorporated into these membranes to provide proton conduction in the absence of water. Although the incorporation of an IL shows a plasticizing effect on the Nafion membrane, the S-POSS composite membrane with an IL shows a higher modulus at high temperatures compared to Nafion 117 and a Nafion-IL membrane, with significantly higher proton conductivity (5 mS/cm at 150 degrees C with 20% IL). This shows the ability of the multifunctional POSS and IL to work symbiotically to achieve the desirable proton conductivity and mechanical properties of such membranes by enhancing the ionic interaction within the material.

  11. Styrene oligomerization as a molecular probe reaction for Brønsted acidity at the nanoscale.

    Science.gov (United States)

    Aramburo, Luis R; Wirick, Sue; Miedema, Piter S; Buurmans, Inge L C; de Groot, Frank M F; Weckhuysen, Bert M

    2012-05-21

    The Brønsted acid-catalyzed oligomerization of 4-fluorostyrene has been studied on a series of H-ZSM-5 zeolite powders, steamed under different conditions, with a combination of UV-Vis micro-spectroscopy and Scanning Transmission X-ray Microscopy (STXM). UV-Vis micro-spectroscopy and STXM have been used to monitor the relative formation of cyclic and linear dimeric carbocations as a function of the steaming post-treatment (i.e., parent vs. steaming at 600, 700 and 800 °C). It was found that the UV-Vis band intensity ratios of linear to cyclic dimeric species increase from 0.79 (parent H-ZSM-5) over 1.41 (H-ZSM-5 steamed at 600 °C) and 1.88 (H-ZSM-5 steamed at 700 °C) to 2.33 (H-ZSM-5 steamed at 800 °C). STXM confirms this trend in reaction product selectivity, as the relative intensities of the transitions attributed to the presence of the cyclic dimer in the carbon K-edge spectra decrease with increasing severity of the steaming post-treatment. Furthermore, STXM reveals spatial heterogeneities in reaction product formation within the H-ZSM-5 zeolite powders at the nanoscale. More specifically, a shrinking carbon core-shell distribution was detected within the zeolite aggregates, in which the relative amount of cyclic dimeric species is higher in the core relative to the shell of the zeolite aggregate and the relative amount of cyclic dimeric species in the zeolite core gradually decreases with increasing severity of the steaming post-treatment. These differences are rationalized in terms of spatial differences in Brønsted acidity within H-ZSM-5 zeolite powders as well as by changes in the formation process of linear and dimeric carbocations within H-ZSM-5 micro- and mesopores.

  12. Spontaneous Activation of Antigen-presenting Cells by Genes Encoding Truncated Homo-Oligomerizing Derivatives of CD40.

    Science.gov (United States)

    Levin, Noam; Pato, Aviad; Cafri, Gal; Eisenberg, Galit; Peretz, Tamar; Margalit, Alon; Lotem, Michal; Gross, Gideon

    The interaction between the CD40 receptor on antigen-presenting cells (APCs) and its trimeric ligand on CD4 T cells is essential for the initiation and progression of the adaptive immune response. Here we undertook to endow CD40 with the capacity to trigger spontaneous APC activation through ligand-independent oligomerization. To this end we exploited the GCN4 yeast transcriptional activator, which contains a leucine zipper DNA-binding motif that induces homophilic interactions. We incorporated GCN4 variants forming homodimers, trimers, or tetramers at the intracellular domain of human and mouse CD40 and replaced the extracellular portion with peptide-β2m or other peptide tags. In parallel we examined similarly truncated CD40 monomers lacking a GCN4 motif. The oligomeric products appeared to arrange in high-molecular-weight aggregates and were considerably superior to the monomer in their ability to trigger nuclear factor kB signaling, substantiating the anticipated constitutively active (ca) phenotype. Cumulative results in human and mouse APC lines transfected with caCD40 mRNA revealed spontaneous upregulation of CD80, IL-1β, TNFα, IL-6, and IL-12, which could be further enhanced by caTLR4 mRNA. In mouse bone-marrow-derived dendritic cells caCD40 upregulated CD80, CD86, MHC-II, and IL-12 and in human monocyte-derived dendritic cells it elevated surface CD80, CD83 CD86, CCR7, and HLA-DR. Oligomeric products carrying the peptide-β2m extracellular portion could support MHC-I presentation of the linked peptide up to 4 days post-mRNA transfection. These findings demonstrate that the expression of a single caCD40 derivative in APCs can exert multiple immunostimulatory effects, offering a new powerful tool in the design of gene-based cancer vaccines.

  13. BRET and Time-resolved FRET strategy to study GPCR oligomerization: from cell lines toward native tissues

    Directory of Open Access Journals (Sweden)

    Martin eCottet

    2012-07-01

    Full Text Available The concept of oligomerization of G protein-coupled receptor (GPCR opens new perspectives regarding physiological function regulation. The capacity of one GPCR to modify its binding and coupling properties by interacting with a second one can be at the origin of regulations unsuspected two decades ago. Although the concept is interesting, its validation at a physiological level is challenging and probably explains why receptor oligomerization is still a matter of debate.Demonstrating the direct interactions between two proteins is not trivial since few techniques present a spatial resolution allowing to conclude to close interactions. Resonance energy transfer (RET strategies are actually the most convenient ones. During the last two decades, two of them, the Bioluminescent Resonance Energy Transfer (BRET and Time-resolved Fluorescence Energy Transfer (TR-FRET have been widely used since they exhibit high signal-to-noise ratio. Most of the experiments based on GPCR labeling have been performed in cell lines and it has been shown that all GPCRs have the propensity to form homo- or hetero-oligomers. However, whether these data can be extrapolated to GPCRs expressed in native tissues and explain receptor functioning in real life, remains an open question. Native tissues impose different constraints since GPCR sequences cannot be modified. Recently a fluorescent ligand-based GPCR labeling strategy combined to TR-FRET approach has been successfully used to prove the existence of GPCR oligomerization in native tissues.Although the RET based strategies are generally quite simple to implement, precautions have to be taken before concluding to the absence or the existence of specific interactions between receptors. For example, one should exclude the possibility of collision of receptors diffusing throughout the membrane leading to a specific FRET signal. We will review the advantages and the limits of different approaches and discuss the consequent

  14. Synthesis and characterization of polyhedral oligomeric titanized silsesquioxane: A new biocompatible cage like molecule for biomedical application

    Energy Technology Data Exchange (ETDEWEB)

    Yahyaei, Hossein [Department of Polymer Engineering and Color Technology, Amirkabir University of Technology, P.O. Box 15875-4413, Tehran (Iran, Islamic Republic of); Mohseni, Mohsen, E-mail: mmohseni@aut.ac.ir [Department of Polymer Engineering and Color Technology, Amirkabir University of Technology, P.O. Box 15875-4413, Tehran (Iran, Islamic Republic of); Ghanbari, Hossein [Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences (TUMS), Tehran (Iran, Islamic Republic of); Messori, Massimo [Dipartimento di Ingegneria ‘Enzo Ferrari’, Università di Modena e Reggio Emilia, Modena (Italy)

    2016-04-01

    Organic–inorganic hybrid materials have shown improved properties to be used as biocompatible coating in biomedical applications. Polyhedral oligomeric silsesquioxane (POSS) containing coatings are among hybrid materials showing promising properties for these applications. In this work an open cage POSS has been reacted with a titanium alkoxide to end cap the POSS molecule with titanium atom to obtain a so called polyhedral oligomeric metalized silsesquioxane (POMS). The synthesized POMS was characterized by FTIR, RAMAN and UV–visible spectroscopy as well as {sup 29}Si NMR and matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) techniques. Appearance of peaks at 920 cm{sup −1} in FTIR and 491 cm{sup −1} and 1083 cm{sup −1} in Raman spectra confirmed Si–O–Ti linkage formation. It was also demonstrated that POMS was in a monomeric form. To evaluate the biocompatibility of hybrids films, pristine POSS and synthesized POMS were used in synthesis of a polycarbonate urethane polymer. Results revealed that POMS containing hybrid, not only had notable thermal and mechanical stability compared to POSS containing one, as demonstrated by DSC and DMTA analysis, they also showed controlled surface properties in such a manner that hydrophobicity and biocompatibility were both reachable to give rise to improved cell viability in presence of human umbilical vein endothelial cells (HUVEC) and MRC-5 cells. - Highlight: • Polyhedral Oligomeric Metalized Silsesquioxane (POMS) based on titanium was synthesized. • POMS can improve mechanical properties of polyurethane. • POMS increases hydrophobicity of polyurethane. • POMS is a unique nanocage to enhance biocompatibility of polyurethane.

  15. Attempted nonenzymatic template-directed oligomerizations on a polyadenylic acid template: implications for the nature of the first genetic material

    Science.gov (United States)

    Stribling, R.; Miller, S. L.

    1991-01-01

    Previous attempts to produce nonenzymatic template-directed oligomerizations of activated pyrimidines on polypurine templates have been unsuccessful. The only efficient reactions are those where the template is composed primarily of pyrimidines, especially cytosine. Because molecular evolution requires that a synthesized daughter polynucleotide be capable of acting as a template for the synthesis of the original polynucleotide, the one-way replication achieved thus far is inadequate to initiate an evolving system. Several uracil analogs were used in this investigation in order to search for possible replacements for uracil. The monomers used in this investigation were the imidazolides of UMP, xanthosine 5'-monophosphate, the bis-monophosphates of the acyclic nucleosides of uracil, and 2,4-quinazolinedione. The concentrations of various salts, buffers, pH, and temperature were among the different variables investigated in attempts to find conditions that would permit template-directed oligomerizations. Although the different monomers in this study demonstrated varying abilities to form very short oligomers, we were unable to detect any enhancement of this oligomerization that could be attributed to the poly(A) template. Although special conditions might be found that would allow purine-rich templates to work, these reactions cannot be considered robust. The results of our experiments suggest that pyrimidines were not part of the original replicating system on the primitive Earth. It has already been shown that ribose is an unlikely component of the first replicating systems, and we now suggest that phosphate was absent as well. This is due to the low solubility of phosphate in the present ocean (3 x 10(-6) M), as well as the difficulty of prebiotic activation of phosphates.

  16. Mechanical property and thermal stability of polyurethane composites reinforced with polyhedral oligomeric silsesquioxanes and inorganic flame retardant filler.

    Science.gov (United States)

    Kim, Ho-Joong; Kwon, Younghwan; Kim, Chang Kee

    2014-08-01

    Mechanical properties and thermal stability of polyurethane composites were investigated with a combination of polyhedral oligomeric silsesquioxane (POSS) molecules and inorganic barium sulfate. These hybrid composites were prepared using one-step method through the incorporation of flexible hydroxyl-terminated polybutadiene prepolymer, reactive POSS nanoparticles, and barium sulfate under isophorone diisocyanate curative system. In polyurethane composites, POSS and inorganic barium sulfate were utilized for mechanical reinforcement and flame retardant filler, respectively. The decomposition of POSS molecules during oxyacetylene torch exposure resulted in the formation of silica-based nanosized droplets, contributing on ablation behavior.

  17. On the simulation of enzymatic digest patterns: the fragmentation of oligomeric and polymeric galacturonides by endo-polygalacturonase II

    CERN Document Server

    Hunt, J J; Williams, M A K; Hunt, Jonathan J; Cameron, Randall G; Williams, Martin A.K.

    2006-01-01

    A simulation methodology for predicting the time-course of enzymatic digestions is described. The model is based solely on the enzyme's subsite architecture and concomitant binding energies. This allows subsite binding energies to be used to predict the evolution of the relative amounts of different products during the digestion of arbitrary mixtures of oligomeric or polymeric substrates. The methodology has been specifically demonstrated by studying the fragmentation of a population of oligogalacturonides of varying degrees of polymerization, when digested by endo-polygalacturonase II (endo-PG II) from Aspergillus niger.

  18. Functionalization of polyhedral oligomeric silsesquioxanes with bis(hydroxyethyl) ester and preparation of the corresponding degradable nanohybrids

    Institute of Scientific and Technical Information of China (English)

    Bing Tao Wang; Yan Zhang; Ping Zhang; Zheng Ping Fang

    2012-01-01

    One novel difunctionalized polyhedral oligomeric silsesquioxanes (POSS) derivative was designed and synthesized by a convenient method with high yield.1H NMR and FT-IR characterizations suggested that Michael addition reaction successfully took place between 2-hydroxyethyl acrylate (HEA) and aminopropylisobutyl POSS (POSS-NH2) under mild conditions,which finally formed bi(hydroxyethyl) ester-functionalized POSS derivatives (BH-POSS).Due to the similar functional groups and high reactivity,BH-POSS could be easily incorporated into the main-chain of biodegradable aliphatic-aromatic copolyesters PBTL via in situ melt polycondensation to prepare corresponding degradable nanohybrids with high mechanical properties.

  19. Structural analysis of β-glucosidase mutants derived from a hyperthermophilic tetrameric structure

    Energy Technology Data Exchange (ETDEWEB)

    Nakabayashi, Makoto; Kataoka, Misumi; Mishima, Yumiko; Maeno, Yuka; Ishikawa, Kazuhiko, E-mail: kazu-ishikawa@aist.go.jp [National Institute of Advanced Industrial Science, 3-11-32, Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046 (Japan)

    2014-03-01

    Substitutive mutations that convert a tetrameric β-glucosidase into a dimeric state lead to improvement of its crystal quality. β-Glucosidase from Pyrococcus furiosus (BGLPf) is a hyperthermophilic tetrameric enzyme which can degrade cellooligosaccharides to glucose under hyperthermophilic conditions and thus holds promise for the saccharification of lignocellulosic biomass at high temperature. Prior to the production of large amounts of this enzyme, detailed information regarding the oligomeric structure of the enzyme is required. Several crystals of BGLPf have been prepared over the past ten years, but its crystal structure had not been solved until recently. In 2011, the first crystal structure of BGLPf was solved and a model was constructed at somewhat low resolution (2.35 Å). In order to obtain more detailed structural data on BGLPf, the relationship between its tetrameric structure and the quality of the crystal was re-examined. A dimeric form of BGLPf was constructed and its crystal structure was solved at a resolution of 1.70 Å using protein-engineering methods. Furthermore, using the high-resolution crystal structural data for the dimeric form, a monomeric form of BGLPf was constructed which retained the intrinsic activity of the tetrameric form. The thermostability of BGLPf is affected by its oligomeric structure. Here, the biophysical and biochemical properties of engineered dimeric and monomeric BGLPfs are reported, which are promising prototype models to apply to the saccharification reaction. Furthermore, details regarding the oligomeric structures of BGLPf and the reasons why the mutations yielded improved crystal structures are discussed.

  20. Thermal decomposition studies of the polyhedral oligomeric silsesquioxane, POSSh, and when it is impregnated with the metallocene bis(eta5-cyclopentadienyl)zirconium (IV) dichloride or immobilized on silica.

    Science.gov (United States)

    Bianchini, D; Butler, I S; Barsan, M M; Martens, W; Frost, R L; Galland, G B; dos Santos, J H Z

    2008-11-01

    Thermal decomposition studies of the free polyhedral oligomeric silsesquioxane, POSSh, and when this compound has been impregnated with Cp2ZrCl2 (Cp = eta5-C5H5) or immobilized on SiO2 were conducted using infrared emission spectroscopy (IES) over a 100-1000 degrees C temperature range and by thermogravimetric analysis (TGA). The organic groups in POSS(h) apparently decompose thermally into Si-CH3, Si-H and other fragments. Upon impregnation with Cp2ZrCl2, however, a different thermal decomposition pathway was followed and new infrared emission bands appeared in the 1000-900 cm(-1) region suggesting the formation of Si-O-Zr moieties. When immobilized on SiO2 and subjected to thermal decomposition, the POSSh compound lost its organic groups and the inorganic structure remaining was incorporated into the SiO2 framework.

  1. Predicting success of oligomerized pool engineering (OPEN for zinc finger target site sequences

    Directory of Open Access Journals (Sweden)

    Goodwin Mathew J

    2010-11-01

    Full Text Available Abstract Background Precise and efficient methods for gene targeting are critical for detailed functional analysis of genomes and regulatory networks and for potentially improving the efficacy and safety of gene therapies. Oligomerized Pool ENgineering (OPEN is a recently developed method for engineering C2H2 zinc finger proteins (ZFPs designed to bind specific DNA sequences with high affinity and specificity in vivo. Because generation of ZFPs using OPEN requires considerable effort, a computational method for identifying the sites in any given gene that are most likely to be successfully targeted by this method is desirable. Results Analysis of the base composition of experimentally validated ZFP target sites identified important constraints on the DNA sequence space that can be effectively targeted using OPEN. Using alternate encodings to represent ZFP target sites, we implemented Naïve Bayes and Support Vector Machine classifiers capable of distinguishing "active" targets, i.e., ZFP binding sites that can be targeted with a high rate of success, from those that are "inactive" or poor targets for ZFPs generated using current OPEN technologies. When evaluated using leave-one-out cross-validation on a dataset of 135 experimentally validated ZFP target sites, the best Naïve Bayes classifier, designated ZiFOpT, achieved overall accuracy of 87% and specificity+ of 90%, with an ROC AUC of 0.89. When challenged with a completely independent test set of 140 newly validated ZFP target sites, ZiFOpT performance was comparable in terms of overall accuracy (88% and specificity+ (92%, but with reduced ROC AUC (0.77. Users can rank potentially active ZFP target sites using a confidence score derived from the posterior probability returned by ZiFOpT. Conclusion ZiFOpT, a machine learning classifier trained to identify DNA sequences amenable for targeting by OPEN-generated zinc finger arrays, can guide users to target sites that are most likely to function

  2. Stable oligomeric clusters of gold nanoparticles: preparation, size distribution, derivatization, and physical and biological properties.

    Science.gov (United States)

    Smithies, Oliver; Lawrence, Marlon; Testen, Anze; Horne, Lloyd P; Wilder, Jennifer; Altenburg, Michael; Bleasdale, Ben; Maeda, Nobuyo; Koklic, Tilen

    2014-11-11

    Reducing dilute aqueous HAuCl4 with NaSCN under alkaline conditions produces 2-3 nm diameter yellow nanoparticles without the addition of extraneous capping agents. We here describe two very simple methods for producing highly stable oligomeric grape-like clusters (oligoclusters) of these small nanoparticles. The oligoclusters have well-controlled diameters ranging from ∼5 to ∼30 nm, depending mainly on the number of subunits in the cluster. Our first ["delay-time"] method controls the size of the oligoclusters by varying from seconds to hours the delay time between making the HAuCl4 alkaline and adding the reducing agent, NaSCN. Our second ["add-on"] method controls size by using yellow nanoparticles as seeds onto which varying amounts of gold derived from "hydroxylated gold", Na(+)[Au(OH4-x)Clx](-), are added-on catalytically in the presence of NaSCN. Possible reaction mechanisms and a simple kinetic model fitting the data are discussed. The crude oligocluster preparations have narrow size distributions, and for most purposes do not require fractionation. The oligoclusters do not aggregate after ∼300-fold centrifugal-filter concentration, and at this high concentration are easily derivatized with a variety of thiol-containing reagents. This allows rare or expensive derivatizing reagents to be used economically. Unlike conventional glutathione-capped nanoparticles of comparable gold content, large oligoclusters derivatized with glutathione do not aggregate at high concentrations in phosphate-buffered saline (PBS) or in the circulation when injected into mice. Mice receiving them intravenously show no visible signs of distress. Their sizes can be made small enough to allow their excretion in the urine or large enough to prevent them from crossing capillary basement membranes. They are directly visible in electron micrographs without enhancement, and can model the biological fate of protein-like macromolecules with controlled sizes and charges. The ease of

  3. Crystal structures of resorcin[4]arene and pyrogallol[4]arene complexes with DL-pipecolinic acid. Model compounds for the recognition of the pipecolinyl ring, a key fragment of FK506, through C-H⋯π interaction

    Science.gov (United States)

    Fujisawa, Ikuhide; Kitamura, Yuji; Kato, Ryo; Murayama, Kazutaka; Aoki, Katsuyuki

    2014-01-01

    Resorcin[4]arene (resorcinol cyclic tetramer, abbreviated as RCT) or pyrogallol[4]arene (pyrogallol cyclic tetramer, PCT) form host-guest 1:1 complexes with DL-pipecolinic acid (DL-pipeH), RCT·DL-pipeH·EtOH·8H2O (1), PCT DL-pipeH·EtOH·4H2O (2), and PCT·DL-pipeH·3H2O (3), whose crystal structures have been determined. In each complex, the pipeH ligand is incorporated into the bowl-shaped cavity of the RCT or PCT host molecules through C-H⋯π interactions between alkyl protons of the piperidine ring of pipeH and π-rings of RCT or PCT, forming an [(RCT/PCT)·pipeH] structural fragment. In 1 and 3, two [(RCT/PCT) pipeH] fragments self-associate across an inversion center to form a guest-mediated, obliquely declined dimeric structure [(RCT/PCT)·L-pipeH·D-pipeH (RCT/PCT)]. In 2, each PCT-capped pipeH ligand bridges to two adjacent PCT molecules to form guest-mediated, optically-discrete helical polymers [PCT·L-pipeH]n or [PCT·D-pipeH]n. An 1H NMR experiment shows that the complexation through C-H⋯π interaction between the piperidine ring of pipeH and π-rings of RCT or PCT occurs also in solution, with the binding constants of 9.7 ± 0.6 M-1 for RCT and 26.5 ± 1.5 M-1 for PCT. These complexes provide a synthetic model for the recognition of the pipecolinyl-ring moiety, a key constituent of immunosuppressant drugs such as FK506, FK520 or rapamycin, by their binding proteins through C-H⋯π interaction.

  4. Cartilage oligomeric matrix protein neoepitope in the synovial fluid of horses with acute lameness: A new biomarker for the early stages of osteoarthritis.

    Science.gov (United States)

    Skiöldebrand, E; Ekman, S; Mattsson Hultén, L; Svala, E; Björkman, K; Lindahl, A; Lundqvist, A; Önnerfjord, P; Sihlbom, C; Rüetschi, U

    2017-09-01

    Clinical tools to diagnose the early changes of osteoarthritis (OA) that occur in the articular cartilage are lacking. We sought to identify and quantify a novel cartilage oligomeric matrix protein (COMP) neoepitope in the synovial fluid from the joints of healthy horses and those with different stages of OA. In vitro quantitative proteomics and assay development with application in synovial fluids samples obtained from biobanks of well-characterised horses. Articular cartilage explants were incubated with or without interleukin-1β for 25 days. Media were analysed via quantitative proteomics. Synovial fluid was obtained from either normal joints (n = 15) or joints causing lameness (n = 17) or with structural OA lesions (n = 7) and analysed for concentrations of the COMP neoepitope using a custom-developed inhibition enzyme-linked immunosorbent assay (ELISA). Explants were immunostained with polyclonal antibodies against COMP and the COMP neoepitopes. Semitryptic COMP peptides were identified and quantified in cell culture media from cartilage explants. A rabbit polyclonal antibody was raised against the neoepitope of the N-terminal portion of one COMP fragment (sequence SGPTHEGVC). An inhibition ELISA was developed to quantify the COMP neoepitope in synovial fluid. The mean concentration of the COMP neoepitope significantly increased in the synovial fluid from the joints responsible for acute lameness compared with normal joints and the joints of chronically lame horses and in joints with chronic structural OA. Immunolabelling for the COMP neoepitope revealed a pericellular staining in the interleukin-1β-stimulated explants. The ELISA is based on polyclonal antisera rather than a monoclonal antibody. The increase in the COMP neoepitope in the synovial fluid from horses with acute lameness suggests that this neoepitope has the potential to be a unique candidate biomarker for the early molecular changes in articular cartilage associated with OA. © 2017 The Authors

  5. Identification of MDP (muramyl dipeptide)-binding key domains in NOD2 (nucleotide-binding and oligomerization domain-2) receptor of Labeo rohita.

    Science.gov (United States)

    Maharana, Jitendra; Swain, Banikalyan; Sahoo, Bikash R; Dikhit, Manas R; Basu, Madhubanti; Mahapatra, Abhijit S; Jayasankar, Pallipuram; Samanta, Mrinal

    2013-08-01

    In lower eukaryotes-like fish, innate immunity contributed by various pattern recognition receptor (PRR) plays an essential role in protection against diseases. Nucleotide-binding and oligomerization domain (NOD)-2 is a cytoplasmic PRR that recognizes MDP (muramyl dipeptide) of the Gram positive and Gram negative bacteria as ligand and activates signalling to induce innate immunity. Hypothesizing a similar NOD2 signalling pathway of higher eukaryotes, the peripheral blood leucocytes (PBLs) of rohu (Labeo rohita) was stimulated with MDP. The data of quantitative real-time PCR (qRT-PCR) revealed MDP-mediated inductive expression of NOD2 and its down-stream molecule RICK/RIP2 (receptor-interacting serine-threonine protein kinase-2). This observation suggested the existence of MDP-binding sites in rohu NOD2 (rNOD2). To investigate it, 3D model of ligand-binding leucine-rich repeat (LRR) region of rNOD2 (rNOD2-LRR) was constructed following ab initio and threading approaches in I-TASSER web server. Structural refinement of the model was performed by energy minimization, and MD (molecular dynamics) simulation was performed in GROMACS (Groningen Machine for Chemical Simulations). The refined model of rNOD2-LRR was validated through SAVES, ProSA, ProQ, WHAT IF and MolProbity servers, and molecular docking with MDP was carried out in GOLD 4.1. The result of docking identified LRR3-7 comprising Lys820, Phe821, Asn822, Arg847, Gly849, Trp877, Trp901 and Trp931 as MDP-binding critical amino acids in rNOD2. This is the first study in fish to provide an insight into the 3D structure of NOD2-LRR region and its important motifs that are expected to be engaged in MDP binding and innate immunity.

  6. Coating of Carbon Fiber with Polyhedral Oligomeric Silsesquioxane (POSS to Enhance Mechanical Properties and Durability of Carbon/Vinyl Ester Composites

    Directory of Open Access Journals (Sweden)

    Mujib Khan

    2011-09-01

    Full Text Available Our continuing quest to improve the performance of polymer composites under moist and saltwater environments has gained momentum in recent years with the reinforcement of inorganic nanoparticles into the polymer. The key to mitigate degradation of composites under such environments is to maintain the integrity of the fiber/matrix (F/M interface. In this study, the F/M interface of carbon/vinyl ester composites has been modified by coating the carbon fiber with polyhedral oligomeric silsesquioxane (POSS. POSS is a nanostructured inorganic-organic hybrid particle with a cubic structure having silicon atoms at the core and linked to oxygen atoms. The advantage of using POSS is that the silicon atoms can be linked to a substituent that can be almost any chemical group known in organic chemistry. Cubic silica cores are ‘hard particles’ and are about 0.53 nm in diameter. The peripheral organic unit is a sphere of about 1–3 nm in diameter. Further, cubic structure of POSS remains intact during the polymerization process and therefore with appropriate functional groups, if installed on the fiber surface, would provide a stable and strong F/M interface. Two POSS systems with two different functional groups; namely, octaisobutyl and trisilanolphenyl have been investigated. A set of chemical and mechanical procedures has been developed to coat carbon fibers with POSS, and to fabricate layered composites with vinyl ester resin. Interlaminar shear and low velocity impact tests have indicated around 17–38% improvement in mechanical properties with respect to control samples made without the POSS coating. Saltwater and hygrothermal tests at various environmental conditions have revealed that coating with POSS reduces water absorption by 20–30% and retains the composite properties.

  7. The N-terminal Domain of Escherichia coli Assimilatory NADPH-Sulfite Reductase Hemoprotein Is an Oligomerization Domain That Mediates Holoenzyme Assembly*

    Science.gov (United States)

    Askenasy, Isabel; Pennington, Joseph M.; Tao, Yeqing; Marshall, Alan G.; Young, Nicolas L.; Shang, Weifeng; Stroupe, M. Elizabeth

    2015-01-01

    Assimilatory NADPH-sulfite reductase (SiR) from Escherichia coli is a structurally complex oxidoreductase that catalyzes the six-electron reduction of sulfite to sulfide. Two subunits, one a flavin-binding flavoprotein (SiRFP, the α subunit) and the other an iron-containing hemoprotein (SiRHP, the β subunit), assemble to make a holoenzyme of about 800 kDa. How the two subunits assemble is not known. The iron-rich cofactors in SiRHP are unique because they are a covalent arrangement of a Fe4S4 cluster attached through a cysteine ligand to an iron-containing porphyrinoid called siroheme. The link between cofactor biogenesis and SiR stability is also ill-defined. By use of hydrogen/deuterium exchange and biochemical analysis, we show that the α8β4 SiR holoenzyme assembles through the N terminus of SiRHP and the NADPH binding domain of SiRFP. By use of small angle x-ray scattering, we explore the structure of the SiRHP N-terminal oligomerization domain. We also report a novel form of the hemoprotein that occurs in the absence of its cofactors. Apo-SiRHP forms a homotetramer, also dependent on its N terminus, that is unable to assemble with SiRFP. From these results, we propose that homotetramerization of apo-SiRHP serves as a quality control mechanism to prevent formation of inactive holoenzyme in the case of limiting cellular siroheme. PMID:26088143

  8. The N-terminal Domain of Escherichia coli Assimilatory NADPH-Sulfite Reductase Hemoprotein Is an Oligomerization Domain That Mediates Holoenzyme Assembly.

    Science.gov (United States)

    Askenasy, Isabel; Pennington, Joseph M; Tao, Yeqing; Marshall, Alan G; Young, Nicolas L; Shang, Weifeng; Stroupe, M Elizabeth

    2015-07-31

    Assimilatory NADPH-sulfite reductase (SiR) from Escherichia coli is a structurally complex oxidoreductase that catalyzes the six-electron reduction of sulfite to sulfide. Two subunits, one a flavin-binding flavoprotein (SiRFP, the α subunit) and the other an iron-containing hemoprotein (SiRHP, the β subunit), assemble to make a holoenzyme of about 800 kDa. How the two subunits assemble is not known. The iron-rich cofactors in SiRHP are unique because they are a covalent arrangement of a Fe4S4 cluster attached through a cysteine ligand to an iron-containing porphyrinoid called siroheme. The link between cofactor biogenesis and SiR stability is also ill-defined. By use of hydrogen/deuterium exchange and biochemical analysis, we show that the α8β4 SiR holoenzyme assembles through the N terminus of SiRHP and the NADPH binding domain of SiRFP. By use of small angle x-ray scattering, we explore the structure of the SiRHP N-terminal oligomerization domain. We also report a novel form of the hemoprotein that occurs in the absence of its cofactors. Apo-SiRHP forms a homotetramer, also dependent on its N terminus, that is unable to assemble with SiRFP. From these results, we propose that homotetramerization of apo-SiRHP serves as a quality control mechanism to prevent formation of inactive holoenzyme in the case of limiting cellular siroheme.

  9. Nanomolar oligomerization and selective co-aggregation of α-synuclein pathogenic mutants revealed by single-molecule fluorescence

    Science.gov (United States)

    Sierecki, Emma; Giles, Nichole; Bowden, Quill; Polinkovsky, Mark E.; Steinbeck, Janina; Arrioti, Nicholas; Rahman, Diya; Bhumkar, Akshay; Nicovich, Philip R.; Ross, Ian; Parton, Robert G.; Böcking, Till; Gambin, Yann

    2016-01-01

    Protein aggregation is a hallmark of many neurodegenerative diseases, notably Alzheimer’s and Parkinson’s disease. Parkinson’s disease is characterized by the presence of Lewy bodies, abnormal aggregates mainly composed of α-synuclein. Moreover, cases of familial Parkinson’s disease have been linked to mutations in α-synuclein. In this study, we compared the behavior of wild-type (WT) α-synuclein and five of its pathological mutants (A30P, E46K, H50Q, G51D and A53T). To this end, single-molecule fluorescence detection was coupled to cell-free protein expression to measure precisely the oligomerization of proteins without purification, denaturation or labelling steps. In these conditions, we could detect the formation of oligomeric and pre-fibrillar species at very short time scale and low micromolar concentrations. The pathogenic mutants surprisingly segregated into two classes: one group forming large aggregates and fibrils while the other tending to form mostly oligomers. Strikingly, co-expression experiments reveal that members from the different groups do not generally interact with each other, both at the fibril and monomer levels. Together, this data paints a completely different picture of α-synuclein aggregation, with two possible pathways leading to the development of fibrils. PMID:27892477

  10. Nanomolar oligomerization and selective co-aggregation of α-synuclein pathogenic mutants revealed by single-molecule fluorescence.

    Science.gov (United States)

    Sierecki, Emma; Giles, Nichole; Bowden, Quill; Polinkovsky, Mark E; Steinbeck, Janina; Arrioti, Nicholas; Rahman, Diya; Bhumkar, Akshay; Nicovich, Philip R; Ross, Ian; Parton, Robert G; Böcking, Till; Gambin, Yann

    2016-11-28

    Protein aggregation is a hallmark of many neurodegenerative diseases, notably Alzheimer's and Parkinson's disease. Parkinson's disease is characterized by the presence of Lewy bodies, abnormal aggregates mainly composed of α-synuclein. Moreover, cases of familial Parkinson's disease have been linked to mutations in α-synuclein. In this study, we compared the behavior of wild-type (WT) α-synuclein and five of its pathological mutants (A30P, E46K, H50Q, G51D and A53T). To this end, single-molecule fluorescence detection was coupled to cell-free protein expression to measure precisely the oligomerization of proteins without purification, denaturation or labelling steps. In these conditions, we could detect the formation of oligomeric and pre-fibrillar species at very short time scale and low micromolar concentrations. The pathogenic mutants surprisingly segregated into two classes: one group forming large aggregates and fibrils while the other tending to form mostly oligomers. Strikingly, co-expression experiments reveal that members from the different groups do not generally interact with each other, both at the fibril and monomer levels. Together, this data paints a completely different picture of α-synuclein aggregation, with two possible pathways leading to the development of fibrils.

  11. Fingolimod phosphate attenuates oligomeric amyloid β-induced neurotoxicity via increased brain-derived neurotrophic factor expression in neurons.

    Directory of Open Access Journals (Sweden)

    Yukiko Doi

    Full Text Available The neurodegenerative processes that underlie Alzheimer's disease are mediated, in part, by soluble oligomeric amyloid β, a neurotoxic protein that inhibits hippocampal long-term potentiation, disrupts synaptic plasticity, and induces the production of reactive oxygen species. Here we show that the sphingosine-1-phosphate (S1P receptor (S1PR agonist fingolimod phosphate (FTY720-P-a new oral drug for multiple sclerosis-protects neurons against oligomeric amyloid β-induced neurotoxicity. We confirmed that primary mouse cortical neurons express all of the S1P receptor subtypes and FTY720-P directly affects the neurons. Treatment with FTY720-P enhanced the expression of brain-derived neurotrophic factor (BDNF in neurons. Moreover, blocking BDNF-TrkB signaling with a BDNF scavenger, TrkB inhibitor, or ERK1/2 inhibitor almost completely ablated these neuroprotective effects. These results suggested that the neuroprotective effects of FTY720-P are mediated by upregulated neuronal BDNF levels. Therefore, FTY720-P may be a promising therapeutic agent for neurodegenerative diseases, such as Alzheimer's disease.

  12. A Simple Method for the Size Controlled Synthesis of Stable Oligomeric Clusters of Gold Nanoparticles under Ambient Conditions.

    Science.gov (United States)

    Lawrence, Marlon; Testen, Anze; Koklic, Tilen; Smithies, Oliver

    2016-02-05

    Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like oligomeric clusters of these yellow nanoparticles of narrow size distribution are synthesized under ambient conditions via two methods. The delay-time method controls the number of subunits in the oligoclusters by varying the time between the addition of HAuCl₄ to alkaline solution and the subsequent addition of reducing agent, NaSCN. The yellow oligoclusters produced range in size from ~3 to ~25 nm. This size range can be further extended by an add-on method utilizing hydroxylated gold chloride (Na(+)[Au(OH₄-x)Clx](-)) to auto-catalytically increase the number of subunits in the as-synthesized oligocluster nanoparticles, providing a total range of 3 nm to 70 nm. The crude oligocluster preparations display narrow size distributions and do not require further fractionation for most purposes. The oligoclusters formed can be concentrated >300 fold without aggregation and the crude reaction mixtures remain stable for weeks without further processing. Because these oligomeric clusters can be concentrated before derivatization they allow expensive derivatizing agents to be used economically. In addition, we present two models by which predictions of particle size can be made with great accuracy.

  13. Protective Effects of Testosterone on Presynaptic Terminals against Oligomeric β-Amyloid Peptide in Primary Culture of Hippocampal Neurons

    Directory of Open Access Journals (Sweden)

    Chi-Fai Lau

    2014-01-01

    Full Text Available Increasing lines of evidence support that testosterone may have neuroprotective effects. While observational studies reported an association between higher bioavailable testosterone or brain testosterone levels and reduced risk of Alzheimer’s disease (AD, there is limited understanding of the underlying neuroprotective mechanisms. Previous studies demonstrated that testosterone could alleviate neurotoxicity induced by β-amyloid (Aβ, but these findings mainly focused on neuronal apoptosis. Since synaptic dysfunction and degeneration are early events during the pathogenesis of AD, we aim to investigate the effects of testosterone on oligomeric Aβ-induced synaptic changes. Our data suggested that exposure of primary cultured hippocampal neurons to oligomeric Aβ could reduce the length of neurites and decrease the expression of presynaptic proteins including synaptophysin, synaptotagmin, and synapsin-1. Aβ also disrupted synaptic vesicle recycling and protein folding machinery. Testosterone preserved the integrity of neurites and the expression of presynaptic proteins. It also attenuated Aβ-induced impairment of synaptic exocytosis. By using letrozole as an aromatase antagonist, we further demonstrated that the effects of testosterone on exocytosis were unlikely to be mediated through the estrogen receptor pathway. Furthermore, we showed that testosterone could attenuate Aβ-induced reduction of HSP70, which suggests a novel mechanism that links testosterone and its protective function on Aβ-induced synaptic damage. Taken together, our data provide further evidence on the beneficial effects of testosterone, which may be useful for future drug development for AD.

  14. Mathematical Models for Quantitative Assessment of Bioluminescence Resonance Energy Transfer (BRET: Application to Seven Transmembrane Receptors (7TMRs Oligomerization

    Directory of Open Access Journals (Sweden)

    Luka eDrinovec

    2012-08-01

    Full Text Available The idea that seven transmembrane receptors (7TMRs; also designated G-protein coupled receptors (GPCRs might form dimers or higher order oligomeric complexes was formulated more than 20 years ago and has been intensively studied since then. In the last decade, bioluminescence resonance energy transfer (BRET has been one of the most frequently used biophysical methods for studying 7TMRs oligomerization. This technique enables monitoring physical interactions between protein partners in living cells fused to donor and acceptor moieties. It relies on non-radiative transfer of energy between donor and acceptor, depending on their intermolecular distance (1–10 nm and relative orientation. Results derived from BRET-based techniques are very persuasive; however, they need appropriate controls and critical interpretation. To overcome concerns about the specificity of BRET-derived results, a set of experiments has been proposed, including negative control with a non-interacting receptor or protein, BRET dilution, saturation and competition assays. This article presents the theoretical background behind BRET assays, then outlines mathematical models for quantitative interpretation of BRET saturation and competition assay results, gives examples of their utilization and discusses the possibilities of quantitative analysis of data generated with other RET-based techniques.

  15. Characterization of oligomeric forms from mammalian F0F1ATP synthase by BN-PAGE: the role of detergents.

    Science.gov (United States)

    Bisetto, Elena; Giorgio, Valentina; Di Pancrazio, Francesca; Mavelli, Irene; Lippe, Giovanna

    2007-12-01

    It is now widely accepted that F0F1ATPsynthase is present in membrane, beside as monomers, in homo-dimeric and higher homo-oligomeric forms, which probably play critical roles in determining mitochondrial morphology. One-step mild detergent extraction followed by blue native electrophoresis (BN-PAGE) is a very interesting tool for studying the native membrane protein assemblies which can be associated with second/third-dimensional SDS-PAGE, immunoblotting, in-gel enzyme activity staining and mass spectrometry analyses. By combining these techniques, we resolved monomers and higher oligomeric forms of ATPsynthase from bovine heart mitochondria. However, a critical point is the choice of the detergents, which strongly influence the protein pattern of BN-PAGE. By using Triton X-100 we obtained that, in spite of the same subunit composition, monomers have a much lower specific activity than dimers and the two forms have a different pattern of tyrosine phosphorylation, suggesting that monomers and dimers are functionally distinct in membrane. In addition, enzyme self-association appeared to occur independently from the binding to ATPsynthase of the inhibitor protein IF1. Dodecylmaltoside was optimal to extract the enzyme from single biopsy samples, allowing us to demonstrate that IF1 plays a central role in regulating the enzyme activity in heart in vivo. Only low concentration of digitonin maintained significant amounts of ATPsynthase oligomers, which seemed to retain intact their native catalytic properties.

  16. Protein phosphatase 2a (PP2A binds within the oligomerization domain of striatin and regulates the phosphorylation and activation of the mammalian Ste20-Like kinase Mst3

    Directory of Open Access Journals (Sweden)

    Jones Candace A

    2011-10-01

    Full Text Available Abstract Background Striatin, a putative protein phosphatase 2A (PP2A B-type regulatory subunit, is a multi-domain scaffolding protein that has recently been linked to several diseases including cerebral cavernous malformation (CCM, which causes symptoms ranging from headaches to stroke. Striatin association with the PP2A A/C (structural subunit/catalytic subunit heterodimer alters PP2A substrate specificity, but targets and roles of striatin-associated PP2A are not known. In addition to binding the PP2A A/C heterodimer to form a PP2A holoenzyme, striatin associates with cerebral cavernous malformation 3 (CCM3 protein, the mammalian Mps one binder (MOB homolog, Mob3/phocein, the mammalian sterile 20-like (Mst kinases, Mst3, Mst4 and STK25, and several other proteins to form a large signaling complex. Little is known about the molecular architecture of the striatin complex and the regulation of these sterile 20-like kinases. Results To help define the molecular organization of striatin complexes and to determine whether Mst3 might be negatively regulated by striatin-associated PP2A, a structure-function analysis of striatin was performed. Two distinct regions of striatin are capable of stably binding directly or indirectly to Mob3--one N-terminal, including the coiled-coil domain, and another more C-terminal, including the WD-repeat domain. In addition, striatin residues 191-344 contain determinants necessary for efficient association of Mst3, Mst4, and CCM3. PP2A associates with the coiled-coil domain of striatin, but unlike Mob3 and Mst3, its binding appears to require striatin oligomerization. Deletion of the caveolin-binding domain on striatin abolishes striatin family oligomerization and PP2A binding. Point mutations in striatin that disrupt PP2A association cause hyperphosphorylation and activation of striatin-associated Mst3. Conclusions Striatin orchestrates the regulation of Mst3 by PP2A. It binds Mst3 likely as a dimer with CCM3 via

  17. Dissecting a Hub for Immune Response: Modeling the Structure of MyD88.

    Science.gov (United States)

    Naro, Chiara; Sette, Claudio

    2016-03-01

    Immune cells sense foreign organisms through the evolutionarily conserved family of Toll-like receptors. Signaling from these receptors relies on oligomerization of adaptor molecules. In this issue of Structure, Vynke et al. (2016) shed light on the dynamical structure of the homo- and hetero-dimerization domain of MyD88, the main adaptor utilized by Toll-like receptors.

  18. Structure and elevator mechanism of the Na(+)-citrate transporter CitS

    NARCIS (Netherlands)

    Lolkema, Juke S; Slotboom, Dirk Jan

    2016-01-01

    The recently determined crystal structure of the bacterial Na(+)-citrate symporter CitS provides unexpected structural and mechanistic insights. The protein has a fold that has not been seen in other proteins, but the oligomeric state, domain organization and proposed transport mechanism strongly re

  19. Homo- and hetero-oligomerization of PDZ-RhoGEF, LARG and p115RhoGEF by their C-terminal region regulates their in vivo Rho GEF activity and transforming potential.

    Science.gov (United States)

    Chikumi, Hiroki; Barac, Ana; Behbahani, Babak; Gao, Yuan; Teramoto, Hidemi; Zheng, Yi; Gutkind, J Silvio

    2004-01-08

    PDZ-RhoGEF, LARG, and p115RhoGEF are members of a newly identified family of Rho-guanine nucleotide exchange factors (GEFs) exhibiting a unique structural feature consisting of the presence of an area of similarity to regulators of G protein signaling (RGS). This RGS-like (RGL) domain provides a functional motif by which Galpha(12) and Galpha(13) can bind and regulate the activity of these RhoGEFs, thus providing a direct link from these heterotrimeric G proteins to Rho. PDZ-RhoGEF and LARG can also be phosphorylated by tyrosine kinases, including FAK, and associate with Plexin B, a semaphorin receptor, which controls axon guidance during development, through their PDZ domain, thereby stimulating Rho. Interestingly, while characterizing a PDZ-RhoGEF antiserum, we found that a transfected PDZ-RhoGEF construct associated with the endogenous PDZ-RhoGEF. Indeed, we observed that PDZ-RhoGEF and LARG can form homo- and hetero-oligomers, whereas p115RhoGEF can only homo-oligomerize, and that this intermolecular interaction was mediated by their unique C-terminal regions. Deletion of the C-terminal tail of PDZ-RhoGEF had no significant effect on the GEF catalytic activity towards Rho in vitro, but resulted in a drastic increase in the ability to stimulate a serum response element reporter and the accumulation of the GTP-bound Rho in vivo. Furthermore, removal of the C-termini of each of the three RGL-containing GEFs unleashed their full transforming potential. Together, these findings suggest the existence of a novel mechanism controlling the activity of PDZ-RhoGEF, LARG, and p115RhoGEF, which involves homo- and hetero-oligomerization through their inhibitory C-terminal region.

  20. Single site mutations in the hetero-oligomeric Mrp antiporter from alkaliphilic Bacillus pseudofirmus OF4 that affect Na+/H+ antiport activity, sodium exclusion, individual Mrp protein levels, or Mrp complex formation.

    Science.gov (United States)

    Morino, Masato; Natsui, Shinsuke; Ono, Tomohiro; Swartz, Talia H; Krulwich, Terry A; Ito, Masahiro

    2010-10-01

    Mrp systems are widely distributed and structurally complex cation/proton antiporters. Antiport activity requires hetero-oligomeric complexes of all six or seven hydrophobic Mrp proteins (MrpA-MrpG). Here, a panel of site-directed mutants in conserved or proposed motif residues was made in the Mrp Na(+)(Li(+))/H(+) antiporter from an alkaliphilic Bacillus. The mutant operons were expressed in antiporter-deficient Escherichia coli KNabc and assessed for antiport properties, support of sodium resistance, membrane levels of each Mrp protein, and presence of monomeric and dimeric Mrp complexes. Antiport did not depend on a VFF motif or a conserved tyrosine pair, but a role for a conserved histidine in a potential quinone binding site of MrpA was supported. The importance of several acidic residues for antiport was confirmed, and the importance of additional residues was demonstrated (e.g. three lysine residues conserved across MrpA, MrpD, and membrane-bound respiratory Complex I subunits (NuoL/M/N)). The results extended indications that MrpE is required for normal membrane levels of other Mrp proteins and for complex formation. Moreover, mutations in several other Mrp proteins lead to greatly reduced membrane levels of MrpE. Thus, changes in either of the two Mrp modules, MrpA-MrpD and MrpE-MrpG, influence the other. Two mutants, MrpB-P37G and MrpC-Q70A, showed a normal phenotype but lacked the MrpA-MrpG monomeric complex while retaining the dimeric hetero-oligomeric complex. Finally, MrpG-P81A and MrpG-P81G mutants exhibited no antiport activity but supported sodium resistance and a low [Na(+)](in). Such mutants could be used to screen hypothesized but uncharacterized sodium efflux functions of Mrp apart from Na(+) (Li(+))/H(+) antiport.

  1. Cartilage oligomeric matrix protein: A novel non-invasivemarker for assessing cirrhosis and risk of hepatocellularcarcinoma

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    AIM To assess serum cartilage oligomeric matrixprotein (COMP) as a marker of cirrhosis and risk ofprogression to hepatocellular carcinoma (HCC).METHODS: A COMP enzyme-linked immunosorbent assay was used to test 187 patients with chronic liverdiseases at the time point of first evaluation. Theselected patients included 72 with chronic hepatitis Binfection, 75 with chronic hepatitis C infection, 22 withprimary biliary cirrhosis, 7 with autoimmune hepatitistype 1, and 11 with alcoholic liver disease. Demographic,biochemical, histological and clinical characteristics ofthe patients were recorded at the first evaluation. Onehundred and forty-seven patients were followed for amedian [interquartile range (IQR)] duration of 96.5 (102)mo. The clinical, biochemical and histological data, aswell as the development of cirrhosis, HCC according tointernationally accepted criteria and in case of death,a liver-related cause during the follow-up period,were recorded at the electronic database of our clinic.COMP determination was also performed in 43 healthyindividuals who served as the control study group.RESULTS: COMP positivity (〉 15 U/L) was detectedin 22%-36% among chronic liver disease groups.Strikingly, almost 83% of COMP-positive patientswere cirrhotic at baseline, independently of cause ofliver disease. Among the patients who developed HCCduring follow-up, 73.7% (14/19) were COMP positiveat baseline. COMP positivity was significantly associatedwith older age (P 〈 0.001), advanced fibrosis (P =0.001) and necroinflammatory activity (P = 0.001),higher aspartate aminotransferase (P 〈 0.001), alanineaminotransferase (P 〈 0.02), γ-glutamyl transpeptidase(P = 0.003), alkaline phosphatase (P = 0.001), bilirubin(P 〈 0.05), international normalized ratio (P = 0.002)and alpha-fetoprotein levels (P 〈 0.02), and loweralbumin (P 〈 0.001), and platelet count (P = 0.008).COMP levels [median

  2. Hexameric oligomerization of mitochondrial peroxiredoxin PrxIIF and formation of an ultrahigh affinity complex with its electron donor thioredoxin Trx-o

    Science.gov (United States)

    Barranco-Medina, Sergio; Krell, Tino; Bernier-Villamor, Laura; Sevilla, Francisca; Lázaro, Juan-José; Dietz, Karl-Josef

    2008-01-01

    Mitochondria from plants, yeast, and animals each contain at least one peroxiredoxin (Prx) that is involved in peroxide detoxification and redox signalling. The supramolecular dynamics of atypical type II Prx targeted to the mitochondrion was addressed in pea. Microcalorimetric (ITC) titrations identified an extremely high-affinity binding between the mitochondrial PsPrxIIF and Trx-o with a KD of 126±14 pM. Binding was driven by a favourable enthalpy change (ΔH= –60.6 kcal mol−1) which was counterbalanced by unfavourable entropy changes (TΔS= –47.1 kcal mol−1). This is consistent with the occurrence of large conformational changes during binding which was abolished upon site-directed mutaganesis of the catalytic C59S and C84S. The redox-dependent interaction was confirmed by gel filtration of mitochondrial extracts and co-immunoprecipitation from extracts. The heterocomplex of PsPrxIIF and Trx-o reduced peroxide substrates more efficiently than free PsPrxIIF suggesting that Trx-o serves as an efficient and specific electron donor to PsPrxIIF in vivo. Other Trx-s tested by ITC analysis failed to interact with PsPrxIIF indicating a specific recognition of PsPrxIIF by Trx-o. PsPrxIIF exists primarily as a dimer or a hexamer depending on the redox state. In addition to the well-characterized oligomerization of classical 2-Cys Prx the results also show that atypical Prx undergo large structural reorganization with implications for protein–protein interaction and function. PMID:18632730

  3. Effect of the electrostatic surface potential on the oligomerization of full-length human recombinant prion protein at single-molecule level

    Science.gov (United States)

    Wang, Bin; Lou, Zhichao; Zhang, Haiqian; Xu, Bingqian

    2016-03-01

    The electrostatic surface potential (ESP) of prion oligomers has critical influences on the aggregating processes of the prion molecules. The atomic force microscopy (AFM) and structural simulation were combined to investigate the molecular basis of the full-length human recombinant prion oligomerization on mica surfaces. The high resolution non-intrusive AFM images showed that the prion oligomers formed different patterns on mica surfaces at different buffer pH values. The basic binding units for the large oligomers were determined to be prion momoners (Ms), dimers (Ds), and trimers (Ts). The forming of the D and T units happened through the binding of hydrophobic β-sheets of the M units. In contrast, the α-helices of these M, D, and T units were the binding areas for the formation of large oligomers. At pH 4.5, the binding units M, D, and T showed clear polarized ESP distributions on the surface domains, while at pH 7.0, they showed more evenly distributed ESPs. Based on the conformations of oligomers observed from AFM images, the D and T units were more abundantly on mica surface at pH 4.5 because the ESP re-distribution of M units helped to stabilize these larger oligomers. The amino acid side chains involved in the binding interfaces were stabilized by hydrogen bonds and electrostatic interactions. The detailed analysis of the charged side chains at pH 4.5 indicated that the polarized ESPs induced the aggregations among M, D, and T to form larger oligomers. Therefore, the hydrogen bonds and electrostatic interactions worked together to form the stabilized prion oligomers.

  4. Computational study of pH-dependent oligomerization and ligand binding in Alt a 1, a highly allergenic protein with a unique fold

    Science.gov (United States)

    Garrido-Arandia, María; Bretones, Jorge; Gómez-Casado, Cristina; Cubells, Nuria; Díaz-Perales, Araceli; Pacios, Luis F.

    2016-05-01

    Alt a 1 is a highly allergenic protein from Alternaria fungi responsible for several respiratory diseases. Its crystal structure revealed a unique β-barrel fold that defines a new family exclusive to fungi and forms a symmetrical dimer in a butterfly-like shape as well as tetramers. Its biological function is as yet unknown but its localization in cell wall of Alternaria spores and its interactions in the onset of allergy reactions point to a function to transport ligands. However, at odds with binding features in β-barrel proteins, monomeric Alt a 1 seems unable to harbor ligands because the barrel is too narrow. Tetrameric Alt a 1 is able to bind the flavonoid quercetin, yet the stability of the aggregate and the own ligand binding are pH-dependent. At pH 6.5, which Alt a 1 would meet when secreted by spores in bronchial epithelium, tetramer-quercetin complex is stable. At pH 5.5, which Alt a 1 would meet in apoplast when infecting plants, the complex breaks down. By means of a combined computational study that includes docking calculations, empirical p Ka estimates, Poisson-Boltzmann electrostatic potentials, and Molecular Dynamics simulations, we identified a putative binding site at the dimeric interface between subunits in tetramer. We propose an explanation on the pH-dependence of both oligomerization states and protein-ligand affinity of Alt a 1 in terms of electrostatic variations associated to distinct protonation states at different pHs. The uniqueness of this singular protein can thus be tracked in the combination of all these features.

  5. Ethylene Oligomerization to Low Carbon Olefins by a Zirconium Complex Incorporating 8-Quinolinolato Ligands at a Low Al/Zr Ratio

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Bis(8-quinolinolato)zirconium dichloride (Ox)2ZrCl2 (Ox- = 8-quinolinolato) was foundactive for ethylene oligomerization with a high selectivity of 84~94% to C4~C10 olefins at70~100℃ under the pressure of 1.8 MPa using Et2AlCl as a co-catalyst (Al/Zr = 60).

  6. Species-specific engagement of human nucleotide oligomerization domain 2 (NOD)2 and Toll-like receptor (TLR) signalling upon intracellular bacterial infection

    DEFF Research Database (Denmark)

    Salem, M; Seidelin, J B; Eickhardt-Dalbøge, Steffen Robert

    2015-01-01

    Recognition of bacterial peptidoglycan-derived muramyl-dipeptide (MDP) by nucleotide oligomerization domain 2 (NOD2) induces crucial innate immune responses. Most bacteria carry the N-acetylated form of MDP (A-MDP) in their cell membranes, whereas N-glycolyl MDP (G-MDP) is typical for mycobacteri...

  7. Constitutive homo- and hetero-oligomerization of TbetaRII-B, an alternatively spliced variant of the mouse TGF-beta type II receptor

    DEFF Research Database (Denmark)

    Krishnaveni, Manda S; Hansen, Jakob Lerche; Seeger, Werner

    2006-01-01

    , but the oligomerization pattern and dynamics of TbetaRII splice variants in live cells has not been demonstrated thus far. Using co-immunoprecipitation and bioluminescence resonance energy transfer (BRET), we demonstrate that the mouse TbetaRII receptor splice variant TbetaRII-B is capable of forming ligand...

  8. Effect of Dimer Dissociation on Activity and Thermostability of the α-Glucuronidase from Geobacillus stearothermophilus: Dissecting the Different Oligomeric Forms of Family 67 Glycoside Hydrolases

    Science.gov (United States)

    Shallom, Dalia; Golan, Gali; Shoham, Gil; Shoham, Yuval

    2004-01-01

    The oligomeric organization of enzymes plays an important role in many biological processes, such as allosteric regulation, conformational stability and thermal stability. α-Glucuronidases are family 67 glycosidases that cleave the α-1,2-glycosidic bond between 4-O-methyl-d-glucuronic acid and xylose units as part of an array of hemicellulose-hydrolyzing enzymes. Currently, two crystal structures of α-glucuronidases are available, those from Geobacillus stearothermophilus (AguA) and from Cellvibrio japonicus (GlcA67A). Both enzymes are homodimeric, but surprisingly their dimeric organization is different, raising questions regarding the significance of dimerization for the enzymes' activity and stability. Structural comparison of the two enzymes suggests several elements that are responsible for the different dimerization organization. Phylogenetic analysis shows that the α-glucuronidases AguA and GlcA67A can be classified into two distinct subfamilies of bacterial α-glucuronidases, where the dimer-forming residues of each enzyme are conserved only within its own subfamily. It seems that the different dimeric forms of AguA and GlcA67A represent the two alternative dimeric organizations of these subfamilies. To study the biological significance of the dimerization in α-glucuronidases, we have constructed a monomeric form of AguA by mutating three of its interface residues (W328E, R329T, and R665N). The activity of the monomer was significantly lower than the activity of the wild-type dimeric AguA, and the optimal temperature for activity of the monomer was around 35°C, compared to 65°C of the wild-type enzyme. Nevertheless, the melting temperature of the monomeric protein, 72.9°C, was almost identical to that of the wild-type, 73.4°C. It appears that the dimerization of AguA is essential for efficient catalysis and that the dissociation into monomers results in subtle conformational changes in the structure which indirectly influence the active site region

  9. Thermal Stability and Ablation Behavior of Modified Polydimethylsiloxane-Based Polyurethane Composites Reinforced with Polyhedral Oligomeric Silsesquioxane.

    Science.gov (United States)

    Han, Zhongyou; Xi, Yukun; Kwon, Younghwan

    2016-02-01

    Series of polydimethylsiloxane (PDMS)-based polyurethane (PU)/polyhedral oligomeric silsesquioxane (POSS) composites are prepared using ether or polyether modified diol/polyol PDMS prepolymers, isophorone diisocyanate (IPDI) and either non-reactive or reactive POSS. The effect of POSS incorporated chemically or physically, number of ethylene oxide units and crosslinking on PDMS based PU is investigated in terms of thermal stability and ablation properties. The ablation property is measured using an oxyacetylene torch test, and the ablation rate is evaluated. The results show that POSS molecules make a considerable influence on the ablative resistance, because they act as protective silica forming precursors under oxyacetylene condition. POSS molecules, especially methyl POSS, in PU matrix leads to the formation of densely accumulated spherical silica layers on the top of the ablated surface, resulting in improved ablation resistance.

  10. Moderate loading of the human osteoarthritic knee joint leads to lowering of intraarticular cartilage oligomeric matrix protein

    DEFF Research Database (Denmark)

    Helmark, Ida C; Petersen, Marie C H; Christensen, Helle E

    2012-01-01

    osteoarthritic joint determined by biochemical markers of cartilage turnover and inflammation in the synovial fluid (SF), serum and urine. Eleven subjects with OA of the knee(s), but with no other joint- or inflammatory disorders, volunteered for the study and had samples of blood, urine and synovial fluid drawn...... both at baseline and following 30-min one-legged knee-extension exercise. Workload: 60% of 1 RM (Repetition Maximum). Determination of cartilage oligomeric matrix protein (COMP), aggrecan, C-terminal collagen II peptide (CTX-II) and interleukin (IL)-6 were performed in synovial fluid (SF), serum...... and urine. A significant decrease was found in SF concentration of COMP following exercise, whereas aggrecan, CTX-II and IL-6 remained unchanged. No differences in any of the tested markers were found in serum and urine between baseline and post-exercise. Thirty minutes of mechanical loading of a single...

  11. Efficient trapping of HIV-1 envelope protein by hetero-oligomerization with an N-helix chimera

    Directory of Open Access Journals (Sweden)

    Silver Jonathan

    2005-08-01

    Full Text Available Abstract Background The N-heptad repeat region of the HIV-1 Transmembrane Envelope protein is a trimerization domain that forms part of a "six helix bundle" crucial to Envelope-mediated membrane fusion. N-heptad repeat peptides have been used as extracellular reagents to inhibit virus fusion. Results When expressed intracellularly with wild-type HIV-1 Envelope protein, the N-heptad repeat domain efficiently hetero-oligomerized with Envelope and trapped it in the endoplasmic reticulum or early Golgi, as indicated by lack of transport to the cell surface, absent proteolytic processing, and aberrant glycosylation. Conclusion Post-translational processing of HIV Envelope is very sensitive to an agent that binds to the N-heptad repeat during synthesis, suggesting that it might be possible to modify drugs that bind to this region to have transport-blocking properties.

  12. Cartilage oligomeric matrix protein deficiency promotes early onset and the chronic development of collagen-induced arthritis

    DEFF Research Database (Denmark)

    Geng, Hui; Carlsen, Stefan; Nandakumar, Kutty;

    2008-01-01

    ABSTRACT: INTRODUCTION: Cartilage oligomeric matrix protein (COMP) is a homopentameric protein in cartilage. The development of arthritis, like collagen-induced arthritis (CIA), involves cartilage as a target tissue. We have investigated the development of CIA in COMP-deficient mice. METHODS: COMP......-deficient mice in the 129/Sv background were backcrossed for 10 generations against B10.Q mice, which are susceptible to chronic CIA. COMP-deficient and wild-type mice were tested for onset, incidence, and severity of arthritis in both the collagen and collagen antibody-induced arthritis models. Serum anti......-collagen II and anti-COMP antibodies as well as serum COMP levels in arthritic and wild-type mice were measured by enzyme-linked immunosorbent assay. RESULTS: COMP-deficient mice showed a significant early onset and increase in the severity of CIA in the chronic phase, whereas collagen II-antibody titers were...

  13. Ethylene Oligomerization by (dppe)MCl2 [M=Fe(Ⅱ), Co(Ⅱ), Ni(Ⅱ)] Complexes/EAO

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The catalytic properties of a series of (dppe)MC12 [M = Fe(Ⅱ), Co(Ⅱ), Ni(Ⅱ)] in combination with EAO (ethylaluminoxane) for ethylene oligomerization have been investigated. Treatment of the bidentate phosphine metal complexes with EAO in toluene generated in situ active catalysts which catalyze oligomerizaton of ethylene to low-carbon olefins. The effects of reaction temperature, ratios of n(A1): n(M) and reaction periods on catalytic activity and product distribution have been studied. The activity of cobalt complex with EAO at 200C was 4021 g/g(Co).h, and the selectivity ofC4-10 olefins is 95.5%.

  14. DFT study on the impact of the methylaluminoxane cocatalyst in ethylene oligomerization using a titanium-based catalyst

    KAUST Repository

    Pasha, Farhan Ahmad

    2015-01-26

    A computational study within the framework of density functional theory is presented on the oligomerization of ethylene to yield 1-hexene using [(η5-C5H4CMe2C6H5)]TiCl3/MAO] catalyst. This study explicitly takes into account a methylaluminoxane (MAO) cocatalyst model, where the MAO cluster has become an anionic species after having abstracted one chloride anion, yielding a cationic activated catalyst. Hence, the reaction profile was calculated using the zwitterionic system, and the potential energy surface has been compared to the cationic catalytic system. Modest differences were found between the two free energy profiles. However, we show for the first time that the use of a realistic zwitterionic model is required to obtain a Brønsted-Evans-Polanyi relationship between the energy barriers and reaction energies.

  15. Radical mechanisms of methyl vinyl ketone oligomerization through aqueous phase OH-oxidation: on the paradoxical role of dissolved molecular oxygen

    Directory of Open Access Journals (Sweden)

    P. Renard

    2013-01-01

    Full Text Available It is now accepted that one of the important pathways of Secondary Organic Aerosol (SOA formation occurs through aqueous phase chemistry in the atmosphere. However, the liquid phase chemical mechanisms leading to macromolecules are still not well understood. For α-dicarbonyl precursors, such as methylglyoxal and glyoxal, radical reactions through OH-oxidation produce oligomers, irreversibly and faster than accretion reactions. Methyl vinyl ketone (MVK was chosen in the present study as it is an α, β-unsaturated carbonyl that can undergo such reaction pathways in the aqueous phase and forms even high molecular weight oligomers. We present here experiments on the aqueous phase OH-oxidation of MVK, performed under atmospheric relevant conditions. Using NMR and UV absorption spectroscopy, high and ultra-high resolution mass spectrometry, we show that the fast formation of oligomers up to 1800 Da is due to radical oligomerization of MVK, and 13 series of oligomers (out of a total of 26 series are identified. The influence of atmospherically relevant parameters such as temperature, initial concentrations of MVK and dissolved oxygen are presented and discussed. In agreement with the experimental observations, we propose a chemical mechanism of OH-oxidation of MVK in the aqueous phase that proceeds via radical oligomerization of MVK on the olefin part of the molecule. This mechanism highlights the paradoxical role of dissolved O2: while it inhibits oligomerization reactions, it contributes to produce oligomerization initiator radicals, which rapidly consume O2, thus leading to the supremacy of oligomerization reactions after several minutes of reaction. These processes, together with the large ranges of initial concentrations investigated (60–656 μM of dissolved O2 and 0.2–20 mM of MVK show the fundamental role that O2 likely plays in atmospheric organic aerosol.

  16. Oligomeric amyloid-{beta} inhibits the proteolytic conversion of brain-derived neurotrophic factor (BDNF), AMPA receptor trafficking, and classical conditioning.

    Science.gov (United States)

    Zheng, Zhaoqing; Sabirzhanov, Boris; Keifer, Joyce

    2010-11-01

    Amyloid-β (Aβ) peptide is thought to have a significant role in the progressive memory loss observed in patients with Alzheimer disease and inhibits synaptic plasticity in animal models of learning. We previously demonstrated that brain-derived neurotrophic factor (BDNF) is critical for synaptic AMPA receptor delivery in an in vitro model of eyeblink classical conditioning. Here, we report that acquisition of conditioned responses was significantly attenuated by bath application of oligomeric (200 nm), but not fibrillar, Aβ peptide. Western blotting revealed that BDNF protein expression during conditioning is significantly reduced by treatment with oligomeric Aβ, as were phosphorylation levels of cAMP-response element-binding protein (CREB), Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV), and ERK. However, levels of PKA and PKCζ/λ were unaffected, as was PDK-1. Protein localization studies using confocal imaging indicate that oligomeric Aβ, but not fibrillar or scrambled forms, suppresses colocalization of GluR1 and GluR4 AMPA receptor subunits with synaptophysin, indicating that trafficking of these subunits to synapses during the conditioning procedure is blocked. In contrast, coapplication of BDNF with oligomeric Aβ significantly reversed these findings. Interestingly, a tolloid-like metalloproteinase in turtle, tTLLs (turtle tolloid-like protein), which normally processes the precursor proBDNF into mature BDNF, was found to degrade oligomeric Aβ into small fragments. These data suggest that an Aβ-induced reduction in BDNF, perhaps due to interference in the proteolytic conversion of proBDNF to BDNF, results in inhibition of synaptic AMPA receptor delivery and suppression of the acquisition of conditioning.

  17. Characterization of SP1, a Stress-Responsive, Boiling-Soluble, Homo-Oligomeric Protein from Aspen1

    Science.gov (United States)

    Wang, Wang-Xia; Pelah, Dan; Alergand, Tal; Shoseyov, Oded; Altman, Arie

    2002-01-01

    sp1 cDNA was isolated from aspen (Populus tremula) plants by immunoscreening an expression library using polyclonal antibodies against BspA protein. BspA, which is a boiling-stable protein, accumulates in aspen plants in response to water stress and abscisic acid application (Pelah et al., 1995). The sp1 cDNA was found to encode a 12.4-kD generally hydrophilic protein with a hydrophobic C terminus, which is different from the BspA protein and was termed SP1 (stable protein 1). Northern-blot analysis revealed that sp1 encodes a small mRNA (about 0.6 kb) that is expressed in aspen plants under non-stress conditions and is accumulated after salt, cold, heat, and desiccation stress, and during the recovery from stress. The SP1 detected in plants remained soluble upon boiling, migrated both as a 12.4-kD band and a much higher mass of 116 kD on a 17% (w/v) Tricine-sodium dodecyl sulfate-polyacrylamide gel. Comparative protease digestion patterns, amino acid analyses, and the N-terminal sequences of the 12.4- and 116-kD proteins revealed that SP1 is homo-oligomeric. Furthermore, gel filtration chromatography analysis indicated that SP1 exists in aspen plants as a complex, composed of 12 subunits of 12.4 kD. A large number of sequences deduced from expressed sequence tags and genomic sequences of other organisms with unknown function show high homology to SP1. Thus, SP1 may represent a new protein family. Here, we present the first report on this putative protein family: the cloning, isolation, and characterization of SP1, a stress-responsive, boiling-soluble, oligomeric protein. PMID:12376651

  18. Structural studies of the oligomeric formation crotoxin and their complexes; Estudos estruturais da formacao oligomerica da crotoxina e seus complexos

    Energy Technology Data Exchange (ETDEWEB)

    Morelli, Humberto A.S.; Fernandes, Carlos A.H.; Magro, Angelo J.; Fontes, Marcos R.M.; Fernandez, Roberto M., E-mail: rmorato@ibb.unesp.br [Universidade Estadual Paulista Julio de Mesquita Filho (UNESP), Botucatu, SP (Brazil). Inst. de Biociencias. Dept. de Fisica e Biofisica; Bicev, Renata N.; Oliveira, Cristiano L.P. de [Universidade de Sao Paulo (USP), Sao Paulo, SP (Brazil). Inst. de Fisica. Dept. de Fisica Experimental; Barioni, Marina B.; Ito, Amando S. [Universidade de Sao Paulo (USP), Ribeirao Preto, SP (Brazil). Fac. de Filosofia, Ciencias e Letras. Dept. de Fisica

    2013-08-15

    The crotoxin is a phospholipase A{sub 2} that is the mainly responsible for the neurotoxic effect of the snakes' venom of the species Crotalus durissus, popularly known as rattlesnakes. In the crotoxin there are two subunits: an acidic, non-toxic and devoid of enzymatic action, (called crotoxin A or CA); and a basic, toxic with enzymatic activity (called crotoxin B or CB). The main objective of this work is to study the CA, CB and the complex CA + CB using the Small-Angle X-ray Scattering (SAXS), Optical absorption spectrometry and static fluorescence techniques. (author)

  19. Glutamine synthetase isoforms in nitrogen-fixing soybean nodules: distinct oligomeric structures and thiol-based regulation.

    Science.gov (United States)

    Masalkar, Pintu D; Roberts, Daniel M

    2015-01-16

    Legume root nodule glutamine synthetase (GS) catalyzes the assimilation of ammonia produced by nitrogen fixation. Two GS isoform subtypes (GS1β and GS1γ) are present in soybean nodules. GS1γ isoforms differ from GS1β isoforms in terms of their susceptibility to reversible inhibition by intersubunit disulfide bond formation between C159 and C92 at the shared active site at subunit interfaces. Although nodule GS enzymes share 86% amino acid sequence identity, analytical ultracentrifugation experiments showed that GS1γ is a dodecamer, whereas the GS1β is a decamer. It is proposed that this difference contributes to the differential thiol sensitivity of each isoform, and that GS1γ1 may be a target of thiol-based regulation.

  20. Analysis of Quaternary Structure of a [LDH-like] Malate Dehydrogenase of Plasmodium falciparum with Oligomeric Mutants

    Science.gov (United States)

    L-Malate dehydrogenase (PfMDH) from Plasmodium falciparum, the causative agent for the most severe form of malaria, has shown remarkable similarities to L-lactate dehydrogenase (PfLDH). PfMDH is more closely related to [LDH-like] MDHs characterized in archea and other prokaryotes. Initial sequence a...

  1. 聚氨酯/低聚倍半硅氧烷复合纤维的制备%Preparation of polyurethane/polyhedral oligomeric silsesquioxane nanocomposite fibers

    Institute of Scientific and Technical Information of China (English)

    肖荣冬; 翁国星

    2015-01-01

    BACKGROUND:Althoughpolyurethane possesses excelent biocompatibility, its inherent inertness leads to its weak interactions with cels. So, its modification is necessary. OBJECTIVE:To prepare the polyurethane/polyhedral oligomeric silsesquioxane nanocomposite fibers with controlable morphology. METHODS:The polyhedral oligomeric silsesquioxane was dispersed in a certain concentration of polyurethane solution to prepare the polyurethane/polyhedral oligomeric silsesquioxane nanocomposite fibers using electrospinning method. Meanwhile, the effects of mass of polyurethane and oligomeric silsesquioxane, spinning voltage and spinning advance velocity on composite fiber morphology were analyzed. The optimum preparing conditions were filtrated. The stability of oligomeric silsesquioxane in polyurethane was determined. The morphology and composition of fibers were analyzed by scanning electron microscopy, infrared spectroscopy, X-ray photoelectron spectroscopy. RESULTS AND CONCLUSION: Oligomeric silsesquioxane stably existed in polyurethane. Polyurethane/polyhedral oligomeric silsesquioxane nanocomposite fibers were the most uniform when the mass fraction of polyurethane in composite fibers accounted for 20%, the mass ratio of polyurethane and oligomeric silsesquioxane was 10: 1, spinning voltage was 15 kV and spinning advance velocity was 0.5 mL/h. Compared with the pure polyurethane, the O/C ratio of polyurethane/polyhedral oligomeric silsesquioxane nanocomposites increased significantly.%背景:聚氨酯具有良好的生物相容性,然而其固有的惰性导致其与细胞之间的相互作用较弱,因此需要对其改性。目的:制备形貌可控的聚氨酯/低聚倍半硅氧烷纳米复合纤维。方法:将多面体低聚倍半硅氧烷分散到一定浓度的聚氨酯溶液中,通过静电纺丝制备聚氨酯/低聚倍半硅氧烷纳米复合纤维,同时分析聚氨酯质量、低聚倍半硅氧烷质量、纺丝电

  2. Thermodynamic Study for Ethylene Oligomerization to α-Olefin%乙烯齐聚制α-烯烃反应体系的热力学研究

    Institute of Scientific and Technical Information of China (English)

    吴建民; 孙启文; 张宗森; 庞利峰

    2013-01-01

    利用Benson基团贡献法计算了乙烯齐聚制α-烯烃反应各物质的标准摩尔生成焓、标准摩尔熵和摩尔定压热容,在298 ~ 700 K温度范围内对乙烯齐聚制α-烯烃反应体系的反应热、吉布斯自由能以及反应平衡常数进行了详尽的计算,分析了不同反应步骤的热力学平衡与限度,对不同反应发生的热力学可能性与顺序进行了判断,考察了反应温度和压力对乙烯齐聚制α-烯烃反应化学平衡的影响,确定了乙烯齐聚制α-烯烃反应体系适宜的工艺条件.结果表明:乙烯齐聚制α-烯烃反应为放热反应;从热力学上看,温度低于546 K时,乙烯齐聚生成直链α-烯烃反应为自发过程,且比α-烯烃异构化和烯烃歧化反应更容易进行;低温、高压有利于乙烯齐聚制α-烯烃反应的进行;乙烯齐聚制α-烯烃反应体系适宜的工艺条件为温度323 ~473 K,压力5~ 20 MPa,且在SHOP法的工艺条件下(温度363 K,压力10.3 MPa),乙烯齐聚生成直链α-烯烃反应的热力学平衡转化率接近于100%.%The standard molar enthalpy of formation,the standard molar entropy and isobaric molar heat capacity of ethylene oligomerization products were estimated with Benson group contribution method.The reaction heat,Gibbs free energy change and equilibrium constant of ethylene oligomerization to α-olefin reaction system were calculated in detail as a function of the temperature from 298 K to 700 K.The thermodynamic equilibrium and the limit of different reaction steps were analyzed.The thermodynamic possibility and formation sequences of ethylene oligomerization reaction system were also judged.The effects of temperature and pressure on the equilibrium conversion were investigated.The appropriate process conditions of ethylene oligomerization to α-olefin reactions were determined according to thermodynamic calculations.The results show that ethylene oligomerization to α-olefin reactions are

  3. Structural Characterization of Monomers and Oligomers of D-Amino Acid-Containing Peptides Using T-Wave Ion Mobility Mass Spectrometry

    Science.gov (United States)

    Pang, Xueqin; Jia, Chenxi; Chen, Zhengwei; Li, Lingjun

    2016-11-01

    The D-residues are crucial to biological function of D-amino acid containing peptides (DAACPs). Previous ion mobility mass spectrometry (IM-MS) studies revealing oligomerization patterns of amyloid cascade demonstrated conversion from native soluble unstructured assembly to fibril ß-sheet oligomers, which has been implicated in amyloid diseases, such as Alzheimer's disease and type 2 diabetes. Although neuropeptides are typically present at very low concentrations in circulation, their local concentrations could be much higher in large dense core vesicles, forming dimers or oligomers. We studied the oligomerization of protonated and metal-adducted achatin I and dermorphin peptide isomers with IM-MS. Our results suggested that dimerization, oligomerization, and metal adduction augment the structural differences between D/L peptide isomers compared to protonated monomers. Dimers and oligomers enhanced the structural differences between D/L peptide isomers in both aqueous and organic solvent system. Furthermore, some oligomer forms were only observed for either D- or L-isomers, indicating the importance of chiral center in oligomerization process. The oligomerization patterns of D/L isomers appear to be similar. Potassium adducts were detected to enlarge the structural differences between D/L isomers.

  4. Structural Characterization of Monomers and Oligomers of D-Amino Acid-Containing Peptides Using T-Wave Ion Mobility Mass Spectrometry

    Science.gov (United States)

    Pang, Xueqin; Jia, Chenxi; Chen, Zhengwei; Li, Lingjun

    2017-01-01

    The D-residues are crucial to biological function of D-amino acid containing peptides (DAACPs). Previous ion mobility mass spectrometry (IM-MS) studies revealing oligomerization patterns of amyloid cascade demonstrated conversion from native soluble unstructured assembly to fibril ß-sheet oligomers, which has been implicated in amyloid diseases, such as Alzheimer's disease and type 2 diabetes. Although neuropeptides are typically present at very low concentrations in circulation, their local concentrations could be much higher in large dense core vesicles, forming dimers or oligomers. We studied the oligomerization of protonated and metal-adducted achatin I and dermorphin peptide isomers with IM-MS. Our results suggested that dimerization, oligomerization, and metal adduction augment the structural differences between D/L peptide isomers compared to protonated monomers. Dimers and oligomers enhanced the structural differences between D/L peptide isomers in both aqueous and organic solvent system. Furthermore, some oligomer forms were only observed for either D- or L-isomers, indicating the importance of chiral center in oligomerization process. The oligomerization patterns of D/L isomers appear to be similar. Potassium adducts were detected to enlarge the structural differences between D/L isomers.

  5. Molecular chirality and chiral capsule-type dimer formation of cyclic triamides via hydrogen-bonding interactions.

    Science.gov (United States)

    Fujimoto, Noriko; Matsumura, Mio; Azumaya, Isao; Nishiyama, Shizuka; Masu, Hyuma; Kagechika, Hiroyuki; Tanatani, Aya

    2012-05-18

    Chiral properties of bowl-shaped cyclic triamides bearing functional groups with hydrogen-bonding ability were examined. Chiral induction of cyclic triamide 3a was observed by addition of chiral amine in solution, and chiral separation was achieved by simple crystallization to afford chiral capsule-type dimer structure of 4a.

  6. Shear Wave Reflection Seismics Image Internal Structure of Quick-Clay Landslides in Sweden

    Science.gov (United States)

    Polom, U.; Krawczyk, C. M.; Malehmir, A.

    2014-12-01

    Covering many different sizes of scale, landslides are widespread and pose a severe hazard in many areas as soon as humans or infrastructure are affected. In order to provide geophysical tools and techniques to better characterize sites prone to sliding, a geophysical assessment working towards a geotechnical understanding of landslides is necessary. As part of a joint project studying clay-related landslides in Nordic countries by a suite of geophysical methods, we therefore tested the use of shear wave reflection seismics to survey shallow structures that are known to be related to quick-clay landslide processes in southern Sweden. On two crossing profiles, a land streamer consisting of 120 SH-geophones with 1 m spacing was deployed, and an ELVIS micro-vibrator was shaking every 4 m to generate the shear wave signal. SH-wave data of high quality were thereby acquired to resolve the gaps between P-wave data and electrical and surface wave based methods of lower resolution. After quality control, correlation, subtractive stack, and geometry setup, single shot gathers already demonstrate the high data quality gained in the region, especially on a gravel road. The migrated depth sections image the structural inventory down to ca. 50 m depth with vertical resolution of less than 1 m. Horizontally layered sediments are visible in the upper 40 m of soft (marine) sediments, followed by top basement with a rough topography varying between ca. 20-40 m depth. The imaged, bowl-shaped basement morphology centres near the profile crossing, and basement is exposed at three sides of the profiles. Three distinct sediment sequences are separated by high-amplitude unconformities. The quick-clay layer may be located above the marked reflection set that lies on top of the more transparent sequence that levels out the basement. Located between 15-20 m depth, this correlates with the height of the last scarp that occurred in the area. In addition, shear wave velocities are determined

  7. Familial Alzheimer's disease mutations differentially alter amyloid β-protein oligomerization.

    Science.gov (United States)

    Gessel, Megan Murray; Bernstein, Summer; Kemper, Martin; Teplow, David B; Bowers, Michael T

    2012-11-21

    Although most cases of Alzheimer's disease (AD) are sporadic, ∼5% of cases are genetic in origin. These cases, known as familial Alzheimer's disease (FAD), are caused by mutations that alter the rate of production or the primary structure of the amyloid β-protein (Aβ). Changes in the primary structure of Aβ alter the peptide's assembly and toxic activity. Recently, a primary working hypothesis for AD has evolved where causation has been attributed to early, soluble peptide oligomer states. Here we posit that both experimental and pathological differences between FAD-related mutants and wild-type Aβ could be reflected in the early oligomer distributions of these peptides. We use ion mobility-based mass spectrometry to probe the structure and early aggregation states of three mutant forms of Aβ40 and Aβ42: Tottori (D7N), Flemish (A21G), and Arctic (E22G). Our results indicate that the FAD-related amino acid substitutions have no noticeable effect on Aβ monomer cross section, indicating there are no major structural changes in the monomers. However, we observe significant changes to the aggregation states populated by the various Aβ mutants, indicating that structural changes present in the monomers are reflected in the oligomers. Moreover, the early oligomer distributions differ for each mutant, suggesting a possible structural basis for the varied pathogenesis of different forms of FAD.

  8. Expanding the Concepts in Protein Structure-Function Relationships and Enzyme Kinetics: Teaching Using Morpheeins

    Science.gov (United States)

    Lawrence, Sarah H.; Jaffe, Eileen K.

    2008-01-01

    A morpheein is a homo-oligomeric protein that can exist as an ensemble of physiologically significant and functionally distinct alternate quaternary assemblies. Morpheeins exist in nature and use conformational equilibria between different tertiary structures to form distinct oligomers as a means of regulating their function. Notably, alternate…

  9. Isolation and Quantification of Oligomeric and Polymeric Procyanidins in the Aerial Parts of St. John's Wort (Hypericum perforatum).

    Science.gov (United States)

    Hellenbrand, Nils; Lechtenberg, Matthias; Petereit, Frank; Sendker, Jandirk; Hensel, Andreas

    2015-08-01

    Proanthocyanidins (condensed tannins) constitute a class of oligomeric and polymeric polyphenols with flavan-3-ols as monomeric building blocks. Despite the high impact of proanthocyanidins, these polyphenols are mostly quantified by colorimetric methods or by chromatographic determination of the flavan-3-ols as cleavage products or low molecular oligomers as lead compounds. For St. John's wort (Hyperici herba) from Hypericum perforatum, a protocol for preparative isolation of oligomeric and polymeric proanthocyanidins from an acetone-water extract by chromatography on Sephadex®LH20 in combination with preparative high-performance liquid chromatography on the diol stationary phase was developed, yielding procyanidin reference clusters with a defined degree of polymerization from 3 to 10. Identity and purity of these clusters was proven by high-performance liquid chromatography (RP18 and diol phase) and mass spectrometry. For identification and quantification of proanthocyanidin clusters from St. John's wort, an ICH-Q2 (International harmonized guideline for analytical validation) validated high-performance liquid chromatography method with fluorimetric detection was developed using an acetone-water extract of the herbal material, purified by solid-phase extraction for the removal of naphthodianthrones. The method enabled the quantification of procyanidin clusters with a degree of polymerization from 2 to 10. Analysis of nine batches of Hyperici herba from different sources indicated a high variability of proanthocyanidin content in the range from 8 to 37 mg/g. In all of the batches investigated, the trimer cluster DP3 was the dominant proanthocyanidin (about 40 %), followed by DP 4 (about 15 %) and DP5 (about 12 %). Monitoring of procyanidin distribution during seasonal growth of fresh plants of H. perforatum indicated the highest proanthocyanidin content in young plants (about 50 mg/g) and a time-dependent decrease during the growing season to about 16

  10. Oligomerization of glycine on clay mineral surface and implication to oligin of life under seafloor hydrothermal conditions

    Science.gov (United States)

    Fuchida, S.; Masuda, H.

    2012-12-01

    The sediments at hydrothermal and/or various parts of the crust has been believed to be good environments to proceed the chemical evolution of life precursor, since minerals promoted oligomerization of amino acids, sugars and lipids on the primitive earth. In this study, the thermal behaviors of glycine (Gly), the simplest amino acid, adsorbed on montmorillonite was observed to evaluate the role of clay minerals and water on the oligomerization under thermal condition of sediments. Gly was adsorbed on montmorillonite was heated at 150 degree C for 3-288 hrs under dry and wet condition. In the latter case, 10 - 60% water was added in the system. The amount of Gly monomer remaining in the montmorillonite exponentially decreased with time; 46% Gly remained in the montmorillonite under dry condition and 74% under wet condition after 288 hrs. The Gly monomer was more stable under hydrothermal condition than dry thermal condtion. FT-IR analysis suggested that the Gly was intercalated in the montmorillonite via hydrogen bond, which is likely to promote to stabilize Gly, between amino group of the Gly and silanol group of the montmorillonite. On the contrast, the yields of peptides were low on motmorillonite after heated under the wet condition: the amounts of glycilglycine (Gly-Gly) and diketopiperazine (DKP) are 0.8% and 0.9%, respectively. The amounts of DKP and GlyGly are 12.9% and 4.8% after heated under the dry condtion. Excessive water would promote to hydrolyze synthesized peptides. New band at 1671cm-1 by FT-IR implies that DKP was condensed on the montmorillonite. DKP was not formed without montmorillonite under the dry condition, although peptide formation is theoretically favorable. Water molecules including in the montmorillonite would act as proton transfer to promote the peptide formation. The peptide formation would be more proceeded under a little wet condition than completely dry condition. Results of this study suggested that deep sediments, where

  11. The Oligomerization Domain of VP3, the Scaffolding Protein of Infectious Bursal Disease Virus, Plays a Critical Role in Capsid Assembly

    Science.gov (United States)

    Maraver, Antonio; Oña, Ana; Abaitua, Fernando; González, Dolores; Clemente, Roberto; Ruiz-Díaz, Jose A.; Castón, Jose R.; Pazos, Florencio; Rodriguez, Jose F.

    2003-01-01

    Infectious bursal disease virus (IBDV) capsids are formed by a single protein layer containing three polypeptides, pVP2, VP2, and VP3. Here, we show that the VP3 protein synthesized in insect cells, either after expression of the complete polyprotein or from a VP3 gene construct, is proteolytically degraded, leading to the accumulation of product lacking the 13 C-terminal residues. This finding led to identification of the VP3 oligomerization domain within a 24-amino-acid stretch near the C-terminal end of the polypeptide, partially overlapping the VP1 binding domain. Inactivation of the VP3 oligomerization domain, by either proteolysis or deletion of the polyprotein gene, abolishes viruslike particle formation. Formation of VP3-VP1 complexes in cells infected with a dual recombinant baculovirus simultaneously expressing the polyprotein and VP1 prevented VP3 proteolysis and led to efficient virus-like particle formation in insect cells. PMID:12743301

  12. Disorder in Milk Proteins: α-Lactalbumin. Part B. A Multifunctional Whey Protein Acting as an Oligomeric Molten Globular "Oil Container" in the Anti-Tumorigenic Drugs, Liprotides.

    Science.gov (United States)

    Uversky, Vladimir N; Permyakov, Serge E; Breydo, Leonid; Redwan, Elrashdy M; Almehdar, Hussein A; Permyakov, Eugene A

    2016-07-15

    This is a second part of the three-part article from a series of reviews on the abundance and roles of intrinsic disorder in milk proteins. We continue to describe α-lactalbumin, a small globular Ca2+-binding protein, which besides being one of the two components of lactose synthase that catalyzes the final step of the lactose biosynthesis in the lactating mammary gland, possesses a multitude of other functions. In fact, recent studies indicated that some partially folded forms of this protein possess noticeable bactericidal activity and other forms might be related to induction of the apoptosis of tumor cells. In its anti-tumorigenic function, oligomeric α-lactalbumin serves as a founding member of a new family of anticancer drugs termed liprotides (for lipids and partially denatured proteins), where an oligomeric molten globular protein acts as an "oil container" or cargo for the delivery of oleic acid to the cell membranes.

  13. A triple-arginine motif in the amino-terminal domain and oligomerization are required for HIV-1 inhibition by human MX2.

    Science.gov (United States)

    Goujon, Caroline; Greenbury, Rebecca A; Papaioannou, Stelios; Doyle, Tomas; Malim, Michael H

    2015-04-01

    We have employed molecular genetic approaches to understand the domain organization of the HIV-1 resistance factor myxovirus resistance 2 (MX2). First, we describe an essential triple-arginine motif in the amino-terminal domain. Second, we demonstrate that this 91-residue domain mediates antiviral activity when appended to heterologous proteins, and we provide genetic evidence that protein oligomerization is required for MX2 function. These insights will facilitate future work aiming to elucidate MX2's mechanism of action.

  14. Oligomerization of the UDP-glucuronosyltransferase 1A proteins: homo- and heterodimerization analysis by fluorescence resonance energy transfer and co-immunoprecipitation.

    Science.gov (United States)

    Operaña, Theresa N; Tukey, Robert H

    2007-02-16

    UDP-glucuronosyltransferases (UGTs) are membrane-bound proteins localized to the endoplasmic reticulum and catalyze the formation of beta-d-glucopyranosiduronic acids (glucuronides) using UDP-glucuronic acid and acceptor substrates such as drugs, steroids, bile acids, xenobiotics, and dietary nutrients. Recent biochemical evidence indicates that the UGT proteins may oligomerize in the membrane, but conclusive evidence is still lacking. In the present study, we have used fluorescence resonance energy transfer (FRET) to study UGT1A oligomerization in live cells. This technique demonstrated that UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10 self-oligomerize (homodimerize). Heterodimer interactions were also explored, and it was determined that UGT1A1 was capable of binding with UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10. In addition to the in vivo FRET analysis, UGT1A protein-protein interactions were demonstrated through co-immunoprecipitation experiments. Co-expression of hemagglutinin-tagged and cyan fluorescent protein-tagged UGT1A proteins, followed by immunoprecipitation with anti-hemagglutinin beads, illustrated the potential of each UGT1A protein to homodimerize. Co-immunoprecipitation results also confirmed that UGT1A1 was capable of forming heterodimer complexes with all of the UGT1A proteins, corroborating the FRET results in live cells. These preliminary studies suggest that the UGT1A family of proteins form oligomerized complexes in the membrane, a property that may influence function and substrate selectivity.

  15. Age- and brain region-dependent α-synuclein oligomerization is attributed to alterations in intrinsic enzymes regulating α-synuclein phosphorylation in aging monkey brains.

    Science.gov (United States)

    Chen, Min; Yang, Weiwei; Li, Xin; Li, Xuran; Wang, Peng; Yue, Feng; Yang, Hui; Chan, Piu; Yu, Shun

    2016-02-23

    We previously reported that the levels of α-syn oligomers, which play pivotal pathogenic roles in age-related Parkinson's disease (PD) and dementia with Lewy bodies, increase heterogeneously in the aging brain. Here, we show that exogenous α-syn incubated with brain extracts from older cynomolgus monkeys and in Lewy body pathology (LBP)-susceptible brain regions (striatum and hippocampus) forms higher amounts of phosphorylated and oligomeric α-syn than that in extracts from younger monkeys and LBP-insusceptible brain regions (cerebellum and occipital cortex). The increased α-syn phosphorylation and oligomerization in the brain extracts from older monkeys and in LBP-susceptible brain regions were associated with higher levels of polo-like kinase 2 (PLK2), an enzyme promoting α-syn phosphorylation, and lower activity of protein phosphatase 2A (PP2A), an enzyme inhibiting α-syn phosphorylation, in these brain extracts. Further, the extent of the age- and brain-dependent increase in α-syn phosphorylation and oligomerization was reduced by inhibition of PLK2 and activation of PP2A. Inversely, phosphorylated α-syn oligomers reduced the activity of PP2A and showed potent cytotoxicity. In addition, the activity of GCase and the levels of ceramide, a product of GCase shown to activate PP2A, were lower in brain extracts from older monkeys and in LBP-susceptible brain regions. Our results suggest a role for altered intrinsic metabolic enzymes in age- and brain region-dependent α-syn oligomerization in aging brains.

  16. Rotavirus NSP4 is secreted from infected cells as an oligomeric lipoprotein and binds to glycosaminoglycans on the surface of non-infected cells

    Directory of Open Access Journals (Sweden)

    Didsbury Alicia

    2011-12-01

    Full Text Available Abstract Background Nonstructural glycoprotein 4 (NSP4 encoded by rotavirus is the only viral protein currently believed to function as an enterotoxin. NSP4 is synthesized as an intracellular transmembrane glycoprotein and as such is essential for virus assembly. Infection of polarized Caco-2 cells with rotavirus also results in the secretion of glycosylated NSP4 apparently in a soluble form despite retention of its transmembrane domain. We have examined the structure, solubility and cell-binding properties of this secreted form of NSP4 to further understand the biochemical basis for its enterotoxic function. We show here that NSP4 is secreted as discrete detergent-sensitive oligomers in a complex with phospholipids and demonstrate that this secreted form of NSP4 can bind to glycosaminoglycans present on the surface of a range of different cell types. Methods NSP4 was purified from the medium of infected cells after ultracentrifugation and ultrafiltration by successive lectin-affinity and ion exchange chromatography. Oligomerisation of NSP4 was examined by density gradient centrifugation and chemical crosslinking and the lipid content was assessed by analytical thin layer chromatography and flame ionization detection. Binding of NSP4 to various cell lines was measured using a flow cytometric-based assay. Results Secreted NSP4 formed oligomers that contained phospholipid but dissociated to a dimeric species in the presence of non-ionic detergent. The purified glycoprotein binds to the surface of various non-infected cells of distinct lineage. Binding of NSP4 to HT-29, a cell line of intestinal origin, is saturable and independent of divalent cations. Complementary biochemical approaches reveal that NSP4 binds to sulfated glycosaminoglycans on the plasma membrane. Conclusion Our study is the first to analyze an authentic (i.e. non-recombinant form of NSP4 that is secreted from virus-infected cells. Despite retention of the transmembrane domain

  17. Determination of oligomeric proanthocyanidins and their antioxidant capacity from different chocolate manufacturing stages using the NP-HPLC-online-DPPH methodology.

    Science.gov (United States)

    Pedan, Vasilisa; Fischer, Norbert; Bernath, Konrad; Hühn, Tilo; Rohn, Sascha

    2017-01-01

    Cocoa beans are a well-known source of antioxidant polyphenols. Especially individual oligomeric proanthocyanidins demonstrated a significant contribution to the total antioxidant activity of cocoa compared to monomeric compounds. An NP-HPLC-online-DPPH assay was developed for separating the homologous series of oligomeric proanthocyanidins and the simultaneous assessment of their antioxidant capacity in relation to the degree of polymerization (DP). The present study describes the influence of the different stages of a lab-scale chocolate manufacturing process on the content of oligomeric proanthocyanidins and their antioxidant capacity. The sum of the total proanthocyanidin content (∑ DP1-DP13) decreased from 30mg epicatechin equivalents per gram non-fat dry matter in raw fresh cocoa beans to 6mg epicatechin equivalents per gram in the final chocolate. The antioxidant capacity decreased accordingly from 25mg epicatechin equivalents per gram non-fat dry matter in raw fresh cocoa beans to 4mg/g in the final chocolate product. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Dominant negative mutants of Bacillus thuringiensis Cry1Ab toxin function as anti-toxins: demonstration of the role of oligomerization in toxicity.

    Directory of Open Access Journals (Sweden)

    Claudia Rodríguez-Almazán

    Full Text Available BACKGROUND: Bacillus thuringiensis Cry toxins, that are used worldwide in insect control, kill insects by a mechanism that depends on their ability to form oligomeric pores that insert into the insect-midgut cells. These toxins are being used worldwide in transgenic plants or spray to control insect pests in agriculture. However, a major concern has been the possible effects of these insecticidal proteins on non-target organisms mainly in ecosystems adjacent to agricultural fields. METHODOLOGY/PRINCIPAL FINDINGS: We isolated and characterized 11 non-toxic mutants of Cry1Ab toxin affected in different steps of the mechanism of action namely binding to receptors, oligomerization and pore-formation. These mutant toxins were analyzed for their capacity to block wild type toxin activity, presenting a dominant negative phenotype. The dominant negative phenotype was analyzed at two levels, in vivo by toxicity bioassays against susceptible Manduca sexta larvae and in vitro by pore formation activity in black lipid bilayers. We demonstrate that some mutations located in helix alpha-4 completely block the wild type toxin activity at sub-stoichiometric level confirming a dominant negative phenotype, thereby functioning as potent antitoxins. CONCLUSIONS/SIGNIFICANCE: This is the first reported case of a Cry toxin dominant inhibitor. These data demonstrate that oligomerization is a fundamental step in Cry toxin action and represent a potential mechanism to protect special ecosystems from the possible effect of Cry toxins on non-target organisms.

  19. A new mode of SAM domain mediated oligomerization observed in the CASKIN2 neuronal scaffolding protein

    KAUST Repository

    Smirnova, Ekaterina

    2016-08-22

    Background: CASKIN2 is a homolog of CASKIN1, a scaffolding protein that participates in a signaling network with CASK (calcium/calmodulin-dependent serine kinase). Despite a high level of homology between CASKIN2 and CASKIN1, CASKIN2 cannot bind CASK due to the absence of a CASK Interaction Domain and consequently, may have evolved undiscovered structural and functional distinctions.

  20. In vivo parameters influencing 2-Cys Prx oligomerization: The role of enzyme sulfinylation.

    Science.gov (United States)

    Noichri, Y; Palais, G; Ruby, V; D'Autreaux, B; Delaunay-Moisan, A; Nyström, T; Molin, M; Toledano, M B

    2015-12-01

    2-Cys Prxs are H2O2-specific antioxidants that become inactivated by enzyme hyperoxidation at elevated H2O2 levels. Although hyperoxidation restricts the antioxidant physiological role of these enzymes, it also allows the enzyme to become an efficient chaperone holdase. The critical molecular event allowing the peroxidase to chaperone switch is thought to be the enzyme assembly into high molecular weight (HMW) structures brought about by enzyme hyperoxidation. How hyperoxidation promotes HMW assembly is not well understood and Prx mutants allowing disentangling its peroxidase and chaperone functions are lacking. To begin addressing the link between enzyme hyperoxidation and HMW structures formation, we have evaluated the in vivo 2-Cys Prxs quaternary structure changes induced by H2O2 by size exclusion chromatography (SEC) on crude lysates, using wild type (Wt) untagged and Myc-tagged S. cerevisiae 2-Cys Prx Tsa1 and derivative Tsa1 mutants or genetic conditions known to inactivate peroxidase or chaperone activity or altering the enzyme sensitivity to hyperoxidation. Our data confirm the strict causative link between H2O2-induced hyperoxidation and HMW formation/stabilization, also raising the question of whether CP hyperoxidation triggers the assembly of HMW structures by the stacking of decamers, which is the prevalent view of the literature, or rather, the stabilization of preassembled stacked decamers.

  1. Crystal engineering of giant molecules based on perylene diimide conjugated polyhedral oligomeric silsesquioxane nano-atom

    Science.gov (United States)

    Ren, He

    Molecular architectures and topologies are found contributing to the formation of supramolecular structures of giant molecules. Dr. Cheng's research group developed a diverse of giant molecules via precisely controlled chemistry synthetic routes. These giant molecules can be categorized into several different families, namely giant surfactants, giant shape amphiphiles and giant polyhedron. By analyzing the hierarchical structures of these carefully designed and precisely synthesized giant molecules, the structural factors which affect, or even dominates, in some cases, the formation of supramolecular structures are revealed in these intensive researches. The results will further contribute to the understanding of dependence of supramolecular structures on molecular designs as well as molecular topology, and providing a practical solution to the scaling up of microscopic molecular functionalities to macroscopic material properties. Molecular Nano Particles (MNPs), including fullerene (C60), POSS, Polyoxometalate (POM) and proteins etc., is defined and applied as a specific type of building blocks in the design and synthesis of giant molecules. The persistence in shape and symmetry is considered as one of the major properties of MNPs. This persistence will support the construction of giant molecules for further supramolecular structures' study by introducing specific shapes, or precisely located side groups which will facilitate self-assembling behaviors with pre-programmed secondary interactions. Dictating material physical properties by its chemical composition is an attractive yet currently failed approach in the study of materials. However, the pursuit of determining material properties by microscopic molecular level properties is never seized, and found its solution when the idea of crystal engineering is raised: should each atom in the material is located exactly where it is designed to be and is properly bonded, the property of the material is hence determined

  2. CCR5/CD4/CXCR4 oligomerization prevents HIV-1 gp120IIIB binding to the cell surface.

    Science.gov (United States)

    Martínez-Muñoz, Laura; Barroso, Rubén; Dyrhaug, Sunniva Y; Navarro, Gemma; Lucas, Pilar; Soriano, Silvia F; Vega, Beatriz; Costas, Coloma; Muñoz-Fernández, M Ángeles; Santiago, César; Rodríguez Frade, José Miguel; Franco, Rafael; Mellado, Mario

    2014-05-13

    CCR5 and CXCR4, the respective cell surface coreceptors of R5 and X4 HIV-1 strains, both form heterodimers with CD4, the principal HIV-1 receptor. Using several resonance energy transfer techniques, we determined that CD4, CXCR4, and CCR5 formed heterotrimers, and that CCR5 coexpression altered the conformation of both CXCR4/CXCR4 homodimers and CD4/CXCR4 heterodimers. As a result, binding of the HIV-1 envelope protein gp120IIIB to the CD4/CXCR4/CCR5 heterooligomer was negligible, and the gp120-induced cytoskeletal rearrangements necessary for HIV-1 entry were prevented. CCR5 reduced HIV-1 envelope-induced CD4/CXCR4-mediated cell-cell fusion. In nucleofected Jurkat CD4 cells and primary human CD4(+) T cells, CCR5 expression led to a reduction in X4 HIV-1 infectivity. These findings can help to understand why X4 HIV-1 strains infection affect T-cell types differently during AIDS development and indicate that receptor oligomerization might be a target for previously unidentified therapeutic approaches for AIDS intervention.

  3. Saturated Fatty Acid Induces Insulin Resistance Partially Through Nucleotide-binding Oligomerization Domain 1 Signaling Pathway in Adipocytes

    Institute of Scientific and Technical Information of China (English)

    Yi-jun Zhou; Yin-si Tang; Yu-ling Song; Ai Li; Hui Zhou; Yan Li

    2013-01-01

    Objective To investigate the potential role of nucleotide-binding oligomerization domain 1 (NOD1), a component of the innate immune system, in mediating lipid-induced insulin resistance in adipocytes. Methods Adipocytes from Toll-like receptor 4 deficiency mice were used for stimulation experiments. The effect of oleate/palmitate mixture on nuclear factor-κB (NF-κB) activation was analyzed by reporter plasmid assay. The release of proinflammatory chemokine/cytokines production was determined by using real-time PCR. Insulin-stimulated glucose uptake was measured by 2-deoxy-D-[3H] glucose uptake assay. Chemokine/cytokine expression and glucose uptake in adipocytes transfected with small interfering RNA (siRNA) targeting NOD1 upon fatty acids treatment were analyzed. Results Oleate/palmitate mixture activated the NF-κB pathway and induced interleukin-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 mRNA expressions in adipocytes from mice deficient in Toll-like receptor 4, and these effects were blocked by siRNA targeting NOD1. Furthermore, saturated fatty acids decreased the ability of insulin-stimulated glucose uptake. Importantly, siRNA targeting NOD1 partially reversed saturated fatty acid-induced suppression of insulin-induced glucose uptake. Conclusion NOD1 might play an important role in saturated fatty acid-induced insulin resistance in adipocytes, suggesting a mechanism by which reduced NOD1 activity confers beneficial effects on insulin action.

  4. Antimutagenicity and antioxidative DNA damage properties of oligomeric proanthocyanidins from Thai grape seeds in TK6 cells.

    Science.gov (United States)

    Praphasawat, Ratsada; Klungsupya, Prapaipat; Muangman, Thunchanok; Laovitthayanggoon, Sarunya; Arunpairojana, Vullapa; Himakoun, Lakana

    2011-01-01

    Oligomeric proanthocyanidins (OPCs) are found mostly in red grape seeds. Many publications have reported that OPCs possess an excellent anti-oxidant effects. Since it could against cellular damage from reactive oxygen species (ROS) led to reduce the risk of chronic disease and cancers. We carried out this study on the Thai OPCs to evaluate the mutagenicity/ anti-mutagenicity and anti-oxidative DNA damage effects in TK6 cells by micronucleus (MN) and comet assays. In the MN assay, OPCs-treatment of TK6 cells at concentrations ranging from 10-200 ?g/ml (4 and 24 h) did not cause micronucleus induction over the negative control group but revealed a significant reduction the micronucleus frequencies against the known mutagen (mitomycin C). In the comet assay, OPCs-treated TK6 cells at concentrations of 100, 250, 500, and 1,000 ?g/ml could inhibit DNA damage induced by H(2)O(2) as indicated by 18.7, 36.4, 30.6, and 60.1%, respectively. Our results suggest that OPCs possess the anti-mutagenic and anti-oxidative DNA damage effects in TK6 cells under the conditions of this assay.

  5. Non-transferrin-bound iron (NTBI uptake by T lymphocytes: evidence for the selective acquisition of oligomeric ferric citrate species.

    Directory of Open Access Journals (Sweden)

    Joao Arezes

    Full Text Available Iron is an essential nutrient in several biological processes such as oxygen transport, DNA replication and erythropoiesis. Plasma iron normally circulates bound to transferrin. In iron overload disorders, however, iron concentrations exceed transferrin binding capacity and iron appears complexed with low molecular weight molecules, known as non-transferrin-bound iron (NTBI. NTBI is responsible for the toxicity associated with iron-overload pathologies but the mechanisms leading to NTBI uptake are not fully understood. Here we show for the first time that T lymphocytes are able to take up and accumulate NTBI in a manner that resembles that of hepatocytes. Moreover, we show that both hepatocytes and T lymphocytes take up the oligomeric Fe3Cit3 preferentially to other iron-citrate species, suggesting the existence of a selective NTBI carrier. These results provide a tool for the identification of the still elusive ferric-citrate cellular carrier and may also open a new pathway towards the design of more efficient iron chelators for the treatment of iron overload disorders.

  6. Nitration of the tumor suppressor protein, p53, at tyrosine 327 promotes p53 oligomerization and activation

    Science.gov (United States)

    Yakovlev, Vasily A.; Bayden, Alexander S.; Graves, Paul R.; Kellogg, Glen E.; Mikkelsen, Ross B.

    2010-01-01

    Previous studies demonstrate that nitric oxide (NO) promotes p53 transcriptional activity by a classical DNA-damage-responsive mechanism involving activation of ATM/ATR and phosphorylation of p53. These studies intentionally used high doses of NO-donors to achieve the maximum DNA-damage. However, lower concentrations of NO donors also stimulate rapid and unequivocal nuclear retention of p53, but apparently do not require ATM/ATR-dependent p53 phosphorylation or total p53 protein accumulation. To identify possible mechanisms for p53 activation at low NO levels, the role of Tyr nitration in p53 activation was evaluated. Low concentrations of the NO donor, DETA NONOate (nitrate Tyr327 within the tetramerization domain promoting p53 oligomerization, nuclear accumulation and increased DNA-binding activity without p53 Ser15 phosphorylation. Molecular modeling indicates that nitration of one Tyr327 stabilizes the dimer by about 2.67 kcal mol−1. Significant quantitative and qualitative differences in the patterns of p53-target gene modulation by low (50μM), non DNA-damaging and high (500μM), DNA-damaging NO donor concentrations was shown. These results demonstrate a new post-translational mechanism for modulating p53 transcriptional activity responsive to low NO concentrations and independent of DNA damage signaling. PMID:20499882

  7. Fouling behaviors of polybenzimidazole (PBI)-polyhedral oligomeric silsesquioxane (POSS)/polyacrylonitrile (PAN) hollow fiber membranes for engineering osmosis processes

    KAUST Repository

    Chen, Sicong

    2014-02-01

    This paper investigated the individual effects of reverse salt flux and permeate flux on fouling behaviors of as-spun and annealed polybenzimidazole (PBI)-polyhedral oligomeric silsesquioxane (POSS)/polyacrylonitrile (PAN) hollow fiber membranes under forward osmosis (FO) and pressure retarded osmosis (PRO) processes. Two types of membrane fouling had been studied; namely, inorganic fouling (CaSO4·2H2O gypsum scaling) during FO operations and organic fouling (sodium alginate fouling) during PRO operations. It is found that gypsum scaling on the membrane surface may be inhibited and even eliminated with an increase in reverse MgCl2 flux due to competitive formations of MgSO4° and CaSO4·2H2O. In contrast, the increase of reverse NaCl flux exhibits a slight enhancement on alginate fouling in both FO and PRO processes. Comparing to the reverse salt flux, the permeate flux always plays a dominant role in fouli