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Sample records for inter-peptide disulfide bonds

  1. Additional disulfide bonds in insulin

    DEFF Research Database (Denmark)

    Vinther, Tine N; Pettersson, Ingrid; Huus, Kasper

    2015-01-01

    The structure of insulin, a glucose homeostasis-controlling hormone, is highly conserved in all vertebrates and stabilized by three disulfide bonds. Recently, we designed a novel insulin analogue containing a fourth disulfide bond located between positions A10-B4. The N-terminus of insulin's B......-chain is flexible and can adapt multiple conformations. We examined how well disulfide bond predictions algorithms could identify disulfide bonds in this region of insulin. In order to identify stable insulin analogues with additional disulfide bonds, which could be expressed, the Cβ cut-off distance had...... in comparison to analogues with additional disulfide bonds that were more difficult to predict. In contrast, addition of the fourth disulfide bond rendered all analogues resistant to fibrillation under stress conditions and all stable analogues bound to the insulin receptor with picomolar affinities. Thus...

  2. Analysis of Disulfide Bond Formation

    NARCIS (Netherlands)

    Braakman, Ineke; Lamriben, Lydia; van Zadelhoff, Guus; Hebert, Daniel N.

    2017-01-01

    In this unit, protocols are provided for detection of disulfide bond formation in cultures of intact cells and in an in vitro translation system containing isolated microsomes or semi-permeabilized cells. First, the newly synthesized protein of interest is biosynthetically labeled with radioactive

  3. Amino Acid Patterns around Disulfide Bonds

    Directory of Open Access Journals (Sweden)

    Brett Drury

    2010-11-01

    Full Text Available Disulfide bonds provide an inexhaustible source of information on molecular evolution and biological specificity. In this work, we described the amino acid composition around disulfide bonds in a set of disulfide-rich proteins using appropriate descriptors, based on ANOVA (for all twenty natural amino acids or classes of amino acids clustered according to their chemical similarities and Scheffé (for the disulfide-rich proteins superfamilies statistics. We found that weakly hydrophilic and aromatic amino acids are quite abundant in the regions around disulfide bonds, contrary to aliphatic and hydrophobic amino acids. The density distributions (as a function of the distance to the center of the disulfide bonds for all defined entities presented an overall unimodal behavior: the densities are null at short distances, have maxima at intermediate distances and decrease for long distances. In the end, the amino acid environment around the disulfide bonds was found to be different for different superfamilies, allowing the clustering of proteins in a biologically relevant way, suggesting that this type of chemical information might be used as a tool to assess the relationship between very divergent sets of disulfide-rich proteins.

  4. On the photostability of the disulfide bond

    DEFF Research Database (Denmark)

    Stephansen, Anne Boutrup; Larsen, Martin Alex Bjørn; Klein, Liv Bærenholdt

    2014-01-01

    Photostability is an essential property of molecular building blocks of nature. Disulfides are central in the structure determination of proteins, which is in striking contradiction to the result that the S-S bond is a photochemically labile structural entity that cleaves to form free radicals upon...... on a sub 50 fs timescale without further ado. In a cyclic motif resembling the cysteine-disulfide bond in proteins, light can perturb the S-S bond to generate short-lived diradicaloid species, but the sulfur atoms are conformationally restricted by the ring that prevents the sulfur atoms from flying apart...... the photostability of disulfide-bonds must be ascribed a cyclic structural arrangement....

  5. Characterization of cyclic peptides containing disulfide bonds

    OpenAIRE

    Johnson, Mindy; Liu, Mingtao; Struble, Elaine; Hettiarachchi, Kanthi

    2015-01-01

    Unlike linear peptides, analysis of cyclic peptides containing disulfide bonds is not straightforward and demands indirect methods to achieve a rigorous proof of structure. Three peptides that belong to this category, p-Cl-Phe-DPDPE, DPDPE, and CTOP, were analyzed and the results are presented in this paper. The great potential of two dimensional NMR and ESI tandem mass spectrometry was harnessed during the course of peptide characterizations. A new RP-HPLC method for the analysis of trifluor...

  6. Widespread Disulfide Bonding in Proteins from Thermophilic Archaea

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    Julien Jorda

    2011-01-01

    Full Text Available Disulfide bonds are generally not used to stabilize proteins in the cytosolic compartments of bacteria or eukaryotic cells, owing to the chemically reducing nature of those environments. In contrast, certain thermophilic archaea use disulfide bonding as a major mechanism for protein stabilization. Here, we provide a current survey of completely sequenced genomes, applying computational methods to estimate the use of disulfide bonding across the Archaea. Microbes belonging to the Crenarchaeal branch, which are essentially all hyperthermophilic, are universally rich in disulfide bonding while lesser degrees of disulfide bonding are found among the thermophilic Euryarchaea, excluding those that are methanogenic. The results help clarify which parts of the archaeal lineage are likely to yield more examples and additional specific data on protein disulfide bonding, as increasing genomic sequencing efforts are brought to bear.

  7. Widespread disulfide bonding in proteins from thermophilic archaea.

    Science.gov (United States)

    Jorda, Julien; Yeates, Todd O

    2011-01-01

    Disulfide bonds are generally not used to stabilize proteins in the cytosolic compartments of bacteria or eukaryotic cells, owing to the chemically reducing nature of those environments. In contrast, certain thermophilic archaea use disulfide bonding as a major mechanism for protein stabilization. Here, we provide a current survey of completely sequenced genomes, applying computational methods to estimate the use of disulfide bonding across the Archaea. Microbes belonging to the Crenarchaeal branch, which are essentially all hyperthermophilic, are universally rich in disulfide bonding while lesser degrees of disulfide bonding are found among the thermophilic Euryarchaea, excluding those that are methanogenic. The results help clarify which parts of the archaeal lineage are likely to yield more examples and additional specific data on protein disulfide bonding, as increasing genomic sequencing efforts are brought to bear.

  8. Widespread Disulfide Bonding in Proteins from Thermophilic Archaea

    OpenAIRE

    Jorda, Julien; Yeates, Todd O.

    2011-01-01

    Disulfide bonds are generally not used to stabilize proteins in the cytosolic compartments of bacteria or eukaryotic cells, owing to the chemically reducing nature of those environments. In contrast, certain thermophilic archaea use disulfide bonding as a major mechanism for protein stabilization. Here, we provide a current survey of completely sequenced genomes, applying computational methods to estimate the use of disulfide bonding across the Archaea. Microbes belonging to the Crenarchaea...

  9. Alpha-cyclodextrins reversibly capped with disulfide bonds

    Czech Academy of Sciences Publication Activity Database

    Kumprecht, Lukáš; Buděšínský, Miloš; Bouř, Petr; Kraus, Tomáš

    2010-01-01

    Roč. 34, č. 10 (2010), s. 2254-2260 ISSN 1144-0546 R&D Projects: GA AV ČR IAA400550810 Institutional research plan: CEZ:AV0Z40550506 Keywords : cyclodextrins * disulfide bond * dynamic covalent bond Subject RIV: CC - Organic Chemistry Impact factor: 2.631, year: 2010

  10. UV Photofragmentation Dynamics of Protonated Cystine: Disulfide Bond Rupture.

    Science.gov (United States)

    Soorkia, Satchin; Dehon, Christophe; Kumar, S Sunil; Pedrazzani, Mélanie; Frantzen, Emilie; Lucas, Bruno; Barat, Michel; Fayeton, Jacqueline A; Jouvet, Christophe

    2014-04-03

    Disulfide bonds (S-S) play a central role in stabilizing the native structure of proteins against denaturation. Experimentally, identification of these linkages in peptide and protein structure characterization remains challenging. UV photodissociation (UVPD) can be a valuable tool in identifying disulfide linkages. Here, the S-S bond acts as a UV chromophore and absorption of one UV photon corresponds to a σ-σ* transition. We have investigated the photodissociation dynamics of protonated cystine, which is a dimer of two cysteines linked by a disulfide bridge, at 263 nm (4.7 eV) using a multicoincidence technique in which fragments coming from the same fragmentation event are detected. Two types of bond cleavages are observed corresponding to the disulfide (S-S) and adjacent C-S bond ruptures. We show that the S-S cleavage leads to three different fragment ions via three different fragmentation mechanisms. The UVPD results are compared to collision-induced dissociation (CID) and electron-induced dissociation (EID) studies.

  11. Disulfide Linkage Characterization of Disulfide Bond-Containing Proteins and Peptides by Reducing Electrochemistry and Mass Spectrometry

    DEFF Research Database (Denmark)

    Cramer, Christian N; Haselmann, Kim F; Olsen, Jesper V

    2016-01-01

    in protein sequencing by tandem MS (MS/MS). Electrochemical (EC) reduction of disulfide bonds has recently been demonstrated to provide efficient reduction efficiencies, significantly enhancing sequence coverages in online coupling with MS characterization. In this study, the potential use of EC disulfide...... link between parent disulfide-linked fragments and free reduced peptides in an LC-EC-MS platform of nonreduced proteolytic protein digestions. Here we report the successful use of EC as a partial reduction approach in mapping of disulfide bonds of intact human insulin (HI) and lysozyme. In addition, we...... established a LC-EC-MS platform advantageous in disulfide characterization of complex and highly disulfide-bonded proteins such as human serum albumin (HSA) by online EC reduction of nonreduced proteolytic digestions....

  12. Recent mass spectrometry-based techniques and considerations for disulfide bond characterization in proteins.

    Science.gov (United States)

    Lakbub, Jude C; Shipman, Joshua T; Desaire, Heather

    2018-04-01

    Disulfide bonds are important structural moieties of proteins: they ensure proper folding, provide stability, and ensure proper function. With the increasing use of proteins for biotherapeutics, particularly monoclonal antibodies, which are highly disulfide bonded, it is now important to confirm the correct disulfide bond connectivity and to verify the presence, or absence, of disulfide bond variants in the protein therapeutics. These studies help to ensure safety and efficacy. Hence, disulfide bonds are among the critical quality attributes of proteins that have to be monitored closely during the development of biotherapeutics. However, disulfide bond analysis is challenging because of the complexity of the biomolecules. Mass spectrometry (MS) has been the go-to analytical tool for the characterization of such complex biomolecules, and several methods have been reported to meet the challenging task of mapping disulfide bonds in proteins. In this review, we describe the relevant, recent MS-based techniques and provide important considerations needed for efficient disulfide bond analysis in proteins. The review focuses on methods for proper sample preparation, fragmentation techniques for disulfide bond analysis, recent disulfide bond mapping methods based on the fragmentation techniques, and automated algorithms designed for rapid analysis of disulfide bonds from liquid chromatography-MS/MS data. Researchers involved in method development for protein characterization can use the information herein to facilitate development of new MS-based methods for protein disulfide bond analysis. In addition, individuals characterizing biotherapeutics, especially by disulfide bond mapping in antibodies, can use this review to choose the best strategies for disulfide bond assignment of their biologic products. Graphical Abstract This review, describing characterization methods for disulfide bonds in proteins, focuses on three critical components: sample preparation, mass

  13. A single disulfide bond disruption in the β3 integrin subunit promotes thiol/disulfide exchange, a molecular dynamics study.

    Directory of Open Access Journals (Sweden)

    Lihie Levin

    Full Text Available The integrins are a family of membrane receptors that attach a cell to its surrounding and play a crucial function in cell signaling. The combination of internal and external stimuli alters a folded non-active state of these proteins to an extended active configuration. The β3 subunit of the platelet αIIbβ3 integrin is made of well-structured domains rich in disulfide bonds. During the activation process some of the disulfides are re-shuffled by a mechanism requiring partial reduction of some of these bonds; any disruption in this mechanism can lead to inherent blood clotting diseases. In the present study we employed Molecular Dynamics simulations for tracing the sequence of structural fluctuations initiated by a single cysteine mutation in the β3 subunit of the receptor. These simulations showed that in-silico protein mutants exhibit major conformational deformations leading to possible disulfide exchange reactions. We suggest that any mutation that prevents Cys560 from reacting with one of the Cys(567-Cys(581 bonded pair, thus disrupting its ability to participate in a disulfide exchange reaction, will damage the activation mechanism of the integrin. This suggestion is in full agreement with previously published experiments. Furthermore, we suggest that rearrangement of disulfide bonds could be a part of a natural cascade of thiol/disulfide exchange reactions in the αIIbβ3 integrin, which are essential for the native activation process.

  14. In-Depth Characterization of Protein Disulfide Bonds by Online Liquid Chromatography-Electrochemistry-Mass Spectrometry

    Science.gov (United States)

    Switzar, Linda; Nicolardi, Simone; Rutten, Julie W.; Oberstein, Saskia A. J. Lesnik; Aartsma-Rus, Annemieke; van der Burgt, Yuri E. M.

    2016-01-01

    Disulfide bonds are an important class of protein post-translational modifications, yet this structurally crucial modification type is commonly overlooked in mass spectrometry (MS)-based proteomics approaches. Recently, the benefits of online electrochemistry-assisted reduction of protein S-S bonds prior to MS analysis were exemplified by successful characterization of disulfide bonds in peptides and small proteins. In the current study, we have combined liquid chromatography (LC) with electrochemistry (EC) and mass analysis by Fourier transform ion cyclotron resonance (FTICR) MS in an online LC-EC-MS platform to characterize protein disulfide bonds in a bottom-up proteomics workflow. A key advantage of a LC-based strategy is the use of the retention time in identifying both intra- and interpeptide disulfide bonds. This is demonstrated by performing two sequential analyses of a certain protein digest, once without and once with electrochemical reduction. In this way, the "parent" disulfide-linked peptide detected in the first run has a retention time-based correlation with the EC-reduced peptides detected in the second run, thus simplifying disulfide bond mapping. Using this platform, both inter- and intra-disulfide-linked peptides were characterized in two different proteins, ß-lactoglobulin and ribonuclease B. In order to prevent disulfide reshuffling during the digestion process, proteins were digested at a relatively low pH, using (a combination of) the high specificity proteases trypsin and Glu-C. With this approach, disulfide bonds in ß-lactoglobulin and ribonuclease B were comprehensively identified and localized, showing that online LC-EC-MS is a useful tool for the characterization of protein disulfide bonds.

  15. Thermodynamic and mechanical effects of disulfide bonds in CXCLl7 chemokine

    Science.gov (United States)

    Singer, Christopher

    Chemokines are a family of signaling proteins mainly responsible for the chemotaxis of leukocytes, where their biological activity is modulated by their oligomerization state. Here, the dynamics and thermodynamic stability are characterized in monomer and homodimer structures of CXCL7, one of the most abundant platelet chemokines. The effects of dimerization and disulfide bond formation are investigated using computational methods that include molecular dynamics (MD) simulations and the Distance Constraint Model (DCM). A consistent picture emerges for the effect of dimerization and role of the Cys5-Cys31 and Cys7- Cys47 disulfide bonds. Surprisingly, neither disulfide bond is critical for maintaining structural stability in the monomer or dimer, although the monomer is destabilized more than the dimer upon removal of disulfide bonds. Instead, it is found that disulfide bonds influence the native state dynamics as well as modulates the relative stability between monomer and dimer. The combined analysis elucidates how CXCL7 is mechanically stable as a monomer, and how upon dimerization flexibly correlated motions are induced between the 30s and 50s loop within each monomer and across the dimer interface. Interestingly, the greatest gain in flexibility upon dimerization occurs when both disulfide bonds are present in each domain, and the homodimer is least stable relative to its two monomers. These results suggest the highly conserved disulfide bonds in chemokines facilitate a structural mechanism for distinguishing functional characteristics between monomer and dimer.

  16. Modulation of Thiol-Disulfide Oxidoreductases for Increased Production of Disulfide-Bond-Containing Proteins in Bacillus subtilis

    NARCIS (Netherlands)

    Kouwen, Thijs R. H. M.; Dubois, Jean-Yves F.; Freudl, Roland; Quax, Wim J.; van Dijl, Jan Maarten

    2008-01-01

    Disulfide bonds are important for the correct folding, structural integrity, and activity of many biotechnologically relevant proteins. For synthesis and subsequent secretion of these proteins in bacteria, such as the well-known "cell factory" Bacillus subtilis, it is often the correct formation of

  17. Solvent Induced Disulfide Bond Formation in 2,5-dimercapto-1,3,4-thiadiazole

    OpenAIRE

    Palanisamy Kalimuthu; Palraj Kalimuthu; S. Abraham John

    2007-01-01

    Disulfide bond formation is the decisive event in the protein folding to determine the conformation and stability of protein. To achieve this disulfide bond formation in vitro, we took 2,5-dimercapto-1,3,4-thiadiazole (DMcT) as a model compound. We found that disulfide bond formation takes place between two sulfhydryl groups of DMcT molecules in methanol. UV-Vis, FT-IR and mass spectroscopic as well as cyclic voltammetry were used to monitor the course of reaction. We proposed a mechanism for...

  18. Protein disulfide bond generation in Escherichia coli DsbB–DsbA

    International Nuclear Information System (INIS)

    Inaba, Kenji

    2008-01-01

    The crystal structure of the DsbB–DsbA–ubiquinone ternary complex has revealed a mechanism of protein disulfide bond generation in Escherichia coli. Protein disulfide bond formation is catalyzed by a series of Dsb enzymes present in the periplasm of Escherichia coli. The crystal structure of the DsbB–DsbA–ubiquinone ternary complex provided important insights into mechanisms of the de novo disulfide bond generation cooperated by DsbB and ubiquinone and of the disulfide bond shuttle from DsbB to DsbA. The structural basis for prevention of the crosstalk between the DsbA–DsbB oxidative and the DsbC–DsbD reductive pathways has also been proposed

  19. Insulin analog with additional disulfide bond has increased stability and preserved activity

    DEFF Research Database (Denmark)

    Vinther, Tine N.; Norrman, Mathias; Ribel, Ulla

    2013-01-01

    Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogs have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide...... bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use. To address this hypothesis, we designed insulin with an additional interchain disulfide bond in positions A10/B4 based on Cα-Cα distances, solvent exposure, and side-chain orientation in human insulin...... (HI) structure. This insulin analog had increased affinity for the insulin receptor and apparently augmented glucodynamic potency in a normal rat model compared with HI. Addition of the disulfide bond also resulted in a 34.6°C increase in melting temperature and prevented insulin fibril formation...

  20. Positions of disulfide bonds in rye (Secale cereale) seed chitinase-a.

    Science.gov (United States)

    Yamagami, T; Funatsu, G; Ishiguro, M

    2000-06-01

    The positions of disulfide bonds of rye seed chitinase-a (RSC-a) were identified by the isolation of disulfide-containing peptides produced with enzymatic and/or chemical cleavages of RSC-a, followed by sequencing them. An unequivocal assignment of disulfide bonds in this enzyme was as follows: Cys3-Cysl8, Cys12-Cys24, Cys15-Cys42, Cys17-Cys31, and Cys35-Cys39 in the chitin-binding domain (CB domain), Cys82-Cys144, Cys156-Cys164, and Cys282-Cys295 in the catalytic domain (Cat domain), and Cys263 was a free form.

  1. The significance of disulfide bonding in biological activity of HB-EGF, a mutagenesis approach

    OpenAIRE

    Hoskins, J.T.; Zhou, Z.; Harding, P.A.

    2008-01-01

    A site-directed mutagenesis approach was taken to disrupt each of 3 disulfide bonds within human HB-EGF by substituting serine for both cysteine residues that contribute to disulfide bonding. Each HB-EGF disulfide analogue (HB-EGF-Cys/Ser108/121, HB-EGF-Cys/Ser116/132, and HB-EGF-Cys/Ser134/143) was cloned under the regulation of the mouse metallothionein (MT) promoter and stably expressed in mouse fibroblasts. HB-EGF immunoreactive proteins with Mr of 6.5, 21 and 24kDa were observed from lys...

  2. Preventing disulfide bond formation weakens non-covalent forces among lysozyme aggregates.

    Directory of Open Access Journals (Sweden)

    Vijay Kumar Ravi

    Full Text Available Nonnative disulfide bonds have been observed among protein aggregates in several diseases like amyotrophic lateral sclerosis, cataract and so on. The molecular mechanism by which formation of such bonds promotes protein aggregation is poorly understood. Here in this work we employ previously well characterized aggregation of hen eggwhite lysozyme (HEWL at alkaline pH to dissect the molecular role of nonnative disulfide bonds on growth of HEWL aggregates. We employed time-resolved fluorescence anisotropy, atomic force microscopy and single-molecule force spectroscopy to quantify the size, morphology and non-covalent interaction forces among the aggregates, respectively. These measurements were performed under conditions when disulfide bond formation was allowed (control and alternatively when it was prevented by alkylation of free thiols using iodoacetamide. Blocking disulfide bond formation affected growth but not growth kinetics of aggregates which were ∼50% reduced in volume, flatter in vertical dimension and non-fibrillar in comparison to control. Interestingly, single-molecule force spectroscopy data revealed that preventing disulfide bond formation weakened the non-covalent interaction forces among monomers in the aggregate by at least ten fold, thereby stalling their growth and yielding smaller aggregates in comparison to control. We conclude that while constrained protein chain dynamics in correctly disulfide bonded amyloidogenic proteins may protect them from venturing into partial folded conformations that can trigger entry into aggregation pathways, aberrant disulfide bonds in non-amyloidogenic proteins (like HEWL on the other hand, may strengthen non-covalent intermolecular forces among monomers and promote their aggregation.

  3. A novel engineered interchain disulfide bond in the constant region enhances the thermostability of adalimumab Fab.

    Science.gov (United States)

    Nakamura, Hitomi; Oda-Ueda, Naoko; Ueda, Tadashi; Ohkuri, Takatoshi

    2018-01-01

    We constructed a system for expressing the Fab of the therapeutic human monoclonal antibody adalimumab at a yield of 20 mg/L in the methylotrophic yeast Pichia pastoris. To examine the contribution of interchain disulfide bonds to conformational stability, we prepared adalimumab Fab from which the interchain disulfide bond at the C-terminal region at both the CH 1 and CL domains was deleted by substitution of Cys with Ala (Fab ΔSS ). DSC measurements showed that the Tm values of Fab ΔSS were approximately 5 °C lower than those of wild-type Fab, suggesting that the interchain disulfide bond contributes to conformational thermostability. Using computer simulations, we designed a novel interchain disulfide bond outside the C-terminal region to increase the stability of Fab ΔSS . The resulting Fab (mutSS Fab ΔSS ) had the mutations H:V177C and L:Q160C in Fab ΔSS , confirming the formation of the disulfide bond between CH 1 and CL. The thermostability of mutSS Fab ΔSS was approximately 5 °C higher than that of Fab ΔSS . Therefore, the introduction of the designed interchain disulfide bond enhanced the thermostability of Fab ΔSS and mitigated the destabilization caused by partial reduction of the interchain disulfide bond at the C-terminal region, which occurs in site-specific modification such as PEGylation. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Enhancing Protein Disulfide Bond Cleavage by UV Excitation and Electron Capture Dissociation for Top-Down Mass Spectrometry

    OpenAIRE

    Wongkongkathep, Piriya; Li, Huilin; Zhang, Xing; Loo, Rachel R. Ogorzalek; Julian, Ryan R.; Loo, Joseph A.

    2015-01-01

    The application of ion pre-activation with 266 nm ultraviolet (UV) laser irradiation combined with electron capture dissociation (ECD) is demonstrated to enhance top-down mass spectrometry sequence coverage of disulfide bond containing proteins. UV-based activation can homolytically cleave a disulfide bond to yield two separated thiol radicals. Activated ECD experiments of insulin and ribonuclease A containing three and four disulfide bonds, respectively, were performed. UV-activation in comb...

  5. Radiation-induced cleavage of disulfide bonds in proteins. Clivage radiolytique des ponts disulfure des proteines

    Energy Technology Data Exchange (ETDEWEB)

    Favaudon, V; Tourbez, H; Lhoste, J M [Paris-11 Univ., 91 - Orsay (FR); Houee-Levin, C [Paris-5 Univ., 75 (FR)

    1991-06-01

    The reduction of the disulfide bonds in apo-Riboflavin-Binding Protein (apoRBP) by the CO{sub 2}{sup -}{center dot} radical occurred under {gamma}-ray irradiation as a chain reaction whose efficiency increased upon acidification of the medium. Pulse-radiolysis analysis showed a rapid one-electron oxidation of the disulfide bonds yielding the anionic or protonated form of the disulfide radical. The main decay path of this radical under acidic conditions consisted of the rapid formation of a thiyl radical intermediate in equilibrium with the closed, cyclic form. At pH 8 the disulfide radical anion decayed via intramolecular and/or intermolecular routes including disproportionation, protein-protein crosslinking, non-dismutative recombination processes, and reaction with sulfhydryl groups in pre-reduced systems.

  6. Enhancing Protein Disulfide Bond Cleavage by UV Excitation and Electron Capture Dissociation for Top-Down Mass Spectrometry.

    Science.gov (United States)

    Wongkongkathep, Piriya; Li, Huilin; Zhang, Xing; Loo, Rachel R Ogorzalek; Julian, Ryan R; Loo, Joseph A

    2015-11-15

    The application of ion pre-activation with 266 nm ultraviolet (UV) laser irradiation combined with electron capture dissociation (ECD) is demonstrated to enhance top-down mass spectrometry sequence coverage of disulfide bond containing proteins. UV-based activation can homolytically cleave a disulfide bond to yield two separated thiol radicals. Activated ECD experiments of insulin and ribonuclease A containing three and four disulfide bonds, respectively, were performed. UV-activation in combination with ECD allowed the three disulfide bonds of insulin to be cleaved and the overall sequence coverage to be increased. For the larger sized ribonuclease A with four disulfide bonds, irradiation from an infrared laser (10.6 µm) to disrupt non-covalent interactions was combined with UV-activation to facilitate the cleavage of up to three disulfide bonds. Preferences for disulfide bond cleavage are dependent on protein structure and sequence. Disulfide bonds can reform if the generated radicals remain in close proximity. By varying the time delay between the UV-activation and the ECD events, it was determined that disulfide bonds reform within 10-100 msec after their UV-homolytic cleavage.

  7. Disulfide bond within mu-calpain active site inhibits activity and autolysis.

    Science.gov (United States)

    Lametsch, René; Lonergan, Steven; Huff-Lonergan, Elisabeth

    2008-09-01

    Oxidative processes have the ability to influence mu-calpain activity. In the present study the influence of oxidation on activity and autolysis of mu-calpain was examined. Furthermore, LC-MS/MS analysis was employed to identify and characterize protein modifications caused by oxidation. The results revealed that the activity of mu-calpain is diminished by oxidation with H2O2 in a reversible manner involving cysteine and that the rate of autolysis of mu-calpain concomitantly slowed. The LC-MS/MS analysis of the oxidized mu-calpain revealed that the amino acid residues 105-133 contained a disulfide bond between Cys(108) and Cys(115). The finding that the active site cysteine in mu-calpain is able to form a disulfide bond has, to our knowledge, not been reported before. This could be part of a unique oxidation mechanism for mu-calpain. The results also showed that the formation of the disulfide bond is limited in the control (no oxidant added), and further limited in a concentration-dependent manner when beta-mercaptoethanol is added. However, the disulfide bond is still present to some extent in all conditions indicating that the active site cysteine is potentially highly susceptible to the formation of this intramolecular disulfide bond.

  8. Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein

    DEFF Research Database (Denmark)

    Østergaard, H.; Henriksen, A.; Hansen, Flemming G.

    2001-01-01

    To visualize the formation of disulfide bonds in living cells, a pair of redox-active cysteines was introduced into the yellow fluorescent variant of green fluorescent protein. Formation of a disulfide bond between the two cysteines was fully reversible and resulted in a >2-fold decrease...... in the intrinsic fluorescence. Inter conversion between the two redox states could thus be followed in vitro as well as in vivoby non- invasive fluorimetric measurements. The 1.5 Angstrom crystal structure of the oxidized protein revealed a disulfide bond- induced distortion of the beta -barrel, as well...... the physiological range for redox-active cysteines. In the cytoplasm of Escherichia coli, the protein was a sensitive probe for the redox changes that occur upon disruption of the thioredoxin reductive pathway....

  9. The road to the first, fully active and more stable human insulin variant with an additional disulfide bond

    DEFF Research Database (Denmark)

    Vinther, Tine N.; Kjeldsen, Thomas B.; Jensen, Knud Jørgen

    2015-01-01

    Insulin, a small peptide hormone, is crucial in maintaining blood glucose homeostasis. The stability and activity of the protein is directed by an intricate system involving disulfide bonds to stabilize the active monomeric species and by their non-covalent oligomerization. All known insulin...... variants in vertebrates consist of two peptide chains and have six cysteine residues, which form three disulfide bonds, two of them link the two chains and a third is an intra-chain bond in the A-chain. This classical insulin fold appears to have been conserved over half a billion years of evolution. We...... addressed the question whether a human insulin variant with four disulfide bonds could exist and be fully functional. In this review, we give an overview of the road to engineering four-disulfide bonded insulin analogs. During our journey, we discovered several active four disulfide bonded insulin analogs...

  10. Enhanced production of a single domain antibody with an engineered stabilizing extra disulfide bond.

    Science.gov (United States)

    Liu, Jinny L; Goldman, Ellen R; Zabetakis, Dan; Walper, Scott A; Turner, Kendrick B; Shriver-Lake, Lisa C; Anderson, George P

    2015-10-09

    Single domain antibodies derived from the variable region of the unique heavy chain antibodies found in camelids yield high affinity and regenerable recognition elements. Adding an additional disulfide bond that bridges framework regions is a proven method to increase their melting temperature, however often at the expense of protein production. To fulfill their full potential it is essential to achieve robust protein production of these stable binding elements. In this work, we tested the hypothesis that decreasing the isoelectric point of single domain antibody extra disulfide bond mutants whose production fell due to the incorporation of the extra disulfide bond would lead to recovery of the protein yield, while maintaining the favorable melting temperature and affinity. Introduction of negative charges into a disulfide bond mutant of a single domain antibody specific for the L1 antigen of the vaccinia virus led to approximately 3.5-fold increase of protein production to 14 mg/L, while affinity and melting temperature was maintained. In addition, refolding following heat denaturation improved from 15 to 70 %. It also maintained nearly 100 % of its binding function after heating to 85 °C for an hour at 1 mg/mL. Disappointingly, the replacement of neutral or positively charged amino acids with negatively charged ones to lower the isoelectric point of two anti-toxin single domain antibodies stabilized with a second disulfide bond yielded only slight increases in protein production. Nonetheless, for one of these binders the charge change itself stabilized the structure equivalent to disulfide bond addition, thus providing an alternative route to stabilization which is not accompanied by loss in production. The ability to produce high affinity, stable single domain antibodies is critical for their utility. While the addition of a second disulfide bond is a proven method for enhancing stability of single domain antibodies, it frequently comes at the cost of reduced

  11. Role of the Disulfide Bond in Prion Protein Amyloid Formation: A Thermodynamic and Kinetic Analysis.

    Science.gov (United States)

    Honda, Ryo

    2018-02-27

    Prion diseases are associated with the structural conversion of prion protein (PrP) to a β-sheet-rich aggregate, PrP Sc . Previous studies have indicated that a reduction of the disulfide bond linking C179 and C214 of PrP yields an amyloidlike β-rich aggregate in vitro. To gain mechanistic insights into the reduction-induced aggregation, here I characterized how disulfide bond reduction modulates the protein folding/misfolding landscape of PrP, by examining 1) the equilibrium stabilities of the native (N) and aggregated states relative to the unfolded (U) state, 2) the transition barrier separating the U and aggregated states, and 3) the final structure of amyloidlike misfolded aggregates. Kinetic and thermodynamic experiments revealed that disulfide bond reduction decreases the equilibrium stabilities of both the N and aggregated states by ∼3 kcal/mol, without changing either the amyloidlike aggregate structure, at least at the secondary structural level, or the transition barrier of aggregation. Therefore, disulfide bond reduction modulates the protein folding/misfolding landscape by entropically stabilizing disordered states, including the U and transition state of aggregation. This also indicates that the equilibrium stability of the N state, but not the transition barrier of aggregation, is the dominant factor determining the reduction-induced aggregation of PrP. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  12. Disulfide bonds in folding and transport of the mouse hepatitis virus glycoproteins

    NARCIS (Netherlands)

    Horzinek, M.C.; Opstelten, D.-J.E.; Groote, P. de; Vennema, H.; Rottier, P.J.M.

    1993-01-01

    We have analyzed the effects of reducing conditions on the folding of the spike (S) protein and on the intracellular transport of the membrane (M) protein of the mouse hepatitis coronavirus. These proteins differ in their potential to form disulfide bonds in the lumen of the endoplasmic reticulum

  13. Per-2,3-O-alkylated beta-cyclodextrin duplexes connected with disulfide bonds

    Czech Academy of Sciences Publication Activity Database

    Tatar, Ameneh; Grishina, Anastasia; Buděšínský, Miloš; Kraus, Tomáš

    2017-01-01

    Roč. 29, č. 1 (2017), s. 40-48 ISSN 1061-0278 R&D Projects: GA MŠk LD12019 Grant - others:COST(XE) CM1005 Institutional support: RVO:61388963 Keywords : cyclodextrins * inclusion complexes * disulfide bonds Subject RIV: CC - Organic Chemistry OBOR OECD: Organic chemistry Impact factor: 1.264, year: 2016

  14. The significance of disulfide bonding in biological activity of HB-EGF, a mutagenesis approach

    International Nuclear Information System (INIS)

    Hoskins, J.T.; Zhou, Z.; Harding, P.A.

    2008-01-01

    A site-directed mutagenesis approach was taken to disrupt each of 3 disulfide bonds within human HB-EGF by substituting serine for both cysteine residues that contribute to disulfide bonding. Each HB-EGF disulfide analogue (HB-EGF-Cys/Ser 108/121 , HB-EGF-Cys/Ser 116/132 , and HB-EGF-Cys/Ser 134/143 ) was cloned under the regulation of the mouse metallothionein (MT) promoter and stably expressed in mouse fibroblasts. HB-EGF immunoreactive proteins with M r of 6.5, 21 and 24 kDa were observed from lysates of HB-EGF and each HB-EGF disulfide analogue. HB-EGF immunohistochemical analyses of each HB-EGF stable cell line demonstrated ubiquitous protein expression except HB-EGF-Cys/Ser 108/121 and HB-EGF-Cys/Ser 116/132 stable cell lines which exhibited accumulated expression immediately outside the nucleus. rHB-EGF, HB-EGF, and HB-EGF 134/143 proteins competed with 125 I-EGF in an A431 competitive binding assay, whereas HB-EGF-Cys/Ser 108/121 and HB-EGF-Cys/Ser 116/132 failed to compete. Each HB-EGF disulfide analogue lacked the ability to stimulate tyrosine phosphorylation of the 170 kDa EGFR. These results suggest that HB-EGF-Cys/Ser 134/143 antagonizes EGFRs

  15. Structural characterization of PTX3 disulfide bond network and its multimeric status in cumulus matrix organization.

    Science.gov (United States)

    Inforzato, Antonio; Rivieccio, Vincenzo; Morreale, Antonio P; Bastone, Antonio; Salustri, Antonietta; Scarchilli, Laura; Verdoliva, Antonio; Vincenti, Silvia; Gallo, Grazia; Chiapparino, Caterina; Pacello, Lucrezia; Nucera, Eleonora; Serlupi-Crescenzi, Ottaviano; Day, Anthony J; Bottazzi, Barbara; Mantovani, Alberto; De Santis, Rita; Salvatori, Giovanni

    2008-04-11

    PTX3 is an acute phase glycoprotein that plays key roles in resistance to certain pathogens and in female fertility. PTX3 exerts its functions by interacting with a number of structurally unrelated molecules, a capacity that is likely to rely on its complex multimeric structure stabilized by interchain disulfide bonds. In this study, PAGE analyses performed under both native and denaturing conditions indicated that human recombinant PTX3 is mainly composed of covalently linked octamers. The network of disulfide bonds supporting this octameric assembly was resolved by mass spectrometry and Cys to Ser site-directed mutagenesis. Here we report that cysteine residues at positions 47, 49, and 103 in the N-terminal domain form three symmetric interchain disulfide bonds stabilizing four protein subunits in a tetrameric arrangement. Additional interchain disulfide bonds formed by the C-terminal domain cysteines Cys(317) and Cys(318) are responsible for linking the PTX3 tetramers into octamers. We also identified three intrachain disulfide bonds within the C-terminal domain that we used as structural constraints to build a new three-dimensional model for this domain. Previously it has been shown that PTX3 is a key component of the cumulus oophorus extracellular matrix, which forms around the oocyte prior to ovulation, because cumuli from PTX3(-/-) mice show defective matrix organization. Recombinant PTX3 is able to restore the normal phenotype ex vivo in cumuli from PTX3(-/-) mice. Here we demonstrate that PTX3 Cys to Ser mutants, mainly assembled into tetramers, exhibited wild type rescue activity, whereas a mutant, predominantly composed of dimers, had impaired functionality. These findings indicate that protein oligomerization is essential for PTX3 activity within the cumulus matrix and implicate PTX3 tetramers as the functional molecular units required for cumulus matrix organization and stabilization.

  16. A novel disulfide bond in the SH2 Domain of the C-terminal Src kinase controls catalytic activity.

    Science.gov (United States)

    Mills, Jamie E; Whitford, Paul C; Shaffer, Jennifer; Onuchic, Jose N; Adams, Joseph A; Jennings, Patricia A

    2007-02-02

    The SH2 domain of the C-terminal Src kinase [Csk] contains a unique disulfide bond that is not present in other known SH2 domains. To investigate whether this unusual disulfide bond serves a novel function, the effects of disulfide bond formation on catalytic activity of the full-length protein and on the structure of the SH2 domain were investigated. The kinase activity of full-length Csk decreases by an order of magnitude upon formation of the disulfide bond in the distal SH2 domain. NMR spectra of the fully oxidized and fully reduced SH2 domains exhibit similar chemical shift patterns and are indicative of similar, well-defined tertiary structures. The solvent-accessible disulfide bond in the isolated SH2 domain is highly stable and far from the small lobe of the kinase domain. However, reduction of this bond results in chemical shift changes of resonances that map to a cluster of residues that extend from the disulfide bond across the molecule to a surface that is in direct contact with the small lobe of the kinase domain in the intact molecule. Normal mode analyses and molecular dynamics calculations suggest that disulfide bond formation has large effects on residues within the kinase domain, most notably within the active-site cleft. Overall, the data indicate that reversible cross-linking of two cysteine residues in the SH2 domain greatly impacts catalytic function and interdomain communication in Csk.

  17. Dissecting the role of disulfide bonds on the amyloid formation of insulin

    International Nuclear Information System (INIS)

    Li, Yang; Gong, Hao; Sun, Yue; Yan, Juan; Cheng, Biao; Zhang, Xin; Huang, Jing; Yu, Mengying; Guo, Yu; Zheng, Ling; Huang, Kun

    2012-01-01

    Highlights: ► We dissect how individual disulfide bond affects the amyloidogenicity of insulin. ► A controlled reduction system for insulin is established in this study. ► Disulfide breakage is associated with unfolding and increased amyloidogenicity. ► Breakage of A6-A11 is associated with significantly increased cytotoxicity. ► Analogs without A6-A11 have a higher potency to form high order toxic oligomers. -- Abstract: Disulfide bonds play a critical role in the stability and folding of proteins. Here, we used insulin as a model system, to investigate the role of its individual disulfide bond during the amyloid formation of insulin. Tris(2-carboxyethyl)phosphine (TCEP) was applied to reduce two of the three disulfide bonds in porcine insulin and the reduced disulfide bonds were then alkylated by iodoacetamide. Three disulfide bond-modified insulin analogs, INS-2 (lack of A6-A11), INS-3 (lack of A7-B7) and INS-6 (lack of both A6-A11 and A7-B7), were obtained. Far-UV circular dichroism (CD) spectroscopy results indicated that the secondary structure of INS-2 was the closest to insulin under neutral conditions, followed by INS-3 and INS-6, whereas in an acidic solution all analogs were essentially unfolded. To test how these modifications affect the amyloidogenicity of insulin, thioflavin-T (ThT) fluorescence and transmission electronic microscopy (TEM) were performed. Our results showed that all analogs were more prone to aggregation than insulin, with the order of aggregation rates being INS-6 > INS-3 > INS-2. Cross-linking of unmodified proteins (PICUP) assay results showed that analogs without A6-A11 (INS-2 and INS-6) have a higher potential for oligomerization than insulin and INS-3, which is accompanied with a higher cytotoxicity as the hemolytic assays of human erythrocytes suggested. The results indicated that breakage of A7-B7 induced more unfolding of the insulin structure and a higher amyloidogenicity than breakage of A6-A11, but breakage of A6

  18. Increased Functional Half-life of Fibroblast Growth Factor-1 by Recovering a Vestigial Disulfide Bond

    Directory of Open Access Journals (Sweden)

    Jihun Lee

    2010-12-01

    Full Text Available The fibroblast growth factor (FGF family of proteins contains an absolutely conserved Cys residue at position 83 that is present as a buried free cysteine. We have previously shown that mutation of the structurally adjacent residue, Ala66, to cysteine results in the formation of a stabilizing disulfide bond in FGF-1. This result suggests that the conserved free cysteine residue at position 83 in the FGF family of proteins represents a vestigial half-cystine. Here, we characterize the functional half-life and mitogenic activity of the oxidized form of the Ala66Cys mutation to identify the effect of the recovered vestigial disulfide bond between Cys83 and Cys66 upon the cellular function of FGF-1. The results show that the mitogenic activity of this mutant is significantly increased and that its functional half-life is greatly extended. These favorable effects are conferred by the formation of a disulfide bond that simultaneously increases thermodynamic stability of the protein and removes a reactive buried thiol at position 83. Recovering this vestigial disulfide by introducing a cysteine at position 66 is a potentially useful protein engineering strategy to improve the functional half-life of other FGF family members.

  19. Conformational analysis by quantitative NOE measurements of the β-proton pairs across individual disulfide bonds in proteins

    International Nuclear Information System (INIS)

    Takeda, Mitsuhiro; Terauchi, Tsutomu; Kainosho, Masatsune

    2012-01-01

    NOEs between the β-protons of cysteine residues across disulfide bonds in proteins provide direct information on the connectivities and conformations of these important cross-links, which are otherwise difficult to investigate. With conventional [U- 13 C, 15 N]-proteins, however, fast spin diffusion processes mediated by strong dipolar interactions between geminal β-protons prohibit the quantitative measurements and thus the analyses of long-range NOEs across disulfide bonds. We describe a robust approach for alleviating such difficulties, by using proteins selectively labeled with an equimolar mixture of (2R, 3S)-[β- 13 C; α,β- 2 H 2 ] Cys and (2R, 3R)-[β- 13 C; α,β- 2 H 2 ] Cys, but otherwise fully deuterated. Since either one of the prochiral methylene protons, namely β2 (proS) or β3 (proR), is always replaced with a deuteron and no other protons remain in proteins prepared by this labeling scheme, all four of the expected NOEs for the β-protons across disulfide bonds could be measured without any spin diffusion interference, even with long mixing times. Therefore, the NOEs for the β2 and β3 pairs across each of the disulfide bonds could be observed at high sensitivity, even though they are 25% of the theoretical maximum for each pair. With the NOE information, the disulfide bond connectivities can be unambiguously established for proteins with multiple disulfide bonds. In addition, the conformations around disulfide bonds, namely χ 2 and χ 3 , can be determined based on the precise proton distances of the four β-proton pairs, by quantitative measurements of the NOEs across the disulfide bonds. The feasibility of this method is demonstrated for bovine pancreatic trypsin inhibitor, which has three disulfide bonds.

  20. Photo-reduction on the rupture of disulfide bonds and the related protein assembling

    Science.gov (United States)

    Wang, Wei

    It has been found that many proteins can self-assemble into nanoscale assemblies when they unfold or partially unfold under harsh conditions, such as low pH, high temperature, or the presence of denaturants, and so on. These nanoscale assemblies can have some applications such as the drug-delivery systems (DDSs). Here we report a study that a very physical way, the UV illumination, can be used to facilitate the formation of protein fibrils and nanoparticles under native conditions by breaking disulfide bonds in some disulfide-containing proteins. By controlling the intensity of UV light and the illumination time, we realized the preparation of self-assembly nanoparticles which encapsulate the anticancer drug doxorubicin (DOX) and can be used as the DDS for inhibiting the growth of tumor. The formation of fibrillary assemblies was also observed. The rupture of disulfide bonds through photo-reduction process due to the effect of tryptophan and tyrosine was studied, and the physical mechanism of the assembling of the related disulfide-containing proteins was also discussed. We thank the financial support from NSF of China and the 973 project.

  1. Nicotinamidase/pyrazinamidase of Mycobacterium tuberculosis forms homo-dimers stabilized by disulfide bonds.

    Science.gov (United States)

    Rueda, Daniel; Sheen, Patricia; Gilman, Robert H; Bueno, Carlos; Santos, Marco; Pando-Robles, Victoria; Batista, Cesar V; Zimic, Mirko

    2014-12-01

    Recombinant wild-pyrazinamidase from H37Rv Mycobacterium tuberculosis was analyzed by gel electrophoresis under differential reducing conditions to evaluate its quaternary structure. PZAse was fractionated by size exclusion chromatography under non-reducing conditions. PZAse activity was measured and mass spectrometry analysis was performed to determine the identity of proteins by de novo sequencing and to determine the presence of disulfide bonds. This study confirmed that M. tuberculosis wild type PZAse was able to form homo-dimers in vitro. Homo-dimers showed a slightly lower specific PZAse activity compared to monomeric PZAse. PZAse dimers were dissociated into monomers in response to reducing conditions. Mass spectrometry analysis confirmed the existence of disulfide bonds (C72-C138 and C138-C138) stabilizing the quaternary structure of the PZAse homo-dimer. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Determination of Disulfide Bond Connectivity of Cysteine-rich Peptide IpTx{sub a}

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Chul Won; Kim, Jim Il [Chonnam National Univ., Gwangju (Korea, Republic of); Sato, Kazuki [Fukuoka Women' s Univ., Fukuoka (Japan)

    2013-06-15

    Cysteine-rich peptides stabilized by intramolecular disulfide bonds have often been isolated from venoms of microbes, animals and plants. These peptides typically have much higher stability and improved biopharmaceutical properties compared to their linear counterparts. Therefore the correct disulfide bond formation of small proteins and peptides has been extensively studied for a better understanding of their folding mechanism and achieving efficient generation of the naturally occurring biologically active product. Imperatoxin A (IpTx{sub a}), a peptide toxin containing 6 cysteine residues, was isolated from the venom of scorpion Pandinus imperator, selectively binds the ryanodine receptors and activates Ca{sup 2+} release from sarcoplasmic reticulum (SR). IpTx{sub a} increases the binding of ryanodine to ryanodine receptors (RyRs) and encourages reconstituted single channel to induce subconductance states.

  3. Conversion of a disulfide bond into a thioacetal group during echinomycin biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Hotta, Kinya; Keegan, Ronan M.; Ranganathan, Soumya; Fang, Minyi; Bibby, Jaclyn; Winn, Martyn D.; Sato, Michio; Lian, Mingzhu; Watanabe, Kenji; Rigden, Daniel J.; Kim, Chu-Young (Liverpool); (Daresbury); (NU Singapore); (Shizuoka); (RAL)

    2013-12-02

    Echinomycin is a nonribosomal depsipeptide natural product with a range of interesting bioactivities that make it an important target for drug discovery and development. It contains a thioacetal bridge, a unique chemical motif derived from the disulfide bond of its precursor antibiotic triostin A by the action of an S-adenosyl-L-methionine-dependent methyltransferase, Ecm18. The crystal structure of Ecm18 in complex with its reaction products S-adenosyl-L-homocysteine and echinomycin was determined at 1.50 Å resolution. Phasing was achieved using a new molecular replacement package called AMPLE, which automatically derives search models from structure predictions based on ab initio protein modelling. Structural analysis indicates that a combination of proximity effects, medium effects, and catalysis by strain drives the unique transformation of the disulfide bond into the thioacetal linkage.

  4. MLKL forms disulfide bond-dependent amyloid-like polymers to induce necroptosis.

    Science.gov (United States)

    Liu, Shuzhen; Liu, Hua; Johnston, Andrea; Hanna-Addams, Sarah; Reynoso, Eduardo; Xiang, Yougui; Wang, Zhigao

    2017-09-05

    Mixed-lineage kinase domain-like protein (MLKL) is essential for TNF-α-induced necroptosis. How MLKL promotes cell death is still under debate. Here we report that MLKL forms SDS-resistant, disulfide bond-dependent polymers during necroptosis in both human and mouse cells. MLKL polymers are independent of receptor-interacting protein kinase 1 and 3 (RIPK1/RIPK3) fibers. Large MLKL polymers are more than 2 million Da and are resistant to proteinase K digestion. MLKL polymers are fibers 5 nm in diameter under electron microscopy. Furthermore, the recombinant N-terminal domain of MLKL forms amyloid-like fibers and binds Congo red dye. MLKL mutants that cannot form polymers also fail to induce necroptosis efficiently. Finally, the compound necrosulfonamide conjugates cysteine 86 of human MLKL and blocks MLKL polymer formation and subsequent cell death. These results demonstrate that disulfide bond-dependent, amyloid-like MLKL polymers are necessary and sufficient to induce necroptosis.

  5. Photo-responsive liquid crystalline epoxy networks with exchangeable disulfide bonds

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yuzhan [Washington State Univ., Pullman, WA (United States); Zhang, Yuehong [Washington State Univ., Pullman, WA (United States); Rios, Orlando [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Keum, Jong K. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Kessler, Michael R. [Washington State Univ., Pullman, WA (United States); North Dakota State Univ., Fargo, ND (United States)

    2017-07-27

    The increasing demand for intelligent materials has driven the development of polymers with a variety of functionalities. However, combining multiple functionalities within one polymer is still challenging because of the difficulties encountered in coordinating different functional building blocks during fabrication. In this work, we demonstrate the fabrication of a multifunctional liquid crystalline epoxy network (LCEN) using the combination of thermotropic liquid crystals, photo-responsive azobenzene molecules, and exchangeable disulfide bonds. In addition to shape memory behavior enabled by the reversible liquid crystalline phase transition and photo-induced bending behavior resulting from the photo-responsive azobenzene molecules, the introduction of dynamic disulfide bonds into the LCEN resulted in a structurally dynamic network, allowing the reshaping, repairing, and recycling of the material.

  6. Modified electrophoretic and digestion conditions allow a simplified mass spectrometric evaluation of disulfide bonds

    Czech Academy of Sciences Publication Activity Database

    Pompach, Petr; Man, Petr; Kavan, Daniel; Hofbauerová, Kateřina; Kumar, Vinay; Bezouška, Karel; Havlíček, Vladimír; Novák, Petr

    2009-01-01

    Roč. 44, č. 11 (2009), s. 1571-1578 ISSN 1076-5174 R&D Projects: GA AV ČR KJB400200501; GA AV ČR IAA5020403; GA AV ČR KJB500200612; GA MŠk LC545; GA MŠk LC07017 Institutional research plan: CEZ:AV0Z50200510 Keywords : disulfide bond * cystamine * gel electrophoresis Subject RIV: CE - Biochemistry Impact factor: 3.411, year: 2009

  7. Hindered disulfide bonds to regulate release rate of model drug from mesoporous silica.

    Science.gov (United States)

    Nadrah, Peter; Maver, Uroš; Jemec, Anita; Tišler, Tatjana; Bele, Marjan; Dražić, Goran; Benčina, Mojca; Pintar, Albin; Planinšek, Odon; Gaberšček, Miran

    2013-05-01

    With the advancement of drug delivery systems based on mesoporous silica nanoparticles (MSNs), a simple and efficient method regulating the drug release kinetics is needed. We developed redox-responsive release systems with three levels of hindrance around the disulfide bond. A model drug (rhodamine B dye) was loaded into MSNs' mesoporous voids. The pore opening was capped with β-cyclodextrin in order to prevent leakage of drug. Indeed, in absence of a reducing agent the systems exhibited little leakage, while the addition of dithiothreitol cleaved the disulfide bonds and enabled the release of cargo. The release rate and the amount of released dye were tuned by the level of hindrance around disulfide bonds, with the increased hindrance causing a decrease in the release rate as well as in the amount of released drug. Thus, we demonstrated the ability of the present mesoporous systems to intrinsically control the release rate and the amount of the released cargo by only minor structural variations. Furthermore, an in vivo experiment on zebrafish confirmed that the present model delivery system is nonteratogenic.

  8. Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E. coli

    Directory of Open Access Journals (Sweden)

    Hatahet Feras

    2010-09-01

    Full Text Available Abstract Background The formation of native disulfide bonds is a complex and essential post-translational modification for many proteins. The large scale production of these proteins can be difficult and depends on targeting the protein to a compartment in which disulfide bond formation naturally occurs, usually the endoplasmic reticulum of eukaryotes or the periplasm of prokaryotes. It is currently thought to be impossible to produce large amounts of disulfide bond containing protein in the cytoplasm of wild-type bacteria such as E. coli due to the presence of multiple pathways for their reduction. Results Here we show that the introduction of Erv1p, a sulfhydryl oxidase and FAD-dependent catalyst of disulfide bond formation found in the inter membrane space of mitochondria, allows the efficient formation of native disulfide bonds in heterologously expressed proteins in the cytoplasm of E. coli even without the disruption of genes involved in disulfide bond reduction, for example trxB and/or gor. Indeed yields of active disulfide bonded proteins were higher in BL21 (DE3 pLysSRARE, an E. coli strain with the reducing pathways intact, than in the commercial Δgor ΔtrxB strain rosetta-gami upon co-expression of Erv1p. Conclusions Our results refute the current paradigm in the field that disruption of at least one of the reducing pathways is essential for the efficient production of disulfide bond containing proteins in the cytoplasm of E. coli and open up new possibilities for the use of E. coli as a microbial cell factory.

  9. {sup 13}C-NMR studies on disulfide bond isomerization in bovine pancreatic trypsin inhibitor (BPTI)

    Energy Technology Data Exchange (ETDEWEB)

    Takeda, Mitsuhiro [Kumamoto University, Department of Structural BioImaging, Faculty of Life Sciences (Japan); Miyanoiri, Yohei [Nagoya University, Structural Biology Research Center, Graduate School of Science (Japan); Terauchi, Tsutomu [Tokyo Metropolitan University, Graduate School of Science and Engineering (Japan); Kainosho, Masatsune, E-mail: kainosho@tmu.ac.jp [Nagoya University, Structural Biology Research Center, Graduate School of Science (Japan)

    2016-09-15

    Conformational isomerization of disulfide bonds is associated with the dynamics and thus the functional aspects of proteins. However, our understanding of the isomerization is limited by experimental difficulties in probing it. We explored the disulfide conformational isomerization of the Cys14–Cys38 disulfide bond in bovine pancreatic trypsin inhibitor (BPTI), by performing an NMR line-shape analysis of its Cys carbon peaks. In this approach, 1D {sup 13}C spectra were recorded at small temperature intervals for BPTI samples selectively labeled with site-specifically {sup 13}C-enriched Cys, and the recorded peaks were displayed in the order of the temperature after the spectral scales were normalized to a carbon peak. Over the profile of the line-shape, exchange broadening that altered with temperature was manifested for the carbon peaks of Cys14 and Cys38. The Cys14–Cys38 disulfide bond reportedly exists in equilibrium between a high-populated (M) and two low-populated states (m{sub c14} and m{sub c38}). Consistent with the three-site exchange model, biphasic exchange broadening arising from the two processes was observed for the peak of the Cys14 α-carbon. As the exchange broadening is maximized when the exchange rate equals the chemical shift difference in Hz between equilibrating sites, semi-quantitative information that was useful for establishing conditions for {sup 13}C relaxation dispersion experiments was obtained through the carbon line-shape profile. With respect to the m{sub c38} isomerization, the {sup 1}H-{sup 13}C signals at the β-position of the minor state were resolved from the major peaks and detected by exchange experiments at a low temperature.

  10. CD44 Binding to Hyaluronic Acid Is Redox Regulated by a Labile Disulfide Bond in the Hyaluronic Acid Binding Site.

    Directory of Open Access Journals (Sweden)

    Helena Kellett-Clarke

    Full Text Available CD44 is the primary leukocyte cell surface receptor for hyaluronic acid (HA, a component of the extracellular matrix. Enzymatic post translational cleavage of labile disulfide bonds is a mechanism by which proteins are structurally regulated by imparting an allosteric change and altering activity. We have identified one such disulfide bond in CD44 formed by Cys77 and Cys97 that stabilises the HA binding groove. This bond is labile on the surface of leukocytes treated with chemical and enzymatic reducing agents. Analysis of CD44 crystal structures reveal the disulfide bond to be solvent accessible and in the-LH hook configuration characteristic of labile disulfide bonds. Kinetic trapping and binding experiments on CD44-Fc chimeric proteins show the bond is preferentially reduced over the other disulfide bonds in CD44 and reduction inhibits the CD44-HA interaction. Furthermore cells transfected with CD44 no longer adhere to HA coated surfaces after pre-treatment with reducing agents. The implications of CD44 redox regulation are discussed in the context of immune function, disease and therapeutic strategies.

  11. Disulfide bond effects on protein stability: designed variants of Cucurbita maxima trypsin inhibitor-V.

    Science.gov (United States)

    Zavodszky, M; Chen, C W; Huang, J K; Zolkiewski, M; Wen, L; Krishnamoorthi, R

    2001-01-01

    Attempts to increase protein stability by insertion of novel disulfide bonds have not always been successful. According to the two current models, cross-links enhance stability mainly through denatured state effects. We have investigated the effects of removal and addition of disulfide cross-links, protein flexibility in the vicinity of a cross-link, and disulfide loop size on the stability of Cucurbita maxima trypsin inhibitor-V (CMTI-V; 7 kD) by differential scanning calorimetry. CMTI-V offers the advantage of a large, flexible, and solvent-exposed loop not involved in extensive intra-molecular interactions. We have uncovered a negative correlation between retention time in hydrophobic column chromatography, a measure of protein hydrophobicity, and melting temperature (T(m)), an indicator of native state stabilization, for CMTI-V and its variants. In conjunction with the complete set of thermodynamic parameters of denaturation, this has led to the following deductions: (1) In the less stable, disulfide-removed C3S/C48S (Delta Delta G(d)(50 degrees C) = -4 kcal/mole; Delta T(m) = -22 degrees C), the native state is destabilized more than the denatured state; this also applies to the less-stable CMTI-V* (Delta Delta G(d)(50 degrees C) = -3 kcal/mole; Delta T(m) = -11 degrees C), in which the disulfide-containing loop is opened by specific hydrolysis of the Lys(44)-Asp(45) peptide bond; (2) In the less stable, disulfide-inserted E38C/W54C (Delta Delta G(d)(50 degrees C) = -1 kcal/mole; Delta T(m) = +2 degrees C), the denatured state is more stabilized than the native state; and (3) In the more stable, disulfide-engineered V42C/R52C (Delta Delta G(d)(50 degrees C) = +1 kcal/mole; Delta T(m) = +17 degrees C), the native state is more stabilized than the denatured state. These results show that a cross-link stabilizes both native and denatured states, and differential stabilization of the two states causes either loss or gain in protein stability. Removal of hydrogen

  12. SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm

    Directory of Open Access Journals (Sweden)

    Lobstein Julie

    2012-05-01

    Full Text Available Abstract Background Production of correctly disulfide bonded proteins to high yields remains a challenge. Recombinant protein expression in Escherichia coli is the popular choice, especially within the research community. While there is an ever growing demand for new expression strains, few strains are dedicated to post-translational modifications, such as disulfide bond formation. Thus, new protein expression strains must be engineered and the parameters involved in producing disulfide bonded proteins must be understood. Results We have engineered a new E. coli protein expression strain named SHuffle, dedicated to producing correctly disulfide bonded active proteins to high yields within its cytoplasm. This strain is based on the trxB gor suppressor strain SMG96 where its cytoplasmic reductive pathways have been diminished, allowing for the formation of disulfide bonds in the cytoplasm. We have further engineered a major improvement by integrating into its chromosome a signal sequenceless disulfide bond isomerase, DsbC. We probed the redox state of DsbC in the oxidizing cytoplasm and evaluated its role in assisting the formation of correctly folded multi-disulfide bonded proteins. We optimized protein expression conditions, varying temperature, induction conditions, strain background and the co-expression of various helper proteins. We found that temperature has the biggest impact on improving yields and that the E. coli B strain background of this strain was superior to the K12 version. We also discovered that auto-expression of substrate target proteins using this strain resulted in higher yields of active pure protein. Finally, we found that co-expression of mutant thioredoxins and PDI homologs improved yields of various substrate proteins. Conclusions This work is the first extensive characterization of the trxB gor suppressor strain. The results presented should help researchers design the appropriate protein expression conditions using

  13. Engineering an improved IgG4 molecule with reduced disulfide bond heterogeneity and increased Fab domain thermal stability.

    Science.gov (United States)

    Peters, Shirley J; Smales, C Mark; Henry, Alistair J; Stephens, Paul E; West, Shauna; Humphreys, David P

    2012-07-13

    The integrity of antibody structure, stability, and biophysical characterization are becoming increasingly important as antibodies receive increasing scrutiny from regulatory authorities. We altered the disulfide bond arrangement of an IgG4 molecule by mutation of the Cys at the N terminus of the heavy chain constant domain 1 (C(H)1) (Kabat position 127) to a Ser and introduction of a Cys at a variety of positions (positions 227-230) at the C terminus of C(H)1. An inter-LC-C(H)1 disulfide bond is thus formed, which mimics the disulfide bond arrangement found in an IgG1 molecule. The antibody species present in the supernatant following transient expression in Chinese hamster ovary cells were analyzed by immunoblot to investigate product homogeneity, and purified product was analyzed by a thermofluor assay to determine thermal stability. We show that the light chain can form an inter-LC-C(H)1 disulfide bond with a Cys when present at several positions on the upper hinge (positions 227-230) and that such engineered disulfide bonds can consequently increase the Fab domain thermal stability between 3 and 6.8 °C. The IgG4 disulfide mutants displaying the greatest increase in Fab thermal stability were also the most homogeneous in terms of disulfide bond arrangement and antibody species present. Importantly, mutations did not affect the affinity for antigen of the resultant molecules. In combination with the previously described S241P mutation, we present an IgG4 molecule with increased Fab thermal stability and reduced product heterogeneity that potentially offers advantages for the production of IgG4 molecules.

  14. Intra- and inter-subunit disulfide bond formation is nonessential in adeno-associated viral capsids.

    Directory of Open Access Journals (Sweden)

    Nagesh Pulicherla

    Full Text Available The capsid proteins of adeno-associated viruses (AAV have five conserved cysteine residues. Structural analysis of AAV serotype 2 reveals that Cys289 and Cys361 are located adjacent to each other within each monomer, while Cys230 and Cys394 are located on opposite edges of each subunit and juxtaposed at the pentamer interface. The Cys482 residue is located at the base of a surface loop within the trimer region. Although plausible based on molecular dynamics simulations, intra- or inter-subunit disulfides have not been observed in structural studies. In the current study, we generated a panel of Cys-to-Ser mutants to interrogate the potential for disulfide bond formation in AAV capsids. The C289S, C361S and C482S mutants were similar to wild type AAV with regard to titer and transduction efficiency. However, AAV capsid protein subunits with C230S or C394S mutations were prone to proteasomal degradation within the host cells. Proteasomal inhibition partially blocked degradation of mutant capsid proteins, but failed to rescue infectious virions. While these results suggest that the Cys230/394 pair is critical, a C394V mutant was found viable, but not the corresponding C230V mutant. Although the exact nature of the structural contribution(s of Cys230 and Cys394 residues to AAV capsid formation remains to be determined, these results support the notion that disulfide bond formation within the Cys289/361 or Cys230/394 pair appears to be nonessential. These studies represent an important step towards understanding the role of inter-subunit interactions that drive AAV capsid assembly.

  15. Identification and prevention of antibody disulfide bond reduction during cell culture manufacturing.

    Science.gov (United States)

    Trexler-Schmidt, Melody; Sargis, Sandy; Chiu, Jason; Sze-Khoo, Stefanie; Mun, Melissa; Kao, Yung-Hsiang; Laird, Michael W

    2010-06-15

    In the biopharmaceutical industry, therapeutic monoclonal antibodies are primarily produced in mammalian cell culture systems. During the scale-up of a monoclonal antibody production process, we observed excessive mechanical cell shear as well as significant reduction of the antibody's interchain disulfide bonds during harvest operations. This antibody reduction event was catastrophic as the product failed to meet the drug substance specifications and the bulk product was lost. Subsequent laboratory studies have demonstrated that cells subjected to mechanical shear release cellular enzymes that contribute to this antibody reduction phenomenon (manuscript submitted; Kao et al., 2009). Several methods to prevent this antibody reduction event were developed using a lab-scale model to reproduce the lysis and reduction events. These methods included modifications to the cell culture media with chemicals (e.g., cupric sulfate (CuSO(4))), pre- and post-harvest chemical additions to the cell culture fluid (CCF) (e.g., CuSO(4), EDTA, L-cystine), as well as lowering the pH and air sparging of the harvested CCF (HCCF). These methods were evaluated for their effectiveness in preventing disulfide bond reduction and their impact to product quality. Effective prevention methods, which yielded acceptable product quality were evaluated for their potential to be implemented at manufacturing-scale. The work described here identifies numerous effective reduction prevention measures from lab-scale studies; several of these methods were then successfully translated into manufacturing processes. 2010 Wiley Periodicals, Inc.

  16. Coupling gold nanoparticles to silica nanoparticles through disulfide bonds for glutathione detection

    International Nuclear Information System (INIS)

    Shi Yupeng; Zhang Heng; Zhang Zhaomin; Yi Changqing; Yue Zhenfeng; Teng, Kar-Seng; Li Meijin; Yang Mengsu

    2013-01-01

    Advances in the controlled assembly of nanoscale building blocks have resulted in functional devices which can find applications in electronics, biomedical imaging, drug delivery etc. In this study, novel covalent nanohybrid materials based upon [Ru(bpy) 3 ] 2+ -doped silica nanoparticles (SiNPs) and gold nanoparticles (AuNPs), which could be conditioned as OFF–ON probes for glutathione (GSH) detection, were designed and assembled in sequence, with the disulfide bonds as the bridging elements. The structural and optical properties of the nanohybrid architectures were characterized using transmission electron microscopy, UV–vis spectroscopy and fluorescence spectroscopy, respectively. Zeta potential measurements, x-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy were employed to monitor the reaction processes of the SiNPs–S–S–COOH and SiNPs–S–S–AuNPs synthesis. It was found that the covalent nanohybrid architectures were fluorescently dark (OFF state), indicating that SiNPs were effectively quenched by AuNPs. The fluorescence of the OFF–ON probe was resumed (ON state) when the bridge of the disulfide bond was cleaved by reducing reagents such as GSH. This work provides a new platform and strategy for GSH detection using covalent nanohybrid materials. (paper)

  17. Efficient assembly of recombinant major histocompatibility complex class I molecules with preformed disulfide bonds

    DEFF Research Database (Denmark)

    Ostergaard Pedersen, L; Nissen, Mogens Holst; Hansen, N J

    2001-01-01

    The expression of major histocompatibility class I (MHC-I) crucially depends upon the binding of appropriate peptides. MHC-I from natural sources are therefore always preoccupied with peptides complicating their purification and analysis. Here, we present an efficient solution to this problem....... Recombinant MHC-I heavy chains were produced in Escherichia coli and subsequently purified under denaturing conditions. In contrast to common practice, the molecules were not reduced during the purification. The oxidized MHC-I heavy chain isoforms were highly active with respect to peptide binding....... This suggests that de novo folding of denatured MHC-I molecules proceed efficiently if directed by preformed disulfide bond(s). Importantly, these molecules express serological epitopes and stain specific T cells; and they bind peptides specifically. Several denatured MHC-I heavy chains were analyzed and shown...

  18. Characterization of intramolecular disulfide bonds and secondary modifications of the glycoprotein from viral hemorrhagic septicemia virus, a fish rhabdovirus

    DEFF Research Database (Denmark)

    Einer-Jensen, Katja; Nielsen, Thomas Krogh; Roepstorff, Peter

    1998-01-01

    were analyzed by mass spectrometry before and after chemical reduction, and six disulfide bonds were identified: Cys29-Cys339, Cys44-Cys295, Cys90-Cys132, Cys172-Cys177, Cys195-Cys265, and Cys231-CyS236. Mass spectrometric analysis in combination with glycosidases allowed characterization of the glycan...... of the protein, The present study was initiated to identify the disulfide bonds and other structural aspects relevant to vaccine design. The N-terminal amino acid residue was identified as being a pyroglutamic acid, corresponding to Gln21 of the primary transcript, Peptides from endoproteinase-degraded G protein...... cysteine residues are situated at conserved positions, This finding suggests that there might be some common disulfide bonding pattern among the six rhabdoviruses....

  19. Gold nanoparticles physicochemically bonded onto tungsten disulfide nanosheet edges exhibit augmented plasmon damping

    Directory of Open Access Journals (Sweden)

    Gregory T. Forcherio

    2017-07-01

    Full Text Available Augmented plasmonic damping of dipole-resonant gold (Au nanoparticles (NP physicochemically bonded onto edges of tungsten disulfide (WS2 nanosheets, ostensibly due to hot electron injection, is quantified using electron energy loss spectroscopy (EELS. EELS allows single-particle spatial resolution. A measured 0.23 eV bandwidth expansion of the localized surface plasmon resonance upon covalent bonding of 20 nm AuNP to WS2 edges was deemed significant by Welch’s t-test. Approximately 0.19 eV of the measured 0.23 eV expansion went beyond conventional radiative and nonradiative damping mechanisms according to discrete dipole models, ostensibly indicating emergence of hot electron transport from AuNP into the WS2. A quantum efficiency of up to 11±5% spanning a 7 fs transfer process across the optimized AuNP-TMD ohmic junction is conservatively calculated. Putative hot electron transport for AuNP physicochemically bonded to TMD edges exceeded that for AuNP physically deposited onto the TMD basal plane. This arose from contributions due to (i direct physicochemical bond between AuNP and WS2; (ii AuNP deposition at TMD edge sites; and (iii lower intrinsic Schottky barrier. This improves understanding of photo-induced doping of TMD by metal NP which could benefit emerging catalytic and optoelectronic applications.

  20. Gold nanoparticles physicochemically bonded onto tungsten disulfide nanosheet edges exhibit augmented plasmon damping

    Science.gov (United States)

    Forcherio, Gregory T.; Dunklin, Jeremy R.; Backes, Claudia; Vaynzof, Yana; Benamara, Mourad; Roper, D. Keith

    2017-07-01

    Augmented plasmonic damping of dipole-resonant gold (Au) nanoparticles (NP) physicochemically bonded onto edges of tungsten disulfide (WS2) nanosheets, ostensibly due to hot electron injection, is quantified using electron energy loss spectroscopy (EELS). EELS allows single-particle spatial resolution. A measured 0.23 eV bandwidth expansion of the localized surface plasmon resonance upon covalent bonding of 20 nm AuNP to WS2 edges was deemed significant by Welch's t-test. Approximately 0.19 eV of the measured 0.23 eV expansion went beyond conventional radiative and nonradiative damping mechanisms according to discrete dipole models, ostensibly indicating emergence of hot electron transport from AuNP into the WS2. A quantum efficiency of up to 11±5% spanning a 7 fs transfer process across the optimized AuNP-TMD ohmic junction is conservatively calculated. Putative hot electron transport for AuNP physicochemically bonded to TMD edges exceeded that for AuNP physically deposited onto the TMD basal plane. This arose from contributions due to (i) direct physicochemical bond between AuNP and WS2; (ii) AuNP deposition at TMD edge sites; and (iii) lower intrinsic Schottky barrier. This improves understanding of photo-induced doping of TMD by metal NP which could benefit emerging catalytic and optoelectronic applications.

  1. S center dot center dot center dot N chalcogen bonded complexes of carbon disulfide with diazines. Theoretical study

    Czech Academy of Sciences Publication Activity Database

    Zierkiewicz, W.; Fanfrlík, Jindřich; Michalczyk, M.; Michalska, D.; Hobza, Pavel

    2018-01-01

    Roč. 500, Jan 26 (2018), s. 37-44 ISSN 0301-0104 Institutional support: RVO:61388963 Keywords : chalcogen bond * carbon disulfide * diazines * DFT Subject RIV: CF - Physical ; Theoretical Chemistry OBOR OECD: Physical chemistry Impact factor: 1.767, year: 2016

  2. Combining biophysical methods to analyze the disulfide bond in SH2 domain of C-terminal Src kinase.

    Science.gov (United States)

    Liu, Dongsheng; Cowburn, David

    2016-01-01

    The Src Homology 2 (SH2) domain is a structurally conserved protein domain that typically binds to a phosphorylated tyrosine in a peptide motif from the target protein. The SH2 domain of C-terminal Src kinase (Csk) contains a single disulfide bond, which is unusual for most SH2 domains. Although the global motion of SH2 domain regulates Csk function, little is known about the relationship between the disulfide bond and binding of the ligand. In this study, we combined X-ray crystallography, solution NMR, and other biophysical methods to reveal the interaction network in Csk. Denaturation studies have shown that disulfide bond contributes significantly to the stability of SH2 domain, and crystal structures of the oxidized and C122S mutant showed minor conformational changes. We further investigated the binding of SH2 domain to a phosphorylated peptide from Csk-binding protein upon reduction and oxidation using both NMR and fluorescence approaches. This work employed NMR, X-ray cryptography, and other biophysical methods to study a disulfide bond in Csk SH2 domain. In addition, this work provides in-depth understanding of the structural dynamics of Csk SH2 domain.

  3. Mutational analysis of Kex2 recognition sites and a disulfide bond in tannase from Aspergillus oryzae.

    Science.gov (United States)

    Koseki, Takuya; Otsuka, Motohiro; Mizuno, Toshiyuki; Shiono, Yoshihito

    2017-01-22

    Aspergillus oryzae tannase (AoTanA), which contains two Kex2 recognition sites at positions Arg311 and Arg316, consists of two subunits that are generated by the cleavage of tannase gene product by the Kex2 protease. Based on the crystal structure of feruloyl esterase from Aspergillus oryzae (AoFaeB), which has been classified as a member of the fungal tannase family, the catalytic triad residues of AoTanA are predicted to be Ser195, Asp455, and His501, with the serine and histidine residues brought together by a disulfide bond of the neighboring cysteines, Cys194 and Cys502. In this study, we investigated the functional role of the Kex2 recognition sites and disulfide bond between the neighboring cysteines in AoTanA. We constructed a double variant (R311A/R316A), a seven amino-acid deletion variant of region Lys310-Arg316 (ΔKR), and two single variants (C194A and C502A). While the R311A/R316A variant exhibited the two bands similar to wild type by SDS-PAGE after treatment with endoglycosidase H, the ΔKR variant exhibited only one band. R311A/R316A variation had no effect on tannase activity and stability. Meanwhile, the ΔKR variant exhibited higher activity compared to the wild-type. The activities of the C194A and C502A variants decreased considerably (<0.24% of the wild-type) toward methyl gallate. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. The role of short-range Cys171-Cys178 disulfide bond in maintaining cutinase active site integrity: A molecular dynamics simulation

    International Nuclear Information System (INIS)

    Matak, Mehdi Youssefi; Moghaddam, Majid Erfani

    2009-01-01

    Understanding structural determinants in enzyme active site integrity can provide a good knowledge to design efficient novel catalytic machineries. Fusarium solani pisi cutinase with classic triad Ser-His-Asp is a promising enzyme to scrutinize these structural determinants. We performed two MD simulations: one, with the native structure, and the other with the broken Cys171-Cys178 disulfide bond. This disulfide bond stabilizes a turn in active site on which catalytic Asp175 is located. Functionally important H-bonds and atomic fluctuations in catalytic pocket have been changed. We proposed that this disulfide bond within active site can be considered as an important determinant of cutinase active site structural integrity.

  5. Microwave-assisted acid and base hydrolysis of intact proteins containing disulfide bonds for protein sequence analysis by mass spectrometry.

    Science.gov (United States)

    Reiz, Bela; Li, Liang

    2010-09-01

    Controlled hydrolysis of proteins to generate peptide ladders combined with mass spectrometric analysis of the resultant peptides can be used for protein sequencing. In this paper, two methods of improving the microwave-assisted protein hydrolysis process are described to enable rapid sequencing of proteins containing disulfide bonds and increase sequence coverage, respectively. It was demonstrated that proteins containing disulfide bonds could be sequenced by MS analysis by first performing hydrolysis for less than 2 min, followed by 1 h of reduction to release the peptides originally linked by disulfide bonds. It was shown that a strong base could be used as a catalyst for microwave-assisted protein hydrolysis, producing complementary sequence information to that generated by microwave-assisted acid hydrolysis. However, using either acid or base hydrolysis, amide bond breakages in small regions of the polypeptide chains of the model proteins (e.g., cytochrome c and lysozyme) were not detected. Dynamic light scattering measurement of the proteins solubilized in an acid or base indicated that protein-protein interaction or aggregation was not the cause of the failure to hydrolyze certain amide bonds. It was speculated that there were some unknown local structures that might play a role in preventing an acid or base from reacting with the peptide bonds therein. 2010 American Society for Mass Spectrometry. Published by Elsevier Inc. All rights reserved.

  6. Chaperonin GroE-facilitated refolding of disulfide-bonded and reduced Taka-amylase A from Aspergillus oryzae.

    Science.gov (United States)

    Kawata, Y; Hongo, K; Mizobata, T; Nagai, J

    1998-12-01

    The refolding characteristics of Taka-amylase A (TAA) from Aspergillus oryzae in the presence of the chaperonin GroE were studied in terms of activity and fluorescence. Disulfide-bonded (intact) TAA and non-disulfide-bonded (reduced) TAA were unfolded in guanidine hydrochloride and refolded by dilution into buffer containing GroE. The intermediates of both intact and reduced enzymes were trapped by GroEL in the absence of nucleotide. Upon addition of nucleotides such as ATP, ADP, CTP or UTP, the intermediates were released from GroEL and recovery of activity was detected. In both cases, the refolding yields in the presence of GroEL and ATP were higher than spontaneous recoveries. Fluorescence studies of intrinsic tryptophan and a hydrophobic probe, 8-anilinonaphthalene-1-sulfonate, suggested that the intermediates trapped by GroEL assumed conformations with different hydrophobic properties. The presence of protein disulfide isomerase or reduced and oxidized forms of glutathione in addition to GroE greatly enhanced the refolding reaction of reduced TAA. These findings suggest that GroE has an ability to recognize folding intermediates of TAA protein and facilitate refolding, regardless of the existence or absence of disulfide bonds in the protein.

  7. Cleavage of the interchain disulfide bonds in rituximab increases its affinity for FcγRIIIA.

    Science.gov (United States)

    Suzuki, Mami; Yamanoi, Ayaka; Machino, Yusuke; Kobayashi, Eiji; Fukuchi, Kaori; Tsukimoto, Mitsutoshi; Kojima, Shuji; Kohroki, Junya; Akimoto, Kazunori; Masuho, Yasuhiko

    2013-07-05

    The Fc region of human IgG1 mediates effector function via binding to Fcγ receptors and complement activation. The H and L chains of IgG1 antibodies are joined by four interchain disulfide bonds. In this study, these bonds within the therapeutic IgG1 rituximab (RTX) were cleaved either by mild reduction followed by alkylation or by mild S-sulfonation; consequently, two modified RTXs - A-RTX (alkylated) and S-RTX (S-sulfonated) - were formed, and both were almost as potent as unmodified RTX when binding CD20 antigen. Unexpectedly, each modified RTX had a higher binding affinity for FcγRIIIA (CD16A) than did unmodified RTX. However, S-RTX and A-RTX were each less potent than RTX in an assay of antibody-dependent cellular cytotoxicity (ADCC). In this ADCC assay, each modified RTX showed decreased secretion of granzyme B, but no change in perforin secretion, from effector cells. These results provide significant information on the structures within IgG1 that are involved in binding FcγRIIIA, and they may be useful in the development of therapeutic antagonists for FcγRIIIA. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. Self-healing polyurethane/attapulgite nanocomposites based on disulfide bonds and shape memory effect

    International Nuclear Information System (INIS)

    Xu, Yurun; Chen, Dajun

    2017-01-01

    Nanocomposites with remarkable enhanced mechanical properties have attracted great research efforts recently. In this work, a series of self-healing polyurethane/attapulgite nanocomposites were prepared by solution blending. Introducing self-healing ability and attapulgite (AT) reinforcement simultaneously led to prolonged material lifetime and enhanced mechanical properties. Scanning electron microscope (SEM) observation indicated that AT could achieve a uniform dispersion in polyurethane matrix when AT content was relatively low. The influences on mechanical properties were evaluated by tensile test. Results showed that incorporating an appropriate content of AT would lead to an enhanced tensile properties. The interactions between AT and polyurethane matrix were studied by effective cross-linking density calculation and Fourier transform infrared (FTIR) analysis. Results indicated that rich hydrogen bonds were formed between AT and polyurethane matrix. Displacement data was utilized to evaluate the influence on shape memory effect. With the incorporation of AT, deformation of the sample under external force was restrained. Meanwhile, closure of the scratches still can be accomplished during healing process. Results of healing test suggested that incorporating 1% of AT would also promote self-healing property. - Highlights: • Composites with both self-healing and enhanced mechanical property are prepared. • Healing mechanism relies on disulfide exchange reaction and shape memory effect. • Mechanical enhancement is caused by rich hydrogen bonds introduced by attapulgite.

  9. Self-healing polyurethane/attapulgite nanocomposites based on disulfide bonds and shape memory effect

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Yurun; Chen, Dajun, E-mail: cdj@dhu.edu.cn

    2017-07-01

    Nanocomposites with remarkable enhanced mechanical properties have attracted great research efforts recently. In this work, a series of self-healing polyurethane/attapulgite nanocomposites were prepared by solution blending. Introducing self-healing ability and attapulgite (AT) reinforcement simultaneously led to prolonged material lifetime and enhanced mechanical properties. Scanning electron microscope (SEM) observation indicated that AT could achieve a uniform dispersion in polyurethane matrix when AT content was relatively low. The influences on mechanical properties were evaluated by tensile test. Results showed that incorporating an appropriate content of AT would lead to an enhanced tensile properties. The interactions between AT and polyurethane matrix were studied by effective cross-linking density calculation and Fourier transform infrared (FTIR) analysis. Results indicated that rich hydrogen bonds were formed between AT and polyurethane matrix. Displacement data was utilized to evaluate the influence on shape memory effect. With the incorporation of AT, deformation of the sample under external force was restrained. Meanwhile, closure of the scratches still can be accomplished during healing process. Results of healing test suggested that incorporating 1% of AT would also promote self-healing property. - Highlights: • Composites with both self-healing and enhanced mechanical property are prepared. • Healing mechanism relies on disulfide exchange reaction and shape memory effect. • Mechanical enhancement is caused by rich hydrogen bonds introduced by attapulgite.

  10. Crystallographic studies evidencing the high energy tolerance to disrupting the interface disulfide bond of thioredoxin 1 from white leg shrimp Litopenaeus vannamei.

    Science.gov (United States)

    Campos-Acevedo, Adam A; Rudiño-Piñera, Enrique

    2014-12-15

    Thioredoxin (Trx) is a small 12-kDa redox protein that catalyzes the reduction of disulfide bonds in proteins from different biological systems. A recent study of the crystal structure of white leg shrimp thioredoxin 1 from Litopenaeus vannamei (LvTrx) revealed a dimeric form of the protein mediated by a covalent link through a disulfide bond between Cys73 from each monomer. In the present study, X-ray-induced damage in the catalytic and the interface disulfide bond of LvTrx was studied at atomic resolution at different transmission energies of 8% and 27%, 12.8 keV at 100 K in the beamline I-24 at Diamond Light Source. We found that at an absorbed dose of 32 MGy, the X-ray induces the cleavage of the disulfide bond of each catalytic site; however, the interface disulfide bond was cleaved at an X-ray adsorbed dose of 85 MGy; despite being the most solvent-exposed disulfide bond in LvTrx (~50 Å2). This result clearly established that the interface disulfide bond is very stable and, therefore, less susceptible to being reduced by X-rays. In fact, these studies open the possibility of the existence in solution of a dimeric LvTrx.

  11. Crystallographic Studies Evidencing the High Energy Tolerance to Disrupting the Interface Disulfide Bond of Thioredoxin 1 from White Leg Shrimp Litopenaeus vannamei

    Directory of Open Access Journals (Sweden)

    Adam A. Campos-Acevedo

    2014-12-01

    Full Text Available Thioredoxin (Trx is a small 12-kDa redox protein that catalyzes the reduction of disulfide bonds in proteins from different biological systems. A recent study of the crystal structure of white leg shrimp thioredoxin 1 from Litopenaeus vannamei (LvTrx revealed a dimeric form of the protein mediated by a covalent link through a disulfide bond between Cys73 from each monomer. In the present study, X-ray-induced damage in the catalytic and the interface disulfide bond of LvTrx was studied at atomic resolution at different transmission energies of 8% and 27%, 12.8 keV at 100 K in the beamline I-24 at Diamond Light Source. We found that at an absorbed dose of 32 MGy, the X-ray induces the cleavage of the disulfide bond of each catalytic site; however, the interface disulfide bond was cleaved at an X-ray adsorbed dose of 85 MGy; despite being the most solvent-exposed disulfide bond in LvTrx (~50 Å2. This result clearly established that the interface disulfide bond is very stable and, therefore, less susceptible to being reduced by X-rays. In fact, these studies open the possibility of the existence in solution of a dimeric LvTrx.

  12. β-Boomerang Antimicrobial and Antiendotoxic Peptides: Lipidation and Disulfide Bond Effects on Activity and Structure.

    Science.gov (United States)

    Mohanram, Harini; Bhattacharjya, Surajit

    2014-04-21

    Drug-resistant Gram-negative bacterial pathogens and endotoxin- or lipopolysaccharide (LPS)-mediated inflammations are among some of the most  prominent health issues globally. Antimicrobial peptides (AMPs) are eminent molecules that can kill drug-resistant strains and neutralize LPS toxicity. LPS, the outer layer of the outer membrane of Gram-negative bacteria safeguards cell integrity against hydrophobic compounds, including antibiotics and AMPs. Apart from maintaining structural integrity, LPS, when released into the blood stream, also induces inflammatory pathways leading to septic shock. In previous works, we have reported the de novo design of a set of 12-amino acid long cationic/hydrophobic peptides for LPS binding and activity. These peptides adopt β-boomerang like conformations in complex with LPS. Structure-activity studies demonstrated some critical features of the β-boomerang scaffold that may be utilized for the further development of potent analogs. In this work, β-boomerang lipopeptides were designed and structure-activity correlation studies were carried out. These lipopeptides were homo-dimerized through a disulfide bridge to stabilize conformations and for improved activity. The designed peptides exhibited potent antibacterial activity and efficiently neutralized LPS toxicity under in vitro assays. NMR structure of C4YI13C in aqueous solution demonstrated the conserved folding of the lipopeptide with a boomerang aromatic lock stabilized with disulfide bond at the C-terminus and acylation at the N-terminus. These lipo-peptides displaying bacterial sterilization and low hemolytic activity may be useful for future applications as antimicrobial and antiendotoxin molecules.

  13. β-Boomerang Antimicrobial and Antiendotoxic Peptides: Lipidation and Disulfide Bond Effects on Activity and Structure

    Directory of Open Access Journals (Sweden)

    Harini Mohanram

    2014-04-01

    Full Text Available Drug-resistant Gram-negative bacterial pathogens and endotoxin- or lipopolysaccharide (LPS-mediated inflammations are among some of the most  prominent health issues globally. Antimicrobial peptides (AMPs are eminent molecules that can kill drug-resistant strains and neutralize LPS toxicity. LPS, the outer layer of the outer membrane of Gram-negative bacteria safeguards cell integrity against hydrophobic compounds, including antibiotics and AMPs. Apart from maintaining structural integrity, LPS, when released into the blood stream, also induces inflammatory pathways leading to septic shock. In previous works, we have reported the de novo design of a set of 12-amino acid long cationic/hydrophobic peptides for LPS binding and activity. These peptides adopt β-boomerang like conformations in complex with LPS. Structure-activity studies demonstrated some critical features of the β-boomerang scaffold that may be utilized for the further development of potent analogs. In this work, β-boomerang lipopeptides were designed and structure-activity correlation studies were carried out. These lipopeptides were homo-dimerized through a disulfide bridge to stabilize conformations and for improved activity. The designed peptides exhibited potent antibacterial activity and efficiently neutralized LPS toxicity under in vitro assays. NMR structure of C4YI13C in aqueous solution demonstrated the conserved folding of the lipopeptide with a boomerang aromatic lock stabilized with disulfide bond at the C-terminus and acylation at the N-terminus. These lipo-peptides displaying bacterial sterilization and low hemolytic activity may be useful for future applications as antimicrobial and antiendotoxin molecules.

  14. Imbalance of heterologous protein folding and disulfide bond formation rates yields runaway oxidative stress

    Directory of Open Access Journals (Sweden)

    Tyo Keith EJ

    2012-03-01

    Full Text Available Abstract Background The protein secretory pathway must process a wide assortment of native proteins for eukaryotic cells to function. As well, recombinant protein secretion is used extensively to produce many biologics and industrial enzymes. Therefore, secretory pathway dysfunction can be highly detrimental to the cell and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. In this study, we apply a systems biology approach to analyze secretory pathway dysfunctions resulting from heterologous production of a small protein (insulin precursor or a larger protein (α-amylase. Results HAC1-dependent and independent dysfunctions and cellular responses were apparent across multiple datasets. In particular, processes involving (a degradation of protein/recycling amino acids, (b overall transcription/translation repression, and (c oxidative stress were broadly associated with secretory stress. Conclusions Apparent runaway oxidative stress due to radical production observed here and elsewhere can be explained by a futile cycle of disulfide formation and breaking that consumes reduced glutathione and produces reactive oxygen species. The futile cycle is dominating when protein folding rates are low relative to disulfide bond formation rates. While not strictly conclusive with the present data, this insight does provide a molecular interpretation to an, until now, largely empirical understanding of optimizing heterologous protein secretion. This molecular insight has direct implications on engineering a broad range of recombinant proteins for secretion and provides potential hypotheses for the root causes of several secretory-associated diseases.

  15. CO2·- radical induced cleavage of disulfide bonds in proteins. A gamma-ray and pulse radiolysis mechanistic investigation

    International Nuclear Information System (INIS)

    Favaudon, V.; Tourbez, H.; Lhoste, J-M.; Houee-Levin, C.

    1990-01-01

    Disulfide bond reduction by the CO 2 ·- radical was investigated in aponeocarzinostatin, aporiboflavin-binding protein, and bovine immunoglobulin. Protein-bound cysteine free thiols were formed under γ-ray irradiation in the course of a pH-dependent and protein concentration dependent chain reaction. The chain efficiency increased upon acidification of the medium, with an apparent pK a around 5, and decreased abruptly below pH 3.6. It decreased also at neutral pH as cysteine accumulated. From pulse radiolysis analysis, CO 2 ·- proved able to induce rapid one-electron oxidation of thiols and of tyrosine phenolic groups in addition to one-electron donation to exposed disulfide bonds. The bulk rate constant of CO 2 ·- uptake by the native proteins was 5- to 10-fold faster at pH 3 than at pH 8, and the protonated form of the disulfide radical anion, appeared to be the major protein radical species formed under acidic conditions. Formation of the disulfide radical cation, phenoxyl radical Tyr-O · disproportionation, and phenoxyl radical induced oxidation of preformed thiol groups should also be taken into consideration to explain the fate of the oxygen-centered phenoxyl radical

  16. The effect of tensile stress on the conformational free energy landscape of disulfide bonds.

    Directory of Open Access Journals (Sweden)

    Padmesh Anjukandi

    Full Text Available Disulfide bridges are no longer considered to merely stabilize protein structure, but are increasingly recognized to play a functional role in many regulatory biomolecular processes. Recent studies have uncovered that the redox activity of native disulfides depends on their C-C-S-S dihedrals, χ2 and χ'2. Moreover, the interplay of chemical reactivity and mechanical stress of disulfide switches has been recently elucidated using force-clamp spectroscopy and computer simulation. The χ2 and χ'2 angles have been found to change from conformations that are open to nucleophilic attack to sterically hindered, so-called closed states upon exerting tensile stress. In view of the growing evidence of the importance of C-C-S-S dihedrals in tuning the reactivity of disulfides, here we present a systematic study of the conformational diversity of disulfides as a function of tensile stress. With the help of force-clamp metadynamics simulations, we show that tensile stress brings about a large stabilization of the closed conformers, thereby giving rise to drastic changes in the conformational free energy landscape of disulfides. Statistical analysis shows that native TDi, DO and interchain Ig protein disulfides prefer open conformations, whereas the intrachain disulfide bridges in Ig proteins favor closed conformations. Correlating mechanical stress with the distance between the two a-carbons of the disulfide moiety reveals that the strain of intrachain Ig protein disulfides corresponds to a mechanical activation of about 100 pN. Such mechanical activation leads to a severalfold increase of the rate of the elementary redox S(N2 reaction step. All these findings constitute a step forward towards achieving a full understanding of functional disulfides.

  17. Detection and function of an intramolecular disulfide bond in the pH-responsive CadC of Escherichia coli

    Directory of Open Access Journals (Sweden)

    Dönhöfer Alexandra

    2011-04-01

    Full Text Available Abstract Background In an acidic and lysine-rich environment Escherichia coli induces expression of the cadBA operon which encodes CadA, the lysine decarboxylase, and CadB, the lysine/cadaverine antiporter. cadBA expression is dependent on CadC, a membrane-integrated transcriptional activator which belongs to the ToxR-like protein family. Activation of CadC requires two stimuli, lysine and low pH. Whereas lysine is detected by an interplay between CadC and the lysine-specific transporter LysP, pH alterations are sensed by CadC directly. Crystal structural analyses revealed a close proximity between two periplasmic cysteines, Cys208 and Cys272. Results Substitution of Cys208 and/or Cys272 by alanine resulted in CadC derivatives that were active in response to only one stimulus, either lysine or pH 5.8. Differential in vivo thiol trapping revealed a disulfide bond between these two residues at pH 7.6, but not at pH 5.8. When Cys208 and Cys272 were replaced by aspartate and lysine, respectively, virtually wild-type behavior was restored indicating that the disulfide bond could be mimicked by a salt bridge. Conclusion A disulfide bond was found in the periplasmic domain of CadC that supports an inactive state of CadC at pH 7.6. At pH 5.8 disulfide bond formation is prevented which transforms CadC into a semi-active state. These results provide new insights into the function of a pH sensor.

  18. Studies of the activity of cytosol on the mixed disulfide bond formed by proteins and radioprotector mercaptoethylguanidine

    Energy Technology Data Exchange (ETDEWEB)

    Horvath, M [National Inst. of Oncology, Budapest (Hungary); Holland, J [Orszagos Onkologiai Intezet, Budapest (Hungary)

    1979-01-01

    The cytoplasm of normal and tumorous rat liver cells contains a heat-resistant compound with reducing ability to break the mixed disulfide bond of albumin-/sup 14/C-mercaptoethylguanidine. The reducing activity of cytosol is destoryed by 1000 krd /sup 60/Co-gamma-ray doses in diluted solution. In vivo supralethal of rats does not affect the activity of cytosol prepared from liver cells.

  19. Effect of trastuzumab interchain disulfide bond cleavage on Fcγ receptor binding and antibody-dependent tumour cell phagocytosis.

    Science.gov (United States)

    Suzuki, Mami; Yamanoi, Ayaka; Machino, Yusuke; Ootsubo, Michiko; Izawa, Ken-ichi; Kohroki, Junya; Masuho, Yasuhiko

    2016-01-01

    The Fc domain of human IgG1 binds to Fcγ receptors (FcγRs) to induce effector functions such as phagocytosis. There are four interchain disulfide bonds between the H and L chains. In this study, the disulfide bonds within the IgG1 trastuzumab (TRA), which is specific for HER2, were cleaved by mild S-sulfonation or by mild reduction followed by S-alkylation with three different reagents. The cleavage did not change the binding activities of TRA to HER2-bearing SK-BR-3 cells. The binding activities of TRA to FcγRIIA and FcγRIIB were greatly enhanced by modification with mild reduction and S-alkylation with ICH2CONH2 or N-(4-aminophenyl) maleimide, while the binding activities of TRA to FcγRI and FcγRIIIA were decreased by any of the four modifications. However, the interchain disulfide bond cleavage by the different modifications did not change the antibody-dependent cell-mediated phagocytosis (ADCP) of SK-BR-3 cells by activated THP-1 cells. The order of FcγR expression levels on the THP-1 cells was FcγRII > FcγRI > FcγRIII and ADCP was inhibited by blocking antibodies against FcγRI and FcγRII. These results imply that the effect of the interchain disulfide bond cleavage on FcγRs binding and ADCP is dependent on modifications of the cysteine residues and the FcγR isotypes. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  20. Occurrence of protein disulfide bonds in different domains of life: a comparison of proteins from the Protein Data Bank.

    Science.gov (United States)

    Bošnjak, I; Bojović, V; Šegvić-Bubić, T; Bielen, A

    2014-03-01

    Disulfide bonds (SS bonds) are important post-translational modifications of proteins. They stabilize a three-dimensional (3D) structure (structural SS bonds) and also have the catalytic or regulatory functions (redox-active SS bonds). Although SS bonds are present in all groups of organisms, no comparative analyses of their frequency in proteins from different domains of life have been made to date. Using the Protein Data Bank, the number and subcellular locations of SS bonds in Archaea, Bacteria and Eukarya have been compared. Approximately three times higher frequency of proteins with SS bonds in eukaryotic secretory organelles (e.g. endoplasmic reticulum) than in bacterial periplasmic/secretory pathways was calculated. Protein length also affects the SS bond frequency: the average number of SS bonds is positively correlated with the length for longer proteins (>200 amino acids), while for the shorter and less stable proteins (proteins (250-350 amino acids) indicated a high number of SS bonds only in Archaea which could be explained by the need for additional protein stabilization in hyperthermophiles. The results emphasize higher capacity for the SS bond formation and isomerization in Eukarya when compared with Archaea and Bacteria.

  1. Solubilization and folding of a fully active recombinant Gaussia luciferase with native disulfide bonds by using a SEP-Tag.

    Science.gov (United States)

    Rathnayaka, Tharangani; Tawa, Minako; Nakamura, Takashi; Sohya, Shihori; Kuwajima, Kunihiro; Yohda, Masafumi; Kuroda, Yutaka

    2011-12-01

    Gaussia luciferase (GLuc) is the smallest known bioluminescent protein and is attracting much attention as a potential reporter protein. However, its 10 disulfide bond forming cysteines have hampered the efficient production of recombinant GLuc and thus limited its use in bio-imaging application. Here, we demonstrate that the addition of a short solubility enhancement peptide tag (SEP-Tag) to the C-terminus of GLuc (GLuc-C9D) significantly increased the fraction of soluble protein at a standard expression temperature. The expression time was much shorter, and the final yield of GLuc-C9D was significantly higher than with our previous pCold expression system. Reversed phase HPLC indicated that the GLuc-C9D variant folded with a single disulfide bond pattern after proper oxidization. Further, the thermal denaturation of GLuc-C9D was completely reversible, and its secondary structure content remained unchanged until 40°C as assessed by CD spectroscopy. The (1)H-NMR spectrum of GLuc indicated sharp well dispersed peaks typical for natively folded proteins. GLuc-C9D bioluminescence activity was strong and fully retained even after incubation at high temperatures. These results suggest that solubilization using SEP-Tags can be useful for producing large quantities of proteins containing multiple disulfide bonds. Copyright © 2011. Published by Elsevier B.V.

  2. The influence of the Cys46/Cys55 disulfide bond on the redox and spectroscopic properties of human neuroglobin.

    Science.gov (United States)

    Bellei, Marzia; Bortolotti, Carlo Augusto; Di Rocco, Giulia; Borsari, Marco; Lancellotti, Lidia; Ranieri, Antonio; Sola, Marco; Battistuzzi, Gianantonio

    2018-01-01

    Neuroglobin is a monomeric globin containing a six-coordinate heme b, expressed in the nervous system, which exerts an important neuroprotective role. In the human protein (hNgb), Cys46 and Cys55 form an intramolecular disulfide bond under oxidizing conditions, whose cleavage induces a helix-to-strand rearrangement of the CD loop that strengthens the bond between the heme iron and the distal histidine. Hence, it is conceivable that the intramolecular disulfide bridge modulates the functionality of human neuroglobin by controlling exogenous ligand binding. In this work, we investigated the influence of the Cys46/Cys55 disulfide bond on the redox properties and on the pH-dependent conformational equilibria of hNgb, using UV-vis spectroelectrochemistry, cyclic voltammetry, electronic absorption spectroscopy and magnetic circular dichroism (MCD). We found that the SS bridge significantly affects the heme Fe(III) to Fe(II) reduction enthalpy (ΔH°' rc ) and entropy (ΔS°' rc ), mostly as a consequence of changes in the reduction-induced solvent reorganization effects, without affecting the axial ligand-binding interactions and the polarity and electrostatics of the heme environment. Between pH3 and 12, the electronic properties of the heme of ferric hNgb are sensitive to five acid-base equilibria, which are scarcely affected by the Cys46/Cys55 disulfide bridge. The equilibria occurring at extreme pH values induce heme release, while those occurring between pH5 and 10 alter the electronic properties of the heme without modifying its axial coordination and low spin state. They involve the sidechains of non-coordinating aminoacids close to the heme and at least one heme propionate. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Engineered disulfide bonds increase active-site local stability and reduce catalytic activity of a cold-adapted alkaline phosphatase.

    Science.gov (United States)

    Asgeirsson, Bjarni; Adalbjörnsson, Björn Vidar; Gylfason, Gudjón Andri

    2007-06-01

    Alkaline phosphatase is an extracellular enzyme that is membrane-bound in eukaryotes but resides in the periplasmic space of bacteria. It normally carries four cysteine residues that form two disulfide bonds, for instance in the APs of Escherichia coli and vertebrates. An AP variant from a Vibrio sp. has only one cysteine residue. This cysteine is second next to the nucleophilic serine in the active site. We have individually modified seven residues to cysteine that are on two loops predicted to be within a 5 A radius. Four of them formed a disulfide bond to the endogenous cysteine. Thermal stability was monitored by circular dichroism and activity measurements. Global stability was similar to the wild-type enzyme. However, a significant increase in heat-stability was observed for the disulfide-containing variants using activity as a measure, together with a large reduction in catalytic rates (k(cat)) and a general decrease in Km values. The results suggest that a high degree of mobility near the active site and in the helix carrying the endogenous cysteine is essential for full catalytic efficiency in the cold-adapted AP.

  4. Conformational landscape and pathway of disulfide bond reduction of human alpha defensin

    NARCIS (Netherlands)

    Snijder, Joost; Van De Waterbeemd, Michiel; Glover, Matthew S.; Shi, Liuqing; Clemmer, David E.; Heck, Albert J R

    2015-01-01

    Human alpha defensins are a class of antimicrobial peptides with additional antiviral activity. Such antimicrobial peptides constitute a major part of mammalian innate immunity. Alpha defensins contain six cysteines, which form three well defined disulfide bridges under oxidizing conditions.

  5. N-glycosylation and disulfide bonding affects GPRC6A receptor expression, function, and dimerization

    DEFF Research Database (Denmark)

    Nørskov-Lauritsen, Lenea; Jørgensen, Stine; Bräuner-Osborne, Hans

    2015-01-01

    Investigation of post-translational modifications of receptor proteins is important for our understanding of receptor pharmacology and disease physiology. However, our knowledge about post-translational modifications of class C G protein-coupled receptors and how these modifications regulate expr...... covalently linked dimers through cysteine disulfide linkage in the extracellular amino-terminal domain and here we show that GPRC6A indeed is a homodimer and that a disulfide bridge between the C131 residues is formed....

  6. Reduction of disulfide bonds in peptides and proteins. Reduction des groupes disulfure dans les peptides et proteines

    Energy Technology Data Exchange (ETDEWEB)

    Conte, D [Institut Curie, 75 - Paris (France); Houee-Levin, C [Paris-5 Univ., 75 (France)

    1993-04-01

    We have re-examined the mechanism of disulfide bond reduction in oxidized glutathione by C0[sub 2][sup .-] free radicals. The process appears to be a chain reaction whose initial yield depends on pH and on both peptide and formate ion concentrations, but remains independent on the radiation dose rate. Kinetic schemes drawn from studies on dithiothreitol are unable to account for the results obtained with glutathione and proteins, although the disulfide radical anion is the primary intermediate found with all compounds. The rate constant for its formation from C0[sub 2][sup .-] and glutathione is in the same range as those found using proteins, while decay pathways are somewhat different. Hypotheses are proposed to account for these differences. 6 figs., 2 tabs.

  7. Protein redox regulation in the thylakoid lumen: the importance of disulfide bonds for violaxanthin de-epoxidase.

    Science.gov (United States)

    Simionato, Diana; Basso, Stefania; Zaffagnini, Mirko; Lana, Tobia; Marzotto, Francesco; Trost, Paolo; Morosinotto, Tomas

    2015-04-02

    When exposed to saturating light conditions photosynthetic eukaryotes activate the xanthophyll cycle where the carotenoid violaxanthin is converted into zeaxanthin by the enzyme violaxanthin de-epoxidase (VDE). VDE protein sequence includes 13 cysteine residues, 12 of which are strongly conserved in both land plants and algae. Site directed mutagenesis of Arabidopsis thaliana VDE showed that all these 12 conserved cysteines have a major role in protein function and their mutation leads to a strong reduction of activity. VDE is also shown to be active in its completely oxidized form presenting six disulfide bonds. Redox titration showed that VDE activity is sensitive to variation in redox potential, suggesting the possibility that dithiol/disulfide exchange reactions may represent a mechanism for VDE regulation. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  8. Determination of Double Bond Positions and Geometry of Methyl Linoleate Isomers with Dimethyl Disulfide Adducts by GC/MS.

    Science.gov (United States)

    Shibamoto, Shigeaki; Murata, Tasuku; Yamamoto, Kouhei

    2016-09-01

    The dimethyl disulfide (DMDS) adduct method is one of the convenient and effective methods for determining double bond positions of unsaturated fatty acid methyl esters (FAME) except conjugated FAME. When analyzed using gas chromatography/electron ionization-mass spectrometry (GC/EI-MS), unsaturated FAME with DMDS added to the double bonds yields high intensity MS spectra of characteristic ions. The MS spectra of characteristic ions can then be used to easily confirm double bond positions. Here we explore the GC/EI-MS analysis of the DMDS adducts of methyl linoleate geometrical isomers isolated by high performance liquid chromatography (HPLC) with a silver nitrate column. For C18:2-c9, c12 and C18:2-t9, t12, DMDS randomly formed adducts with double bonds at either carbon 9-10 or carbon 12-13, but not both at the same time due to steric hindrance. For C18:2-c9, t12 and C18:2-t9, c12, however, DMDS only formed adducts with the double bond in the cis configuration. Consequently, when analyzing fatty acids with methylene interrupted double bonds, with one double bond in the cis and one in the trans configuration, double bond positions cannot be completely confirmed.

  9. Engineering a disulfide bond in the lid hinge region of Rhizopus chinensis lipase: increased thermostability and altered acyl chain length specificity.

    Directory of Open Access Journals (Sweden)

    Xiao-Wei Yu

    Full Text Available The key to enzyme function is the maintenance of an appropriate balance between molecular stability and structural flexibility. The lid domain which is very important for "interfacial activation" is the most flexible part in the lipase structure. In this work, rational design was applied to explore the relationship between lid rigidity and lipase activity by introducing a disulfide bond in the hinge region of the lid, in the hope of improving the thermostability of R. chinensis lipase through stabilization of the lid domain without interfering with its catalytic performance. A disulfide bridge between F95C and F214C was introduced into the lipase from R. chinensis in the hinge region of the lid according to the prediction of the "Disulfide by Design" algorithm. The disulfide variant showed substantially improved thermostability with an eleven-fold increase in the t(1/2 value at 60°C and a 7°C increase of T(m compared with the parent enzyme, probably contributed by the stabilization of the geometric structure of the lid region. The additional disulfide bond did not interfere with the catalytic rate (k(cat and the catalytic efficiency towards the short-chain fatty acid substrate, however, the catalytic efficiency of the disulfide variant towards pNPP decreased by 1.5-fold probably due to the block of the hydrophobic substrate channel by the disulfide bond. Furthermore, in the synthesis of fatty acid methyl esters, the maximum conversion rate by RCLCYS reached 95% which was 9% higher than that by RCL. This is the first report on improving the thermostability of the lipase from R. chinensis by introduction of a disulfide bond in the lid hinge region without compromising the catalytic rate.

  10. Improvement in the thermostability of chitosanase from Bacillus ehimensis by introducing artificial disulfide bonds.

    Science.gov (United States)

    Sheng, Jun; Ji, Xiaofeng; Zheng, Yuan; Wang, Zhipeng; Sun, Mi

    2016-10-01

    To determine the effects of artificial disulfide bridges on the thermostability and catalytic efficiency of chitosanase EAG1. Five artificial disulfide bridges were designed based on the structural information derived from the three-dimensional (3-D) model of chitosanase EAG1. Two beneficial mutants (G113C/D116C, A207C-L286C) were located in the flexible surface loop region, whereas the similar substitutions introduced in α-helices regions had a negligible effect. Mut5, the most active mutant, had a longer half-life at 50 °C (from 10.5 to 69.3 min) and a 200 % higher catalytic efficiency (K cat/K m) than that of the original EAG1. The contribution of disulfide bridges to enzyme thermostability is mainly dependent on its location within the polypeptide chain. Strategical placement of a disulfide bridge in flexible regions provides a rigid support and creation of a protected microenvironment, which is effective in improving enzyme's thermostability and catalytic efficiency.

  11. Synthesis of Reusable Silica Nanosphere-Supported Pt(IV Complex for Formation of Disulfide Bonds in Peptides

    Directory of Open Access Journals (Sweden)

    Xiaonan Hou

    2017-02-01

    Full Text Available Some peptide-based drugs, including oxytocin, vasopressin, ziconotide, pramlintide, nesiritide, and octreotide, contain one intramolecular disulfide bond. A novel and reusable monodispersed silica nanosphere-supported Pt(IV complex (SiO2@TPEA@Pt(IV; TPEA: N-[3-(trimethoxysilylpropyl]ethylenediamine was synthesized via a four-step procedure and was used for the formation of intramolecular disulfide bonds in peptides. Transmission electron microscopy (TEM and chemical mapping results for the Pt(II intermediates and for SiO2@TPEA@Pt(IV show that the silica nanospheres possess a monodisperse spherical structure and contain uniformly-distributed Si, O, C, N, Cl, and Pt. The valence state of Pt on the silica nanospheres was characterized by X-ray photoelectron spectroscopy (XPS. The Pt(IV loaded on SiO2@TPEA@Pt(IV was 0.15 mmol/g, as determined by UV-VIS spectrometry. The formation of intramolecular disulfides in six dithiol-containing peptides of variable lengths by the use of SiO2@TPEA@Pt(IV was investigated, and the relative oxidation yields were determined by high-performance liquid chromatography (HPLC. In addition, peptide 1 (Ac-CPFC-NH2 was utilized to study the reusability of SiO2@TPEA@Pt(IV. No significant decrease in the relative oxidation yield was observed after ten reaction cycles. Moreover, the structure of SiO2@TPEA@Pt(IV after being used for ten cycles was determined to be similar to its initial one, demonstrating the cycling stability of the complex.

  12. A Disulfide Bond in the Membrane Protein IgaA Is Essential for Repression of the RcsCDB System

    Directory of Open Access Journals (Sweden)

    M. Graciela Pucciarelli

    2017-12-01

    Full Text Available IgaA is an integral inner membrane protein that was discovered as repressor of the RcsCDB phosphorelay system in the intracellular pathogen Salmonella enterica serovar Typhimurium. The RcsCDB system, conserved in many members of the family Enterobacteriaceae, regulates expression of varied processes including motility, biofilm formation, virulence and response to envelope stress. IgaA is an essential protein to which, in response to envelope perturbation, the outer membrane lipoprotein RcsF has been proposed to bind in order to activate the RcsCDB phosphorelay. Envelope stress has also been reported to be sensed by a surface exposed domain of RcsF. These observations support a tight control of the RcsCDB system by RcsF and IgaA via mechanisms that, however, remain unknown. Interestingly, RcsF and IgaA have four conserved cysteine residues in loops exposed to the periplasmic space. Two non-consecutive disulfide bonds were shown to be required for RcsF function. Here, we report mutagenesis studies supporting the presence of one disulfide bond (C404-C425 in the major periplasmic loop of IgaA that is essential for repression of the RcsCDB phosphorelay. Our data therefore suggest that the redox state of the periplasm may be critical for the control of the RcsCDB system by its two upstream regulators, RcsF and IgaA.

  13. A structural model of pestivirus E(rns) based on disulfide bond connectivity and homology modeling reveals an extremely rare vicinal disulfide

    NARCIS (Netherlands)

    Langedijk, J.P.M.; Veelen, van P.A.; Schaaper, W.M.M.; Ru, de A.H.; Meloen, R.H.; Hulst, M.M.

    2002-01-01

    Erns is a pestivirus envelope glycoprotein and is the only known viral surface protein with RNase activity. Erns is a disulfide-linked homodimer of 100 kDa; it is found on the surface of pestivirus-infected cells and is secreted into the medium. In this study, the disulfide arrangement of the nine

  14. Efficient soluble expression of disulfide bonded proteins in the cytoplasm of Escherichia coli in fed-batch fermentations on chemically defined minimal media.

    Science.gov (United States)

    Gąciarz, Anna; Khatri, Narendar Kumar; Velez-Suberbie, M Lourdes; Saaranen, Mirva J; Uchida, Yuko; Keshavarz-Moore, Eli; Ruddock, Lloyd W

    2017-06-15

    The production of recombinant proteins containing disulfide bonds in Escherichia coli is challenging. In most cases the protein of interest needs to be either targeted to the oxidizing periplasm or expressed in the cytoplasm in the form of inclusion bodies, then solubilized and re-folded in vitro. Both of these approaches have limitations. Previously we showed that soluble expression of disulfide bonded proteins in the cytoplasm of E. coli is possible at shake flask scale with a system, known as CyDisCo, which is based on co-expression of a protein of interest along with a sulfhydryl oxidase and a disulfide bond isomerase. With CyDisCo it is possible to produce disulfide bonded proteins in the presence of intact reducing pathways in the cytoplasm. Here we scaled up production of four disulfide bonded proteins to stirred tank bioreactors and achieved high cell densities and protein yields in glucose fed-batch fermentations, using an E. coli strain (BW25113) with the cytoplasmic reducing pathways intact. Even without process optimization production of purified human single chain IgA 1 antibody fragment reached 139 mg/L and hen avidin 71 mg/L, while purified yields of human growth hormone 1 and interleukin 6 were around 1 g/L. Preliminary results show that human growth hormone 1 was also efficiently produced in fermentations of W3110 strain and when glucose was replaced with glycerol as the carbon source. Our results show for the first time that efficient production of high yields of soluble disulfide bonded proteins in the cytoplasm of E. coli with the reducing pathways intact is feasible to scale-up to bioreactor cultivations on chemically defined minimal media.

  15. Significant improvement of thermal stability of glucose 1-dehydrogenase by introducing disulfide bonds at the tetramer interface.

    Science.gov (United States)

    Ding, Haitao; Gao, Fen; Liu, Danfeng; Li, Zeli; Xu, Xiaohong; Wu, Min; Zhao, Yuhua

    2013-12-10

    Rational design was applied to glucose 1-dehydrogenase (LsGDH) from Lysinibacillus sphaericus G10 to improve its thermal stability by introduction of disulfide bridges between subunits. One out of the eleven mutants, designated as DS255, displayed significantly enhanced thermal stability with considerable soluble expression and high specific activity. It was extremely stable at pH ranging from 4.5 to 10.5, as it retained nearly 100% activity after incubating at different buffers for 1h. Mutant DS255 also exhibited high thermostability, having a half-life of 9900min at 50°C, which was 1868-fold as that of its wild type. Moreover, both of the increased free energy of denaturation and decreased entropy of denaturation of DS255 suggested that the enzyme structure was stabilized by the engineered disulfide bonds. On account of its robust stability, mutant DS255 would be a competitive candidate in practical applications of chiral chemicals synthesis, biofuel cells and glucose biosensors. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. The Disulfide Bond Cys255-Cys279 in the Immunoglobulin-Like Domain of Anthrax Toxin Receptor 2 Is Required for Membrane Insertion of Anthrax Protective Antigen Pore.

    Directory of Open Access Journals (Sweden)

    Pedro Jacquez

    Full Text Available Anthrax toxin receptors act as molecular clamps or switches that control anthrax toxin entry, pH-dependent pore formation, and translocation of enzymatic moieties across the endosomal membranes. We previously reported that reduction of the disulfide bonds in the immunoglobulin-like (Ig domain of the anthrax toxin receptor 2 (ANTXR2 inhibited the function of the protective antigen (PA pore. In the present study, the disulfide linkage in the Ig domain was identified as Cys255-Cys279 and Cys230-Cys315. Specific disulfide bond deletion mutants were achieved by replacing Cys residues with Ala residues. Deletion of the disulfide bond C255-C279, but not C230-C315, inhibited the PA pore-induced release of the fluorescence dyes from the liposomes, suggesting that C255-C279 is essential for PA pore function. Furthermore, we found that deletion of C255-C279 did not affect PA prepore-to-pore conversion, but inhibited PA pore membrane insertion by trapping the PA membrane-inserting loops in proteinaceous hydrophobic pockets. Fluorescence spectra of Trp59, a residue adjacent to the PA-binding motif in von Willebrand factor A (VWA domain of ANTXR2, showed that deletion of C255-C279 resulted in a significant conformational change on the receptor ectodomain. The disulfide deletion-induced conformational change on the VWA domain was further confirmed by single-particle 3D reconstruction of the negatively stained PA-receptor heptameric complexes. Together, the biochemical and structural data obtained in this study provides a mechanistic insight into the role of the receptor disulfide bond C255-C279 in anthrax toxin action. Manipulation of the redox states of the receptor, specifically targeting to C255-C279, may become a novel strategy to treat anthrax.

  17. Structure of thrombospondin type 3 repeats in bacterial outer membrane protein A reveals its intra-repeat disulfide bond-dependent calcium-binding capability

    Energy Technology Data Exchange (ETDEWEB)

    Dai, Shuyan; Sun, Cancan; Tan, Kemin; Ye, Sheng; Zhang, Rongguang

    2017-09-01

    Eukaryotic thrombospondin type 3 repeat (TT3R) is an efficient calcium ion (Ca2+) binding motif only found in mammalian thrombospondin family. TT3R has also been found in prokaryotic cellulase Cel5G, which was thought to forfeit the Ca2+-binding capability due to the formation of intra-repeat disulfide bonds, instead of the inter-repeat ones possessed by eukaryotic TT3Rs. In this study, we have identified an enormous number of prokaryotic TT3R-containing proteins belonging to several different protein families, including outer membrane protein A (OmpA), an important structural protein connecting the outer membrane and the periplasmic peptidoglycan layer in gram-negative bacteria. Here, we report the crystal structure of the periplasmic region of OmpA from Capnocytophaga gingivalis, which contains a linker region comprising five consecutive TT3Rs. The structure of OmpA-TT3R exhibits a well-ordered architecture organized around two tightly-coordinated Ca2+ and confirms the presence of abnormal intra-repeat disulfide bonds. Further mutagenesis studies showed that the Ca2+-binding capability of OmpA-TT3R is indeed dependent on the proper formation of intra-repeat disulfide bonds, which help to fix a conserved glycine residue at its proper position for Ca2+ coordination. Additionally, despite lacking inter repeat disulfide bonds, the interfaces between adjacent OmpA-TT3Rs are enhanced by both hydrophobic and conserved aromatic-proline interactions.

  18. Contributions of a disulfide bond and a reduced cysteine side chain to the intrinsic activity of the high-density lipoprotein receptor SR-BI.

    Science.gov (United States)

    Yu, Miao; Lau, Thomas Y; Carr, Steven A; Krieger, Monty

    2012-12-18

    The high-density lipoprotein (HDL) receptor scavenger receptor class B, type I (SR-BI), binds HDL and mediates selective cholesteryl ester uptake. SR-BI's structure and mechanism are poorly understood. We used mass spectrometry to assign the two disulfide bonds in SR-BI that connect cysteines within the conserved Cys(321)-Pro(322)-Cys(323) (CPC) motif and connect Cys(280) to Cys(334). We used site-specific mutagenesis to evaluate the contributions of the CPC motif and the side chain of extracellular Cys(384) to HDL binding and lipid uptake. The effects of CPC mutations on activity were context-dependent. Full wild-type (WT) activity required Pro(322) and Cys(323) only when Cys(321) was present. Reduced intrinsic activities were observed for CXC and CPX, but not XXC, XPX, or XXX mutants (X ≠ WT residue). Apparently, a free thiol side chain at position 321 that cannot form an intra-CPC disulfide bond with Cys(323) is deleterious, perhaps because of aberrant disulfide bond formation. Pro(322) may stabilize an otherwise strained CPC disulfide bond, thus supporting WT activity, but this disulfide bond is not absolutely required for normal activity. C(384)X (X = S, T, L, Y, G, or A) mutants exhibited altered activities that varied with the side chain's size: larger side chains phenocopied WT SR-BI treated with its thiosemicarbazone inhibitor BLT-1 (enhanced binding, weakened uptake); smaller side chains produced almost inverse effects (increased uptake:binding ratio). C(384)X mutants were BLT-1-resistant, supporting the proposal that Cys(384)'s thiol interacts with BLT-1. We discuss the implications of our findings on the functions of the extracellular loop cysteines in SR-BI and compare our results to those presented by other laboratories.

  19. Quantification of thiols and disulfides

    DEFF Research Database (Denmark)

    Winther, Jakob R.; Thorpe, Colin

    2014-01-01

    lengths to regulate thiol-disulfide bond homeostasis, typically with several, apparently redundant, systems working in parallel. Dissecting the extent of oxidation and reduction of disulfides is an ongoing challenge due, in part, to the facility of thiol/disulfide exchange reactions.......Disulfide bond formation is a key posttranslational modification, with implications for structure, function and stability of numerous proteins. While disulfide bond formation is a necessary and essential process for many proteins, it is deleterious and disruptive for others. Cells go to great...

  20. A class III chitinase without disulfide bonds from the fern, Pteris ryukyuensis: crystal structure and ligand-binding studies.

    Science.gov (United States)

    Kitaoku, Yoshihito; Umemoto, Naoyuki; Ohnuma, Takayuki; Numata, Tomoyuki; Taira, Toki; Sakuda, Shohei; Fukamizo, Tamo

    2015-10-01

    We first solved the crystal structure of class III catalytic domain of a chitinase from fern (PrChiA-cat), and found a structural difference between PrChiA-cat and hevamine. PrChiA-cat was found to have reduced affinities to chitin oligosaccharides and allosamidin. Plant class III chitinases are subdivided into enzymes with three disulfide bonds and those without disulfide bonds. We here referred to the former enzymes as class IIIa chitinases and the latter as class IIIb chitinases. In this study, we solved the crystal structure of the class IIIb catalytic domain of a chitinase from the fern Pteris ryukyuensis (PrChiA-cat), and compared it with that of hevamine, a class IIIa chitinase from Hevea brasiliensis. PrChiA-cat was found to adopt an (α/β)8 fold typical of GH18 chitinases in a similar manner to that of hevamine. However, PrChiA-cat also had two large loops that extruded from the catalytic site, and the corresponding loops in hevamine were markedly smaller than those of PrChiA-cat. An HPLC analysis of the enzymatic products revealed that the mode of action of PrChiA-cat toward chitin oligosaccharides, (GlcNAc) n (n = 4-6), differed from those of hevamine and the other class IIIa chitinases. The binding affinities of (GlcNAc)3 and (GlcNAc)4 toward the inactive mutant of PrChiA-cat were determined by isothermal titration calorimetry, and were markedly lower than those toward other members of the GH18 family. The affinity and the inhibitory activity of allosamidin toward PrChiA-cat were also lower than those toward the GH18 chitinases investigated to date. Several hydrogen bonds found in the crystal structure of hevamine-allosamidin complex were missing in the modeled structure of PrChiA-cat-allosamidin complex. The structural findings for PrChiA-cat successfully interpreted the functional data presented.

  1. Rational Design of Disulfide Bonds Increases Thermostability of a Mesophilic 1,3-1,4-β-Glucanase from Bacillus terquilensis.

    Directory of Open Access Journals (Sweden)

    Chengtuo Niu

    Full Text Available 1,3-1,4-β-glucanase is an important biocatalyst in brewing industry and animal feed industry, while its low thermostability often reduces its application performance. In this study, the thermostability of a mesophilic β-glucanase from Bacillus terquilensis was enhanced by rational design and engineering of disulfide bonds in the protein structure. Protein spatial configuration was analyzed to pre-exclude the residues pairs which negatively conflicted with the protein structure and ensure the contact of catalytic center. The changes in protein overall and local flexibility among the wild-type enzyme and the designated mutants were predicted to select the potential disulfide bonds for enhancement of thermostability. Two residue pairs (N31C-T187C and P102C-N125C were chosen as engineering targets and both of them were proved to significantly enhance the protein thermostability. After combinational mutagenesis, the double mutant N31C-T187C/P102C-N125C showed a 48.3% increase in half-life value at 60°C and a 4.1°C rise in melting temperature (Tm compared to wild-type enzyme. The catalytic property of N31C-T187C/P102C-N125C mutant was similar to that of wild-type enzyme. Interestingly, the optimal pH of double mutant was shifted from pH6.5 to pH6.0, which could also increase its industrial application. By comparison with mutants with single-Cys substitutions, the introduction of disulfide bonds and the induced new hydrogen bonds were proved to result in both local and overall rigidification and should be responsible for the improved thermostability. Therefore, the introduction of disulfide bonds for thermostability improvement could be rationally and highly-effectively designed by combination with spatial configuration analysis and molecular dynamics simulation.

  2. UV-Photochemistry of the Disulfide Bond: Evolution of Early Photoproducts from Picosecond X-ray Absorption Spectroscopy at the Sulfur K-Edge.

    Science.gov (United States)

    Ochmann, Miguel; Hussain, Abid; von Ahnen, Inga; Cordones, Amy A; Hong, Kiryong; Lee, Jae Hyuk; Ma, Rory; Adamczyk, Katrin; Kim, Tae Kyu; Schoenlein, Robert W; Vendrell, Oriol; Huse, Nils

    2018-05-30

    We have investigated dimethyl disulfide as the basic moiety for understanding the photochemistry of disulfide bonds, which are central to a broad range of biochemical processes. Picosecond time-resolved X-ray absorption spectroscopy at the sulfur K-edge provides unique element-specific insight into the photochemistry of the disulfide bond initiated by 267 nm femtosecond pulses. We observe a broad but distinct transient induced absorption spectrum which recovers on at least two time scales in the nanosecond range. We employed RASSCF electronic structure calculations to simulate the sulfur-1s transitions of multiple possible chemical species, and identified the methylthiyl and methylperthiyl radicals as the primary reaction products. In addition, we identify disulfur and the CH 2 S thione as the secondary reaction products of the perthiyl radical that are most likely to explain the observed spectral and kinetic signatures of our experiment. Our study underscores the importance of elemental specificity and the potential of time-resolved X-ray spectroscopy to identify short-lived reaction products in complex reaction schemes that underlie the rich photochemistry of disulfide systems.

  3. Aggregation of bovine serum albumin upon cleavage of its disulfide bonds, studied by the time-resolved small-angle X-ray scattering technique with synchrotron radiation

    International Nuclear Information System (INIS)

    Ueki, Tatzuo; Inoko, Yoji; Izumi, Yoshinobu; Tagawa, Hiroyuki; Muroga, Yoshio

    1985-01-01

    A rapid mixing system of the stopped-flow type, used with small-angle X-ray scattering equipment using synchrotron radiation, is described. The process of aggregation of bovine serum albumin was traced with a time interval of 50 s, initiated upon cleavage of its disulfide bonds by reduction with dithiothreitol. The results indicate that a 218-fold molar excess of dithiothreitol over the number of moles of disulfide bonds in bovine serum albumin is sufficient to initiate the reaction immediately after mixing, which reaches equilibrium in about 15 min. On the other hand, half this amount is not sufficient to initiate the reaction, so that the reaction is delayed by about 150 s. Such a single-shot time-resolved experiment showed that experiments with a time interval of 100 ms are possible with repeated multi-shot runs. (Auth.)

  4. Aggregation of bovine serum albumin upon cleavage of its disulfide bonds, studied by the time-resolved small-angle X-ray scattering technique with synchrotron radiation

    Energy Technology Data Exchange (ETDEWEB)

    Ueki, Tatzuo; Inoko, Yoji; Hiragi, Yuzuru; Kataoka, Mikio; Amemiya, Yoshiyuki; Izumi, Yoshinobu; Tagawa, Hiroyuki; Muroga, Yoshio

    1985-11-01

    A rapid mixing system of the stopped-flow type, used with small-angle X-ray scattering equipment using synchrotron radiation, is described. The process of aggregation of bovine serum albumin was traced with a time interval of 50s, initiated upon cleavage of its disulfide bonds by reduction with dithiothreitol. The results indicate that a 218-fold molar excess of dithiothreitol over the number of moles of disulfide bonds in bovine serum albumin is sufficient to initiate the reaction immediately after mixing, which reaches equilibrium in about 15 min. On the other hand, half this amount is not sufficient to initiate the reaction, so that the reaction is delayed by about 150s. Such a single-shot time-resolved experiment showed that experiments with a time interval of 100 ms are possible with repeated multi-shot runs. 26 refs.; 8 figs.

  5. An unusual cysteine VL87 affects the antibody fragment conformations without interfering with the disulfide bond formation.

    Science.gov (United States)

    Attallah, Carolina; Aguilar, María Fernanda; Garay, A Sergio; Herrera, Fernando E; Etcheverrigaray, Marina; Oggero, Marcos; Rodrigues, Daniel E

    2017-10-01

    The Cys residues are almost perfectly conserved in all antibodies. They contribute significantly to the antibody fragment stability. The relevance of two natural contiguous Cys residues of an anti-recombinant human-follicle stimulation hormone (rhFSH) in a format of single-chain variable fragment (scFv) was studied. This scFv contains 5 Cys residues: V H 22 and V H 92 in the variable heavy chain (V H ) and V L 23, V L 87 and V L 88 in the variable light chain (V L ). The influence of two unusual contiguous Cys at positions V L 87 and V L 88 was studied by considering the wild type fragment and mutant variants: V L -C88S, V L -C87S, V L -C87Y. The analysis was carried out using antigen-binding ability measurement by indirect specific ELISA and a detailed molecular modeling that comprises homology methods, long molecular dynamics simulations and docking. We found that V L -C87 affected the antibody fragment stability without interfering with the disulfide bond formation. The effect of mutating the V L -C87 by a usual residue at this position like Tyr caused distant structural changes at the V H region that confers a higher mobility to the V H -CDR2 and V H -CDR3 loops improving the scFv binding to the antigen. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Sulfur dioxide induced aggregation of wine thaumatin-like proteins: Role of disulfide bonds.

    Science.gov (United States)

    Chagas, Ricardo; Laia, César A T; Ferreira, Ricardo B; Ferreira, Luísa M

    2018-09-01

    Aggregation of heat unstable wine proteins is responsible for the economically and technologically detrimental problem called wine protein haze. This is caused by the aggregation of thermally unfolded proteins that can precipitate in bottled wine. To study the influence of SO 2 in this phenomenon, wine proteins were isolated and thaumatins were identified has the most prone to aggregate in the presence of this compound. Isolated wine thaumatins aggregation was followed by dynamic light scattering (DLS), circular dichroism (CD), fluorescence spectroscopy and size exclusion chromatography (SEC). Our experimental results demonstrate that protein thermal unfolding after exposure of the protein to 70 °C does not present differences whether SO 2 is present or not. Conversely, when the protein solution is cooled to 15 °C (after heat stress) significant analytical changes can be observed between samples with and without SO 2 . A remarkable change of circular dichroism spectra in the region 220-230 nm is observed (which can be related to S-S torsion angles), as well as an increase in tryptophan fluorescence intensity (absence of fluorescence quenching by S-S bonds). Formation of covalently-linked dimeric and tetrameric protein species were also detected by SEC. The ability to dissolve the aggregates with 8 M urea seems to indicate that hydrophobic interactions are prevalent in the formed aggregates. Also, the reduction of these aggregates with tris (2-carboxyethyl) phosphine (TCEP) to only monomeric species reveals the presence of intermolecular S-S bonds. Copyright © 2018 Elsevier Ltd. All rights reserved.

  7. Reactivity of hydropersulfides toward the hydroxyl radical unraveled: disulfide bond cleavage, hydrogen atom transfer, and proton-coupled electron transfer.

    Science.gov (United States)

    Anglada, Josep M; Crehuet, Ramon; Adhikari, Sarju; Francisco, Joseph S; Xia, Yu

    2018-02-14

    Hydropersulfides (RSSH) are highly reactive as nucleophiles and hydrogen atom transfer reagents. These chemical properties are believed to be key for them to act as antioxidants in cells. The reaction involving the radical species and the disulfide bond (S-S) in RSSH, a known redox-active group, however, has been scarcely studied, resulting in an incomplete understanding of the chemical nature of RSSH. We have performed a high-level theoretical investigation on the reactions of the hydroxyl radical (˙OH) toward a set of RSSH (R = -H, -CH 3 , -NH 2 , -C(O)OH, -CN, and -NO 2 ). The results show that S-S cleavage and H-atom abstraction are the two competing channels. The electron inductive effect of R induces selective ˙OH substitution at one sulfur atom upon S-S cleavage, forming RSOH and ˙SH for the electron donating groups (EDGs), whereas producing HSOH and ˙SR for the electron withdrawing groups (EWGs). The H-Atom abstraction by ˙OH follows a classical hydrogen atom transfer (hat) mechanism, producing RSS˙ and H 2 O. Surprisingly, a proton-coupled electron transfer (pcet) process also occurs for R being an EDG. Although for RSSH having EWGs hat is the leading channel, S-S cleavage can be competitive or even dominant for the EDGs. The overall reactivity of RSSH toward ˙OH attack is greatly enhanced with the presence of an EDG, with CH 3 SSH being the most reactive species found in this study (overall rate constant: 4.55 × 10 12 M -1 s -1 ). Our results highlight the complexity in RSSH reaction chemistry, the extent of which is closely modulated by the inductive effect of the substituents in the case of the oxidation by hydroxyl radicals.

  8. A Disulfide Bond-forming Machine Is Linked to the Sortase-mediated Pilus Assembly Pathway in the Gram-positive Bacterium Actinomyces oris*

    Science.gov (United States)

    Reardon-Robinson, Melissa E.; Osipiuk, Jerzy; Chang, Chungyu; Wu, Chenggang; Jooya, Neda; Joachimiak, Andrzej; Das, Asis; Ton-That, Hung

    2015-01-01

    Export of cell surface pilins in Gram-positive bacteria likely occurs by the translocation of unfolded precursor polypeptides; however, how the unfolded pilins gain their native conformation is presently unknown. Here, we present physiological studies to demonstrate that the FimA pilin of Actinomyces oris contains two disulfide bonds. Alanine substitution of cysteine residues forming the C-terminal disulfide bridge abrogates pilus assembly, in turn eliminating biofilm formation and polymicrobial interaction. Transposon mutagenesis of A. oris yielded a mutant defective in adherence to Streptococcus oralis, and revealed the essential role of a vitamin K epoxide reductase (VKOR) gene in pilus assembly. Targeted deletion of vkor results in the same defects, which are rescued by ectopic expression of VKOR, but not a mutant containing an alanine substitution in its conserved CXXC motif. Depletion of mdbA, which encodes a membrane-bound thiol-disulfide oxidoreductase, abrogates pilus assembly and alters cell morphology. Remarkably, overexpression of MdbA or a counterpart from Corynebacterium diphtheriae, rescues the Δvkor mutant. By alkylation assays, we demonstrate that VKOR is required for MdbA reoxidation. Furthermore, crystallographic studies reveal that A. oris MdbA harbors a thioredoxin-like fold with the conserved CXXC active site. Consistently, each MdbA enzyme catalyzes proper disulfide bond formation within FimA in vitro that requires the catalytic CXXC motif. Because the majority of signal peptide-containing proteins encoded by A. oris possess multiple Cys residues, we propose that MdbA and VKOR constitute a major folding machine for the secretome of this organism. This oxidative protein folding pathway may be a common feature in Actinobacteria. PMID:26170452

  9. A Disulfide Bond-forming Machine Is Linked to the Sortase-mediated Pilus Assembly Pathway in the Gram-positive Bacterium Actinomyces oris.

    Science.gov (United States)

    Reardon-Robinson, Melissa E; Osipiuk, Jerzy; Chang, Chungyu; Wu, Chenggang; Jooya, Neda; Joachimiak, Andrzej; Das, Asis; Ton-That, Hung

    2015-08-28

    Export of cell surface pilins in Gram-positive bacteria likely occurs by the translocation of unfolded precursor polypeptides; however, how the unfolded pilins gain their native conformation is presently unknown. Here, we present physiological studies to demonstrate that the FimA pilin of Actinomyces oris contains two disulfide bonds. Alanine substitution of cysteine residues forming the C-terminal disulfide bridge abrogates pilus assembly, in turn eliminating biofilm formation and polymicrobial interaction. Transposon mutagenesis of A. oris yielded a mutant defective in adherence to Streptococcus oralis, and revealed the essential role of a vitamin K epoxide reductase (VKOR) gene in pilus assembly. Targeted deletion of vkor results in the same defects, which are rescued by ectopic expression of VKOR, but not a mutant containing an alanine substitution in its conserved CXXC motif. Depletion of mdbA, which encodes a membrane-bound thiol-disulfide oxidoreductase, abrogates pilus assembly and alters cell morphology. Remarkably, overexpression of MdbA or a counterpart from Corynebacterium diphtheriae, rescues the Δvkor mutant. By alkylation assays, we demonstrate that VKOR is required for MdbA reoxidation. Furthermore, crystallographic studies reveal that A. oris MdbA harbors a thioredoxin-like fold with the conserved CXXC active site. Consistently, each MdbA enzyme catalyzes proper disulfide bond formation within FimA in vitro that requires the catalytic CXXC motif. Because the majority of signal peptide-containing proteins encoded by A. oris possess multiple Cys residues, we propose that MdbA and VKOR constitute a major folding machine for the secretome of this organism. This oxidative protein folding pathway may be a common feature in Actinobacteria. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Direct, simple derivatization of disulfide bonds in proteins with organic mercury in alkaline medium without any chemical pre-reducing agents

    Energy Technology Data Exchange (ETDEWEB)

    Campanella, Beatrice; Onor, Massimo [National Research Council of Italy, C.N.R., Istituto di Chimica dei Composti Organo Metallici-ICCOM- UOS Pisa, Area di Ricerca, Via G. Moruzzi 1, 56124 Pisa (Italy); Ferrari, Carlo [National Research Council of Italy, C.N.R., Istituto Nazionale di Ottica, INO-UOS Pisa, Area di Ricerca, Via G. Moruzzi 1, 56124 Pisa (Italy); D’Ulivo, Alessandro [National Research Council of Italy, C.N.R., Istituto di Chimica dei Composti Organo Metallici-ICCOM- UOS Pisa, Area di Ricerca, Via G. Moruzzi 1, 56124 Pisa (Italy); Bramanti, Emilia, E-mail: bramanti@pi.iccom.cnr.it [National Research Council of Italy, C.N.R., Istituto di Chimica dei Composti Organo Metallici-ICCOM- UOS Pisa, Area di Ricerca, Via G. Moruzzi 1, 56124 Pisa (Italy)

    2014-09-16

    Highlights: • A simple procedure for the derivatization of proteins disulfide bonds. • Cysteine groups in several proteins derivatised with pHMB in alkaline media. • 75–100% labelling of cysteines in proteins with pHMB. - Abstract: In this work we have studied the derivatization of protein disulfide bonds with p-Hydroxymercurybenzoate (pHMB) in strong alkaline medium without any preliminary reduction. The reaction has been followed by the determination of the protein–pHMB complex using size exclusion chromatography coupled to a microwave/UV mercury oxidation system for the on-line oxidation of free and protein-complexed pHMB and atomic fluorescence spectrometry (SEC–CVG–AFS) detection. The reaction has been optimized by an experimental design using lysozyme as a model protein and applied to several thiolic proteins. The proposed method reports, for the first time, that it is possible to label 75–100% cysteines of proteins and, thus, to determine thiolic proteins without the need of any reducing step to obtain reduced -SH groups before mercury labelling. We obtained a detection limit of 100 nmol L{sup −1} based on a signal-to-noise ratio of 3 for unbound and complexed pHMB, corresponding to a detection limit of proteins ranged between 3 and 360 nmol L{sup −1}, depending on the number of cysteines in the protein sequence.

  11. Direct, simple derivatization of disulfide bonds in proteins with organic mercury in alkaline medium without any chemical pre-reducing agents

    International Nuclear Information System (INIS)

    Campanella, Beatrice; Onor, Massimo; Ferrari, Carlo; D’Ulivo, Alessandro; Bramanti, Emilia

    2014-01-01

    Highlights: • A simple procedure for the derivatization of proteins disulfide bonds. • Cysteine groups in several proteins derivatised with pHMB in alkaline media. • 75–100% labelling of cysteines in proteins with pHMB. - Abstract: In this work we have studied the derivatization of protein disulfide bonds with p-Hydroxymercurybenzoate (pHMB) in strong alkaline medium without any preliminary reduction. The reaction has been followed by the determination of the protein–pHMB complex using size exclusion chromatography coupled to a microwave/UV mercury oxidation system for the on-line oxidation of free and protein-complexed pHMB and atomic fluorescence spectrometry (SEC–CVG–AFS) detection. The reaction has been optimized by an experimental design using lysozyme as a model protein and applied to several thiolic proteins. The proposed method reports, for the first time, that it is possible to label 75–100% cysteines of proteins and, thus, to determine thiolic proteins without the need of any reducing step to obtain reduced -SH groups before mercury labelling. We obtained a detection limit of 100 nmol L −1 based on a signal-to-noise ratio of 3 for unbound and complexed pHMB, corresponding to a detection limit of proteins ranged between 3 and 360 nmol L −1 , depending on the number of cysteines in the protein sequence

  12. Engineering out motion: introduction of a de novo disulfide bond and a salt bridge designed to close a dynamic cleft on the surface of cytochrome b5.

    Science.gov (United States)

    Storch, E M; Daggett, V; Atkins, W M

    1999-04-20

    A previous molecular dynamics (MD) simulation of cytochrome b5 (cyt b5) at 25 degrees C displayed localized dynamics on the surface of the protein giving rise to the periodic formation of a cleft that provides access to the heme through a protected hydrophobic channel [Storch and Daggett (1995) Biochemistry 34, 9682]. Here we describe the production and testing of mutants designed to prevent the cleft from opening using a combination of experimental and theoretical techniques. Two mutants have been designed to close the surface cleft: S18D to introduce a salt bridge and S18C:R47C to incorporate a disulfide bond. The putative cleft forms between two separate cores of the protein: one is structural in nature and can be monitored through the fluorescence of Trp 22, and the other binds the heme prosthetic group and can be tracked via heme absorbance. An increase in motion localized to the cleft region was observed for each protein, except for the disulfide-containing variant, in MD simulations at 50 degrees C compared to simulations at 25 degrees C. For the disulfide-containing variant, the cleft remained closed. Both urea and temperature denaturation curves were nearly identical for wild-type and mutant proteins when heme absorbance was monitored. In contrast, fluorescence studies revealed oxidized S18C:R47C to be considerably more stable based on the midpoints of the denaturation transitions, Tm and U1/2. Moreover, the fluorescence changes for each protein were complete at approximately 50 degrees C and a urea concentration of approximately 3.9 M, significantly below the temperature and urea concentration (62 degrees C, 5 M urea) required to observe heme release. In addition, solvent accessibility based on acrylamide quenching of Trp 22 was lower in the S18C:R47C mutant, particularly at 50 degrees C, before heme release [presented in the accompanying paper (58)]. The results suggest that a constraining disulfide bond can be designed to inhibit dynamic cleft formation

  13. Disulfide bonds in the ectodomain of anthrax toxin receptor 2 are required for the receptor-bound protective-antigen pore to function.

    Directory of Open Access Journals (Sweden)

    Jianjun Sun

    Full Text Available BACKGROUND: Cell-surface receptors play essential roles in anthrax toxin action by providing the toxin with a high-affinity anchor and self-assembly site on the plasma membrane, mediating the toxin entry into cells through endocytosis, and shifting the pH threshold for prepore-to-pore conversion of anthrax toxin protective antigen (PA to a more acidic pH, thereby inhibiting premature pore formation. Each of the two known anthrax toxin receptors, ANTXR1 and ANTXR2, has an ectodomain comprised of an N-terminal von Willebrand factor A domain (VWA, which binds PA, and an uncharacterized immunoglobulin-like domain (Ig that connects VWA to the membrane-spanning domain. Potential roles of the receptor Ig domain in anthrax toxin action have not been investigated heretofore. METHODOLOGY/PRINCIPAL FINDINGS: We expressed and purified the ANTXR2 ectodomain (R2-VWA-Ig in E. coli and showed that it contains three disulfide bonds: one in R2-VWA and two in R2-Ig. Reduction of the ectodomain inhibited functioning of the pore, as measured by K(+ release from liposomes or Chinese hamster ovary cells or by PA-mediated translocation of a model substrate across the plasma membrane. However, reduction did not affect binding of the ectodomain to PA or the transition of ectodomain-bound PA prepore to the pore conformation. The inhibitory effect depended specifically on reduction of the disulfides within R2-Ig. CONCLUSIONS/SIGNIFICANCE: We conclude that disulfide integrity within R2-Ig is essential for proper functioning of receptor-bound PA pore. This finding provides a novel venue to investigate the mechanism of anthrax toxin action and suggests new strategies for inhibiting toxin action.

  14. An Engineered Disulfide Bond Reversibly Traps the IgE-Fc3-4 in a Closed, Nonreceptor Binding Conformation

    Energy Technology Data Exchange (ETDEWEB)

    Wurzburg, Beth A.; Kim, Beomkyu; Tarchevskaya, Svetlana S.; Eggel, Alexander; Vogel, Monique; Jardetzky, Theodore S. [Bern; (Stanford-MED)

    2013-08-02

    IgE antibodies interact with the high affinity IgE Fc receptor, FcϵRI, and activate inflammatory pathways associated with the allergic response. The IgE-Fc region, comprising the C-terminal domains of the IgE heavy chain, binds FcϵRI and can adopt different conformations ranging from a closed form incompatible with receptor binding to an open, receptor-bound state. A number of intermediate states are also observed in different IgE-Fc crystal forms. To further explore this apparent IgE-Fc conformational flexibility and to potentially trap a closed, inactive state, we generated a series of disulfide bond mutants. Here we describe the structure and biochemical properties of an IgE-Fc mutant that is trapped in the closed, non-receptor binding state via an engineered disulfide at residue 335 (Cys-335). Reduction of the disulfide at Cys-335 restores the ability of IgE-Fc to bind to its high affinity receptor, FcϵRIα. The structure of the Cys-335 mutant shows that its conformation is within the range of previously observed, closed form IgE-Fc structures and that it retains the hydrophobic pocket found in the hinge region of the closed conformation. Locking the IgE-Fc into the closed state with the Cys-335 mutation does not affect binding of two other IgE-Fc ligands, omalizumab and DARPin E2_79, demonstrating selective blocking of the high affinity receptor binding.

  15. Strengthening injectable thermo-sensitive NIPAAm-g-chitosan hydrogels using chemical cross-linking of disulfide bonds as scaffolds for tissue engineering.

    Science.gov (United States)

    Wu, Shu-Wei; Liu, Xifeng; Miller, A Lee; Cheng, Yu-Shiuan; Yeh, Ming-Long; Lu, Lichun

    2018-07-15

    In the present study, we fabricated non-toxic, injectable, and thermo-sensitive NIPAAm-g-chitosan (NC) hydrogels with thiol modification for introduction of disulfide cross-linking strategy. Previously, NIPAAm and chitosan copolymer has been proven to have excellent biocompatibility, biodegradability and rapid phase transition after injection, suitable to serve as cell carriers or implanted scaffolds. However, weak mechanical properties significantly limit their potential for biomedical fields. In order to overcome this issue, we incorporated thiol side chains into chitosan by covalently conjugating N-acetyl-cysteine (NAC) with carbodiimide chemistry to strengthen mechanical properties. After oxidation of thiols into disulfide bonds, modified NC hydrogels did improve the compressive modulus over 9 folds (11.4 kPa). Oscillatory frequency sweep showed a positive correlation between storage modulus and cross-liking density as well. Additionally, there was no cytotoxicity observed to mesenchymal stem cells, fibroblasts and osteoblasts. We suggested that the thiol-modified thermo-sensitive polysaccharide hydrogels are promising to be a cell-laden biomaterial for tissue regeneration. Copyright © 2018 Elsevier Ltd. All rights reserved.

  16. Role for the disulfide-bonded region of human immunodeficiency virus type 1 gp41 in receptor-triggered activation of membrane fusion function

    International Nuclear Information System (INIS)

    Bellamy-McIntyre, Anna K.; Baer, Severine; Ludlow, Louise; Drummer, Heidi E.; Poumbourios, Pantelis

    2010-01-01

    The conserved disulfide-bonded region (DSR) of the human immunodeficiency virus type 1 (HIV-1) fusion glycoprotein, gp41, mediates association with the receptor-binding glycoprotein, gp120. Interactions between gp120, CD4 and chemokine receptors activate the fusion activity of gp41. The introduction of W596L and W610F mutations to the DSR of HIV-1 QH1549.13 blocked viral entry and hemifusion without affecting gp120-gp41 association. The fusion defect correlated with inhibition of CD4-triggered gp41 pre-hairpin formation, consistent with the DSR mutations having decoupled receptor-induced conformational changes in gp120 from gp41 activation. Our data implicate the DSR in sensing conformational changes in the gp120-gp41 complex that lead to fusion activation.

  17. Engineered disulfide bonds restore chaperone-like function of DJ-1 mutants linked to familial Parkinson's disease.

    Science.gov (United States)

    Logan, Todd; Clark, Lindsay; Ray, Soumya S

    2010-07-13

    Loss-of-function mutations such as L166P, A104T, and M26I in the DJ-1 gene (PARK7) have been linked to autosomal-recessive early onset Parkinson's disease (PD). Cellular and structural studies of the familial mutants suggest that these mutations may destabilize the dimeric structure. To look for common dynamical signatures among the DJ-1 mutants, short MD simulations of up to 1000 ps were conducted to identify the weakest region of the protein (residues 38-70). In an attempt to stabilize the protein, we mutated residue Val 51 to cysteine (V51C) to make a symmetry-related disulfide bridge with the preexisting Cys 53 on the opposite subunit. We found that the introduction of this disulfide linkage stabilized the mutants A104T and M26I against thermal denaturation, improved their ability to scavenge reactive oxygen species (ROS), and restored a chaperone-like function of blocking alpha-synuclein aggregation. The L166P mutant was far too unstable to be rescued by introduction of the V51C mutation. The results presented here point to the possible development of pharmacological chaperones, which may eventually lead to PD therapeutics.

  18. Differential effects of the loss of intrachain- versus interchain-disulfide bonds in the cystine-knot domain of von Willebrand factor on the clinical phenotype of von Willebrand disease

    NARCIS (Netherlands)

    Tjernberg, Pernilla; Vos, Hans L.; Spaargaren-van Riel, Caroline C.; Luken, Brenda M.; Voorberg, Jan; Bertina, Rogier M.; Eikenboom, Jeroen C. J.

    2006-01-01

    Von Willebrand factor (VWF) contains a large number of cysteine residues, which all form disulfide bonds. Mutations of cysteines located in the cystine-knot (CK) domain of VWF have been identified in both qualitative type 2A (IID) and quantitative type 3 von Willebrand disease (VWD). Our objective

  19. Natural disulfide bond-disrupted mutants of AVR4 of the tomato pathogen Cladosporium fulvum are sensitive to proteolysis, circumvent Cf-4-mediated resistance, but retain their chitin binding ability.

    NARCIS (Netherlands)

    Burg, van den H.A.; Westerink, N.; Francoijs, C.J.J.; Roth, R.; Woestenenk, E.A.; Boeren, J.A.; Wit, de P.J.G.M.; Joosten, M.H.A.J.; Vervoort, J.J.M.

    2003-01-01

    The extracellular AVR4 elicitor of the pathogenic fungus Cladosporium fulvum induces defense responses in the tomato genotype Cf-4. Here, the four disulfide bonds of AVR4 were identified as Cys-11-41, Cys-21-27, Cys-35-80, and Cys-57-72 by partial reduction with Tris-(2-carboxyethyl)-phosphine

  20. Relative stabilities of IgG1 and IgG4 Fab domains: Influence of the light–heavy interchain disulfide bond architecture

    Science.gov (United States)

    Heads, James T; Adams, Ralph; D'Hooghe, Lena E; Page, Matt J T; Humphreys, David P; Popplewell, Andrew G; Lawson, Alastair D; Henry, Alistair J

    2012-01-01

    The stability of therapeutic antibodies is a prime pharmaceutical concern. In this work we examined thermal stability differences between human IgG1 and IgG4 Fab domains containing the same variable regions using the thermofluor assay. It was found that the IgG1 Fab domain is up to 11°C more stable than the IgG4 Fab domain containing the same variable region. We investigated the cause of this difference with the aim of developing a molecule with the enhanced stability of the IgG1 Fab and the biological properties of an IgG4 Fc. We found that replacing the seven residues, which differ between IgG1 CH1 and IgG4 CH1 domains, while retaining the native IgG1 light-heavy interchain disulfide (L–H) bond, did not affect thermal stability. Introducing the IgG1 type L–H interchain disulfide bond (DSB) into the IgG4 Fab resulted in an increase in thermal stability to levels observed in the IgG1 Fab with the same variable region. Conversely, replacement of the IgG1 L–H interchain DSB with the IgG4 type L–H interchain DSB reduced the thermal stability. We utilized the increased stability of the IgG1 Fab and designed a hybrid antibody with an IgG1 CH1 linked to an IgG4 Fc via an IgG1 hinge. This construct has the expected biophysical properties of both the IgG4 Fc and IgG1 Fab domains and may therefore be a pharmaceutically relevant format. PMID:22761163

  1. Structural and catalytic characterization of a thermally stable and acid-stable variant of human carbonic anhydrase II containing an engineered disulfide bond

    Energy Technology Data Exchange (ETDEWEB)

    Boone, Christopher D.; Habibzadegan, Andrew [University of Florida, PO Box 100245, Gainesville, FL 32610 (United States); Tu, Chingkuang; Silverman, David N. [University of Florida, PO Box 100267, Gainesville, FL 32610 (United States); McKenna, Robert, E-mail: rmckenna@ufl.edu [University of Florida, PO Box 100245, Gainesville, FL 32610 (United States)

    2013-08-01

    The X-ray crystallographic structure of the disulfide-containing HCAII (dsHCAII) has been solved to 1.77 Å resolution and revealed that successful oxidation of the cysteine bond was achieved while also retaining desirable active-site geometry. The carbonic anhydrases (CAs) are a family of mostly zinc metalloenzymes that catalyze the reversible hydration of CO{sub 2} to bicarbonate and a proton. Recently, there has been industrial interest in utilizing CAs as biocatalysts for carbon sequestration and biofuel production. The conditions used in these processes, however, result in high temperatures and acidic pH. This unfavorable environment results in rapid destabilization and loss of catalytic activity in CAs, ultimately resulting in cost-inefficient high-maintenance operation of the system. In order to negate these detrimental industrial conditions, cysteines at residues 23 (Ala23Cys) and 203 (Leu203Cys) were engineered into a wild-type variant of human CA II (HCAII) containing the mutation Cys206Ser. The X-ray crystallographic structure of the disulfide-containing HCAII (dsHCAII) was solved to 1.77 Å resolution and revealed that successful oxidation of the cysteine bond was achieved while also retaining desirable active-site geometry. Kinetic studies utilizing the measurement of {sup 18}O-labeled CO{sub 2} by mass spectrometry revealed that dsHCAII retained high catalytic efficiency, and differential scanning calorimetry showed acid stability and thermal stability that was enhanced by up to 14 K compared with native HCAII. Together, these studies have shown that dsHCAII has properties that could be used in an industrial setting to help to lower costs and improve the overall reaction efficiency.

  2. Removal of a C-terminal serine residue proximal to the inter-chain disulfide bond of a human IgG1 lambda light chain mediates enhanced antibody stability and antibody dependent cell-mediated cytotoxicity

    Science.gov (United States)

    Shen, Yang; Zeng, Lin; Zhu, Aiping; Blanc, Tim; Patel, Dipa; Pennello, Anthony; Bari, Amtul; Ng, Stanley; Persaud, Kris; Kang, Yun (Kenneth); Balderes, Paul; Surguladze, David; Hindi, Sagit; Zhou, Qinwei; Ludwig, Dale L.; Snavely, Marshall

    2013-01-01

    Optimization of biophysical properties is a critical success factor for the developability of monoclonal antibodies with potential therapeutic applications. The inter-domain disulfide bond between light chain (Lc) and heavy chain (Hc) in human IgG1 lends structural support for antibody scaffold stability, optimal antigen binding, and normal Fc function. Recently, human IgG1λ has been suggested to exhibit significantly greater susceptibility to reduction of the inter Lc-Hc disulfide bond relative to the same disulfide bond in human IgG1κ. To understand the molecular basis for this observed difference in stability, the sequence and structure of human IgG1λ and human IgG1κ were compared. Based on this Lc comparison, three single mutations were made in the λ Lc proximal to the cysteine residue, which forms a disulfide bond with the Hc. We determined that deletion of S214 (dS) improved resistance of the association between Lc and Hc to thermal stress. In addition, deletion of this terminal serine from the Lc of IgG1λ provided further benefit, including an increase in stability at elevated pH, increased yield from transient transfection, and improved in vitro antibody dependent cell-mediated cytotoxicity (ADCC). These observations support the conclusion that the presence of the terminal serine of the λ Lc creates a weaker inter-chain disulfide bond between the Lc and Hc, leading to slightly reduced stability and a potential compromise in IgG1λ function. Our data from a human IgG1λ provide a basis for further investigation of the effects of deleting terminal serine from λLc on the stability and function of other human IgG1λ antibodies. PMID:23567210

  3. 1,2,3-Triazole Rings as a Disulfide Bond Mimetic in Chimeric AGRP-Melanocortin Peptides: Design, Synthesis, and Functional Characterization.

    Science.gov (United States)

    Tala, Srinivasa R; Singh, Anamika; Lensing, Cody J; Schnell, Sathya M; Freeman, Katie T; Rocca, James R; Haskell-Luevano, Carrie

    2018-05-16

    The melanocortin system is involved in the regulation of complex physiological functions, including energy and weight homeostasis, feeding behavior, inflammation, sexual function, pigmentation, and exocrine gland function. The five melanocortin receptors that belong to the superfamily of G protein-coupled receptors (GPCRs) are regulated by endogenously expressed agonists and antagonists. The aim of this study was to explore the potential of replacing the disulfide bridge in chimeric AGRP-melanocortin peptide Tyr-c[Cys-His-d-Phe-Arg-Trp-Asn-Ala-Phe-Cys]-Tyr-NH 2 (1) with 1,2,3-triazole moieties. A series of 1,2,3-triazole-bridged peptidomimetics were designed, synthesized, and pharmacologically evaluated at the mouse melanocortin receptors. The ligands possessed nanomolar to micromolar agonist cAMP signaling potency. A key finding was that the disulfide bond in peptide 1 can be replaced with the monotriazole ring with minimal effect on the functional activity at the melanocortin receptors. The 1,5-disubstituted triazole-bridged peptide 6 showed equipotent functional activity at the mMC3R and modest 5-fold decreased agonist potency at the mMC4R compared to those of 1. Interestingly, the 1,4- and 1,5-disubstituted isomers of the triazole ring resulted in different selectivities at the receptor subtypes, indicating subtle structural features that may be exploited in the generation of selective melanocortin ligands. Introducing cyclic and acyclic bis-triazole moieties into chimeric AGRP template 1 generally decreased agonist activity. These results will be useful for the further design of neuronal chemical probes for the melanocortin receptors as well as in other receptor systems.

  4. EThcD Discrimination of Isomeric Leucine/Isoleucine Residues in Sequencing of the Intact Skin Frog Peptides with Intramolecular Disulfide Bond

    Science.gov (United States)

    Samgina, Tatiana Yu; Kovalev, Sergey V.; Tolpina, Miriam D.; Trebse, Polonca; Torkar, Gregor; Lebedev, Albert T.

    2018-05-01

    Our scientific interests involve de novo sequencing of non-tryptic natural amphibian skin peptides including those with intramolecular S-S bond by means of exclusively mass spectrometry. Reliable discrimination of the isomeric leucine/isoleucine residues during peptide sequencing by means of mass spectrometry represents a bottleneck in the workflow for complete automation of the primary structure elucidation of these compounds. MS3 is capable of solving the problem. Earlier we demonstrated the advanced efficiency of ETD-HCD method to discriminate Leu/Ile in individual peptides by consecutive application of ETD to the polyprotonated peptides followed by HCD applied to the manually selected primary z-ions with the targeted isomeric residues at their N-termini and registration of the characteristic w-ions. Later this approach was extended to deal with several (4-7) broad band mass ranges, without special isolation of the primary z-ions. The present paper demonstrates an advanced version of this method when EThcD is applied in the whole mass range to a complex mixture of natural non-tryptic peptides without their separation and intermediate isolation of the targeted z-ions. The proposed EThcD method showed over 81% efficiency for the large natural peptides with intact disulfide ring, while the interfering process of radical site migration is suppressed. Due to higher speed and sensitivity, the proposed EThcD approach facilitates the analytical procedure and allows for the automation of the entire experiment and data processing. Moreover, in some cases it gives a chance to establish the nature of the residues in the intact intramolecular disulfide loops. [Figure not available: see fulltext.

  5. On the Mechanism of the Copper-Mediated C-S Bond Formation in the Intramolecular Disproportionation of Imine Disulfides

    Czech Academy of Sciences Publication Activity Database

    Rokob, Tibor András; Rulíšek, Lubomír; Šrogl, Jiří; Révész, Agnes; Zins, Emilie-Laure; Schröder, Detlef

    2011-01-01

    Roč. 50, č. 20 (2011), s. 9968-9979 ISSN 0020-1669 R&D Projects: GA MŠk LC512 Grant - others:European Research Council(XE) AdG HORIZOMS Institutional research plan: CEZ:AV0Z40550506 Keywords : collision-induced dissociation * DFT calculations * C-S bond formation * Cu(I) catalysis * infrared multiphoton spectroscopy Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 4.601, year: 2011

  6. Selenoglutathione Diselenide: Unique Redox Reactions in the GPx-Like Catalytic Cycle and Repairing of Disulfide Bonds in Scrambled Protein.

    Science.gov (United States)

    Shimodaira, Shingo; Asano, Yuki; Arai, Kenta; Iwaoka, Michio

    2017-10-24

    Selenoglutathione (GSeH) is a selenium analogue of naturally abundant glutathione (GSH). In this study, this water-soluble small tripeptide was synthesized in a high yield (up to 98%) as an oxidized diselenide form, i.e., GSeSeG (1), by liquid-phase peptide synthesis (LPPS). Obtained 1 was applied to the investigation of the glutathione peroxidase (GPx)-like catalytic cycle. The important intermediates, i.e., GSe - and GSeSG, besides GSeO 2 H were characterized by 77 Se NMR spectroscopy. Thiol exchange of GSeSG with various thiols, such as cysteine and dithiothreitol, was found to promote the conversion to GSe - significantly. In addition, disproportionation of GSeSR to 1 and RSSR, which would be initiated by heterolytic cleavage of the Se-S bond and catalyzed by the generated selenolate, was observed. On the basis of these redox behaviors, it was proposed that the heterolytic cleavage of the Se-S bond can be facilitated by the interaction between the Se atom and an amino or aromatic group, which is present at the GPx active site. On the other hand, when a catalytic amount of 1 was reacted with scrambled 4S species of RNase A in the presence of NADPH and glutathione reductase, native protein was efficiently regenerated, suggesting a potential use of 1 to repair misfolded proteins through reduction of the non-native SS bonds.

  7. Phylogeny of the Vitamin K 2,3-Epoxide Reductase (VKOR) Family and Evolutionary Relationship to the Disulfide Bond Formation Protein B (DsbB) Family.

    Science.gov (United States)

    Bevans, Carville G; Krettler, Christoph; Reinhart, Christoph; Watzka, Matthias; Oldenburg, Johannes

    2015-07-29

    In humans and other vertebrate animals, vitamin K 2,3-epoxide reductase (VKOR) family enzymes are the gatekeepers between nutritionally acquired K vitamins and the vitamin K cycle responsible for posttranslational modifications that confer biological activity upon vitamin K-dependent proteins with crucial roles in hemostasis, bone development and homeostasis, hormonal carbohydrate regulation and fertility. We report a phylogenetic analysis of the VKOR family that identifies five major clades. Combined phylogenetic and site-specific conservation analyses point to clade-specific similarities and differences in structure and function. We discovered a single-site determinant uniquely identifying VKOR homologs belonging to human pathogenic, obligate intracellular prokaryotes and protists. Building on previous work by Sevier et al. (Protein Science 14:1630), we analyzed structural data from both VKOR and prokaryotic disulfide bond formation protein B (DsbB) families and hypothesize an ancient evolutionary relationship between the two families where one family arose from the other through a gene duplication/deletion event. This has resulted in circular permutation of primary sequence threading through the four-helical bundle protein folds of both families. This is the first report of circular permutation relating distant a-helical membrane protein sequences and folds. In conclusion, we suggest a chronology for the evolution of the five extant VKOR clades.

  8. Optimal expression of a Fab-effector fusion protein in Escherichia coli by removing the cysteine residues responsible for an interchain disulfide bond of a Fab molecule.

    Science.gov (United States)

    Kang, Hyeon-Ju; Kim, Hye-Jin; Jung, Mun-Sik; Han, Jae-Kyu; Cha, Sang-Hoon

    2017-04-01

    Development of novel bi-functional or even tri-functional Fab-effector fusion proteins would have a great potential in the biomedical sciences. However, the expression of Fab-effector fusion proteins in Escherichia coli is problematic especially when a eukaryotic effector moiety is genetically linked to a Fab due to the lack of proper chaperone proteins and an inappropriate physicochemical environment intrinsic to the microbial hosts. We previously reported that a human Fab molecule, referred to as SL335, reactive to human serum albumin has a prolonged in vivo serum half-life in rats. We, herein, tested six discrete SL335-human growth hormone (hGH) fusion constructs as a model system to define an optimal Fab-effector fusion format for E. coli expression. We found that one variant, referred to as HserG/Lser, outperformed the others in terms of a soluble expression yield and functionality in that HserG/Lser has a functional hGH bioactivity and possesses an serum albumin-binding affinity comparable to SL335. Our results clearly demonstrated that the genetic linkage of an effector domain to the C-terminus of Fd (V H +C H1 ) and the removal of cysteine (Cys) residues responsible for an interchain disulfide bond (IDB) ina Fab molecule optimize the periplasmic expression of a Fab-effector fusion protein in E. coli. We believe that our approach can contribute the development of diverse bi-functional Fab-effector fusion proteins by providing a simple strategy that enables the reliable expression of a functional fusion proteins in E. coli. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  9. Soft Computing Methods for Disulfide Connectivity Prediction.

    Science.gov (United States)

    Márquez-Chamorro, Alfonso E; Aguilar-Ruiz, Jesús S

    2015-01-01

    The problem of protein structure prediction (PSP) is one of the main challenges in structural bioinformatics. To tackle this problem, PSP can be divided into several subproblems. One of these subproblems is the prediction of disulfide bonds. The disulfide connectivity prediction problem consists in identifying which nonadjacent cysteines would be cross-linked from all possible candidates. Determining the disulfide bond connectivity between the cysteines of a protein is desirable as a previous step of the 3D PSP, as the protein conformational search space is highly reduced. The most representative soft computing approaches for the disulfide bonds connectivity prediction problem of the last decade are summarized in this paper. Certain aspects, such as the different methodologies based on soft computing approaches (artificial neural network or support vector machine) or features of the algorithms, are used for the classification of these methods.

  10. Ero1-PDI interactions, the response to redox flux and the implications for disulfide bond formation in the mammalian endoplasmic reticulum

    NARCIS (Netherlands)

    Benham, A.M.; Lith, M. van; Sitia, R.; Braakman, I.|info:eu-repo/dai/nl/073923737

    2013-01-01

    The protein folding machinery of the endoplasmic reticulum (ER) ensures that proteins entering the eukaryotic secretory pathway acquire appropriate post-translational modifications and reach a stably folded state. An important component of this protein folding process is the supply of disulfide

  11. Behavior of the E-E' Bonds (E, E' = S and Se) in Glutathione Disulfide and Derivatives Elucidated by Quantum Chemical Calculations with the Quantum Theory of Atoms-in-Molecules Approach.

    Science.gov (United States)

    Hayashi, Satoko; Tsubomoto, Yutaka; Nakanishi, Waro

    2018-02-17

    The nature of the E-E' bonds (E, E' = S and Se) in glutathione disulfide ( 1 ) and derivatives 2 - 3 , respectively, was elucidated by applying quantum theory of atoms-in-molecules (QTAIM) dual functional analysis (QTAIM-DFA), to clarify the basic contribution of E-E' in the biological redox process, such as the glutathione peroxidase process. Five most stable conformers a - e were obtained, after applying the Monte-Carlo method then structural optimizations. In QTAIM-DFA, total electron energy densities H b ( r c ) are plotted versus H b ( r c ) - V b ( r c )/2 at bond critical points (BCPs), where V b ( r c ) are potential energy densities at BCPs. Data from the fully optimized structures correspond to the static nature. Those containing perturbed structures around the fully optimized one in the plot represent the dynamic nature of interactions. The behavior of E-E' was examined carefully. Whereas E-E' in 1a - 3e were all predicted to have the weak covalent nature of the shared shell interactions, two different types of S-S were detected in 1 , depending on the conformational properties. Contributions from the intramolecular non-covalent interactions to stabilize the conformers were evaluated. An inverse relationship was observed between the stability of a conformer and the strength of E-E' in the conformer, of which reason was discussed.

  12. Behavior of the E–E’ Bonds (E, E’ = S and Se in Glutathione Disulfide and Derivatives Elucidated by Quantum Chemical Calculations with the Quantum Theory of Atoms-in-Molecules Approach

    Directory of Open Access Journals (Sweden)

    Satoko Hayashi

    2018-02-01

    Full Text Available The nature of the E–E’ bonds (E, E’ = S and Se in glutathione disulfide (1 and derivatives 2–3, respectively, was elucidated by applying quantum theory of atoms-in-molecules (QTAIM dual functional analysis (QTAIM-DFA, to clarify the basic contribution of E–E’ in the biological redox process, such as the glutathione peroxidase process. Five most stable conformers a–e were obtained, after applying the Monte-Carlo method then structural optimizations. In QTAIM-DFA, total electron energy densities Hb(rc are plotted versus Hb(rc − Vb(rc/2 at bond critical points (BCPs, where Vb(rc are potential energy densities at BCPs. Data from the fully optimized structures correspond to the static nature. Those containing perturbed structures around the fully optimized one in the plot represent the dynamic nature of interactions. The behavior of E–E’ was examined carefully. Whereas E–E’ in 1a–3e were all predicted to have the weak covalent nature of the shared shell interactions, two different types of S–S were detected in 1, depending on the conformational properties. Contributions from the intramolecular non-covalent interactions to stabilize the conformers were evaluated. An inverse relationship was observed between the stability of a conformer and the strength of E–E’ in the conformer, of which reason was discussed.

  13. Complete Mapping of Complex Disulfide Patterns with Closely-Spaced Cysteines by In-Source Reduction and Data-Dependent Mass Spectrometry

    DEFF Research Database (Denmark)

    Cramer, Christian N; Kelstrup, Christian D; Olsen, Jesper V

    2017-01-01

    bonds are present in complicated patterns. This includes the presence of disulfide bonds in nested patterns and closely spaced cysteines. Unambiguous mapping of such disulfide bonds typically requires advanced MS approaches. In this study, we exploited in-source reduction (ISR) of disulfide bonds during...... the electrospray ionization process to facilitate disulfide bond assignments. We successfully developed a LC-ISR-MS/MS methodology to use as an online and fully automated partial reduction procedure. Postcolumn partial reduction by ISR provided fast and easy identification of peptides involved in disulfide bonding......Mapping of disulfide bonds is an essential part of protein characterization to ensure correct cysteine pairings. For this, mass spectrometry (MS) is the most widely used technique due to fast and accurate characterization. However, MS-based disulfide mapping is challenged when multiple disulfide...

  14. Formation of disulfide bonds and homodimers of the major cat allergen Fel d 1 equivalent to the natural allergen by expression in Escherichia coli.

    Science.gov (United States)

    Grönlund, Hans; Bergman, Tomas; Sandström, Kristofer; Alvelius, Gunvor; Reininger, Renate; Verdino, Petra; Hauswirth, Alexander; Liderot, Karin; Valent, Peter; Spitzauer, Susanne; Keller, Walter; Valenta, Rudolf; van Hage-Hamsten, Marianne

    2003-10-10

    Dander from the domestic cat (Felis domesticus) is one of the most common causes of IgE-mediated allergy. Attempts to produce tetrameric folded major allergen Fel d 1 by recombinant methods with structural features similar to the natural allergen have been only partially successful. In this study, a recombinant folded Fel d 1 with molecular and biological properties similar to the natural counterpart was produced. A synthetic gene coding for direct fusion of the Fel d 1 chain 2 N-terminally to chain 1 was constructed by overlapping oligonucleotides in PCR. Escherichia coli expression resulted in a non-covalently associated homodimer with an apparent molecular mass of 30 kDa defined by size exclusion chromatography. Furthermore, each 19,177-Da subunit displayed a disulfide pattern identical to that found in the natural Fel d 1, i.e. Cys3(1) Cys73(2), Cys44(1)-Cys48(2), Cys70(1)-Cys7(2), as determined by electrospray mass spectrometry after tryptic digestion. Circular dichroism analysis showed identical folds of natural and recombinant Fel d 1. Furthermore, recombinant Fel d l reacted specifically with serum IgE, inducing expression of CD203c on basophils and lymphoproliferative responses in cat-allergic patients. The results show that the overall fold and immunological properties of the recombinant Fel d 1 are very similar to those of natural Fel d 1. Moreover, the recombinant Fel d 1 construct provides a tool for defining the three-dimensional structure of Fel d 1 and represents a reagent for diagnosis and allergen-specific immunotherapy of cat allergy.

  15. Oxidative addition of diphenyl disulfide across a Ta=Ta bond. Preparation and characterization of [TaCl3(Me2S)]2(μ-SPh)2

    International Nuclear Information System (INIS)

    Campbell, G.C.; Canich, J.A.M; Cotton, F.A.; Duraj, S.A.; Haw, J.F.

    1986-01-01

    The tantalum complex (SMe 2 )Cl 2 Ta(μ-Cl) 2 (μ-SMe 2 )TaCl 2 (SMe 2 ), possessing a sigma 2 π 2 Ta=Ta double bond, reacts readily with PhSSPh to give (SMe 2 )Cl 3 Ta(μ-SPh) 2 TaCl 2 (SMe 2 ). In this reaction, the starting material loses the bridging SMe 2 ligand and two chloride bridges are broken while only two new SPh bridges are formed in the final product. This oxidative-addition reaction of the S-S single bond to the Ta=Ta double bond converts the face-sharing bioctahedron structure of the starting compound to an edge-sharing bioctahedron structure in the final dimer, with concomitant change of the oxidation state of tantalum from III to IV. The product is the first example of a d 1 -d 1 ditantalum thiolate-bridged dimer. Important structural data for (SMe 2 )Cl 3 Ta(μ-SPh) 2 TaCl 3 (SMe 2 ), which has an inversion center, are determined. The new compound crystallizes in the monoclinic space group C2/c with a = 17.934 (5) A, b = 12.445 (4) A, c = 11.705 (4) A, β = 92.50 (3) 0 , V = 2610 (2) A 3 , and Z = 4. Solid-state 13 C NMR spectroscopy with cross polarization and magic-angle spinning (CP/MAS) at 28 and -103 0 C provides evidence that this compound is diamagnetic. 12 references, 3 figures, 4 tables

  16. Structures of class A macrophage scavenger receptors. Electron microscopic study of flexible, multidomain, fibrous proteins and determination of the disulfide bond pattern of the scavenger receptor cysteine-rich domain.

    Science.gov (United States)

    Resnick, D; Chatterton, J E; Schwartz, K; Slayter, H; Krieger, M

    1996-10-25

    Structures of secreted forms of the human type I and II class A macrophage scavenger receptors were studied using biochemical and biophysical methods. Proteolytic analysis was used to determine the intramolecular disulfide bonds in the type I-specific scavenger receptor cysteine-rich (SRCR) domain: Cys2-Cys7, Cys3-Cys8, and Cys5-Cys6. This pattern is likely to be shared by the highly homologous domains in the many other members of the SRCR domain superfamily. Electron microscopy using rotary shadowing and negative staining showed that the type I and II receptors are extended molecules whose contour lengths are approximately 440 A. They comprised two adjacent fibrous segments, an alpha-helical coiled-coil ( approximately 230 A, including a contribution from the N-terminal spacer domain) and a collagenous triple helix ( approximately 210 A). The type I molecules also contained a C-terminal globular structure ( approximately 58 x 76 A) composed of three SRCR domains. The fibrous domains were joined by an extremely flexible hinge. The angle between these domains varied from 0 to 180 degrees and depended on the conditions of sample preparation. Unexpectedly, at physiologic pH, the prevalent angle seen using rotary shadowing was 0 degrees , resulting in a structure that is significantly more compact than previously suggested. The apparent juxtaposition of the fibrous domains at neutral pH provides a framework for future structure-function studies of these unusual multiligand receptors.

  17. L-Cysteine supplementation increases adiponectin synthesis and secretion, and GLUT4 and glucose utilization by upregulating disulfide bond A-like protein expression mediated by MCP-1 inhibition in 3T3-L1 adipocytes exposed to high glucose.

    Science.gov (United States)

    Achari, Arunkumar Elumalai; Jain, Sushil K

    2016-03-01

    Adiponectin is an anti-diabetic and anti-atherogenic adipokine; its plasma levels are decreased in obesity, insulin resistance, and type 2 diabetes. An adiponectin-interacting protein named disulfide bond A-like protein (DsbA-L) plays an important role in the assembly of adiponectin. This study examined the hypothesis that L-cysteine (LC) regulates glucose homeostasis through the DsbA-L upregulation and synthesis and secretion of adiponectin in diabetes. 3T3L1 adipocytes were treated with LC (250 and 500 µM, 2 h) and high glucose (HG, 25 mM, 20 h). Results showed that LC supplementation significantly (p L, adiponectin, and GLUT-4 protein expression and glucose utilization in HG-treated adipocytes. LC supplementation significantly (p L expression and adiponectin levels in 3T3-L1 cells. Treatment with LC prevented the decrease in DsbA-L, adiponectin, and GLUT-4 expression in 3T3L1 adipocyte cells exposed to MCP-1. Thus, this study demonstrates that DsbA-L and adiponectin upregulation mediates the beneficial effects of LC on glucose utilization by inhibiting MCP-1 secretion in adipocytes and provides a novel mechanism by which LC supplementation can improve insulin sensitivity in diabetes.

  18. How thioredoxin dissociates its mixed disulfide.

    Directory of Open Access Journals (Sweden)

    Goedele Roos

    2009-08-01

    Full Text Available The dissociation mechanism of the thioredoxin (Trx mixed disulfide complexes is unknown and has been debated for more than twenty years. Specifically, opposing arguments for the activation of the nucleophilic cysteine as a thiolate during the dissociation of the complex have been put forward. As a key model, the complex between Trx and its endogenous substrate, arsenate reductase (ArsC, was used. In this structure, a Cys29(Trx-Cys89(ArsC intermediate disulfide is formed by the nucleophilic attack of Cys29(Trx on the exposed Cys82(ArsC-Cys89(ArsC in oxidized ArsC. With theoretical reactivity analysis, molecular dynamics simulations, and biochemical complex formation experiments with Cys-mutants, Trx mixed disulfide dissociation was studied. We observed that the conformational changes around the intermediate disulfide bring Cys32(Trx in contact with Cys29(Trx. Cys32(Trx is activated for its nucleophilic attack by hydrogen bonds, and Cys32(Trx is found to be more reactive than Cys82(ArsC. Additionally, Cys32(Trx directs its nucleophilic attack on the more susceptible Cys29(Trx and not on Cys89(ArsC. This multidisciplinary approach provides fresh insights into a universal thiol/disulfide exchange reaction mechanism that results in reduced substrate and oxidized Trx.

  19. Kinetic and Thermodynamic Aspects of Cellular Thiol-Disulfide Redox Regulation

    DEFF Research Database (Denmark)

    Jensen, Kristine Steen; Hansen, Rosa Erritzøe; Winther, Jakob R

    2009-01-01

    . In the cytosol regulatory disulfide bonds are typically formed in spite of the prevailing reducing conditions and may thereby function as redox switches. Such disulfide bonds are protected from enzymatic reduction by kinetic barriers and are thus allowed to exist long enough to elicit the signal. Factors......Regulation of intracellular thiol-disulfide redox status is an essential part of cellular homeostasis. This involves the regulation of both oxidative and reductive pathways, production of oxidant scavengers and, importantly, the ability of cells to respond to changes in the redox environment...... that affect the rate of thiol-disulfide exchange and stability of disulfide bonds are discussed within the framework of the underlying chemical foundations. This includes the effect of thiol acidity (pKa), the local electrostatic environment, molecular strain and entropy. Even though a thiol-disulfide...

  20. Domain architecture of protein-disulfide isomerase facilitates its dual role as an oxidase and an isomerase in Ero1p-mediated disulfide formation

    DEFF Research Database (Denmark)

    Kulp, M. S.; Frickel, E. M.; Ellgaard, Lars

    2006-01-01

    reduction/rearrangement of non-native disulfides is poorly understood. We analyzed the role of individual PDI domains in disulfide bond formation in a reaction driven by their natural oxidant, Ero1p. We found that Ero1p oxidizes the isolated PDI catalytic thioredoxin domains, A and A' at the same rate......Native disulfide bond formation in eukaryotes is dependent on protein-disulfide isomerase (PDI) and its homologs, which contain varying combinations of catalytically active and inactive thioredoxin domains. However, the specific contribution of PDI to the formation of new disulfides versus...... catalytic (A) domain. The specific order of thioredoxin domains in PDI is important in establishing the asymmetry in the rate of oxidation of the two active sites thus allowing A and A', two thioredoxin domains that are similar in sequence and structure, to serve opposing functional roles as a disulfide...

  1. The synthesis of unsymmetric disulfides for use as radio-protectives

    International Nuclear Information System (INIS)

    Chang, S.H.H.

    1988-01-01

    Unsymmetric disulfides with radioprotective potential were synthesized by linking biomolecules, and related substances, to known radio-protective aminothiols via a disulfide bond. The biomolecules used in this research include mercaptoalcohols, mercaptopyridines and mercaptophenothiazines. Unsymmetric disulfides were synthesized by reacting two thiols with diethyl azodicarboxylate sequentially at low temperature. The reactions of thiols with thiosulfinate were studied as an alternative for synthesizing disulfides. A cross-linked polystyrene was thiolated by different reagents. The thiolation of polymers is part of a methodological study using solid phase synthesis to synthesize unsymmetric disulfides

  2. [Effects of redox state of disulfide bonds on the intrinsic fluorescence and denaturation of Trx-fused gibberellin-induced cysteine-rich protein from Gymnadnia conopsea].

    Science.gov (United States)

    Zhang, Teng; Feng, Juan; Li, Yang; Chen, Rui; Tang, Li-Xia; Pang, Xiao-Feng; Ren, Zheng-Long

    2010-02-01

    In the present paper, thioredoxin-fused gibberellin-induced cysteine-rich protein from Gymnadnia conopsea, desigated as Trx-GcGASA and expressed prokaryotically, was purified and identified by using Ni(2+) -NTA affinity chromatography column and SDS-PAGE, and then its intrinsic fluorescence was investigated in the absence and presence of dithiothreitol (DTT), oxidized glutathione (GSSG), peroxide and guanidine hydrochloride (GdnHCl) by means of steady-state fluorescence spectroscopic methods. It was found that (1) at the neutral pH Trx-GcGASA had maximum fluorescence emission at 305 nm following excitation at different wavelengths varying from 250 to 280 nm, which was ascribed to the fluorescence emission from tyrosine residues. (2) The reduction of disulphide bonds lead to the changes in the relative fluorescence intensity between tyrosine and tryptophan residues from 0.7 to 1.8. (3) Both Tyr and Trp residues underwent 12%-21% decrease in fluorescence intensity with the addition of 0.5 mmol x L(-1) GSSG or 5 mmol x L(-1) peroxide. The latter was roughly consistent with the antioxidative activity reported in vivo. (4) No matter whether 1 mmol x L(-1) DTT was absent or present, the fusion protein could not be fully unfolded with lambda(max) Trx-GcGASA experienced GdnHCl-induced denaturation process, and the unfolding equilibrium curve could be well fitted by using two-state model, giving the Gibbs free energy change (deltaG) of 3.7 kJ x mol(-1). However, it was not the case for reduced Trx-GcGASA protein. The aforementioned experimental results will not only provide some guides to investigate the effects of fusion partner Trx on the unfolding thermodynamics, kinetics and refolding process of Trx-GcGASA, but also will be useful for further studies on the strucuture of GA-induced cysteine-rich protein with the help of spectroscopic methods.

  3. Low-intensity electromagnetic irradiation of 70.6 and 73 GHz frequencies enhances the effects of disulfide bonds reducer on Escherichia coli growth and affects the bacterial surface oxidation-reduction state

    Energy Technology Data Exchange (ETDEWEB)

    Torgomyan, Heghine [Department of Biophysics of Biology Faculty, Yerevan State University, Yerevan 0025 (Armenia); Trchounian, Armen, E-mail: Trchounian@ysu.am [Department of Biophysics of Biology Faculty, Yerevan State University, Yerevan 0025 (Armenia)

    2011-10-14

    Highlights: {yields} Low intensity 70.6 and 73 GHz electromagnetic irradiation (EMI) strongly suppressed Escherichia coli growth at 73 GHz and pH 7.3. {yields} Reducer DL-dithiothreitol had bactericidal effect and disturbed the SH-groups number. {yields} EMI enhanced E. coli sensitivity toward dithiothreitol. {yields} EMI decreased the SH-groups number of membrane disturbed by ATP and N,N'-dicyclohexycarbodiimide. {yields} The changed membrane oxidation-reduction state could be the primary mechanisms in EMI effects. -- Abstract: Low-intensity electromagnetic irradiation (EMI) of 70.6 and 73 GHz frequencies (flux capacity - 0.06 mW cm{sup -2}) had bactericidal effects on Escherichia coli. This EMI (1 h) exposure suppressed the growth of E. coli K-12({lambda}). The pH value (6.0-8.0) did not significantly affect the growth. The lag-phase duration was prolonged, and the growth specific rate was inhibited, and these effects were more noticeable after 73 GHz irradiation. These effects were enhanced by the addition of DL-dithiothreitol (DTT), a strong reducer of disulfide bonds in surface membrane proteins, which in its turn also has bactericidal effect. Further, the number of accessible SH-groups in membrane vesicles was markedly decreased by EMI that was augmented by N,N'-dicyclohexycarbodiimide and DTT. These results indicate a change in the oxidation-reduction state of bacterial cell membrane proteins that could be the primary membranous mechanism in the bactericidal effects of low-intensity EMI of the 70.6 and 73 GHz frequencies.

  4. Low-intensity electromagnetic irradiation of 70.6 and 73 GHz frequencies enhances the effects of disulfide bonds reducer on Escherichia coli growth and affects the bacterial surface oxidation-reduction state

    International Nuclear Information System (INIS)

    Torgomyan, Heghine; Trchounian, Armen

    2011-01-01

    Highlights: → Low intensity 70.6 and 73 GHz electromagnetic irradiation (EMI) strongly suppressed Escherichia coli growth at 73 GHz and pH 7.3. → Reducer DL-dithiothreitol had bactericidal effect and disturbed the SH-groups number. → EMI enhanced E. coli sensitivity toward dithiothreitol. → EMI decreased the SH-groups number of membrane disturbed by ATP and N,N'-dicyclohexycarbodiimide. → The changed membrane oxidation-reduction state could be the primary mechanisms in EMI effects. -- Abstract: Low-intensity electromagnetic irradiation (EMI) of 70.6 and 73 GHz frequencies (flux capacity - 0.06 mW cm -2 ) had bactericidal effects on Escherichia coli. This EMI (1 h) exposure suppressed the growth of E. coli K-12(λ). The pH value (6.0-8.0) did not significantly affect the growth. The lag-phase duration was prolonged, and the growth specific rate was inhibited, and these effects were more noticeable after 73 GHz irradiation. These effects were enhanced by the addition of DL-dithiothreitol (DTT), a strong reducer of disulfide bonds in surface membrane proteins, which in its turn also has bactericidal effect. Further, the number of accessible SH-groups in membrane vesicles was markedly decreased by EMI that was augmented by N,N'-dicyclohexycarbodiimide and DTT. These results indicate a change in the oxidation-reduction state of bacterial cell membrane proteins that could be the primary membranous mechanism in the bactericidal effects of low-intensity EMI of the 70.6 and 73 GHz frequencies.

  5. The human protein disulfide isomerase gene family

    Directory of Open Access Journals (Sweden)

    Galligan James J

    2012-07-01

    Full Text Available Abstract Enzyme-mediated disulfide bond formation is a highly conserved process affecting over one-third of all eukaryotic proteins. The enzymes primarily responsible for facilitating thiol-disulfide exchange are members of an expanding family of proteins known as protein disulfide isomerases (PDIs. These proteins are part of a larger superfamily of proteins known as the thioredoxin protein family (TRX. As members of the PDI family of proteins, all proteins contain a TRX-like structural domain and are predominantly expressed in the endoplasmic reticulum. Subcellular localization and the presence of a TRX domain, however, comprise the short list of distinguishing features required for gene family classification. To date, the PDI gene family contains 21 members, varying in domain composition, molecular weight, tissue expression, and cellular processing. Given their vital role in protein-folding, loss of PDI activity has been associated with the pathogenesis of numerous disease states, most commonly related to the unfolded protein response (UPR. Over the past decade, UPR has become a very attractive therapeutic target for multiple pathologies including Alzheimer disease, Parkinson disease, alcoholic and non-alcoholic liver disease, and type-2 diabetes. Understanding the mechanisms of protein-folding, specifically thiol-disulfide exchange, may lead to development of a novel class of therapeutics that would help alleviate a wide range of diseases by targeting the UPR.

  6. Identification of thioredoxin target disulfides in proteins released from barley aleurone layers

    DEFF Research Database (Denmark)

    Hägglund, Per; Bunkenborg, J.; Yang, Fen

    2010-01-01

    Thioredoxins are ubiquitous disulfide reductases involved in a wide range of cellular processes including DNA synthesis, oxidative stress response and apoptosis. In cereal seeds thioredoxins are proposed to facilitate the germination process by reducing disulfide bonds in storage proteins and other...

  7. Electrochemical reduction of disulfide-containing proteins for hydrogen/deuterium exchange monitored by mass spectrometry

    DEFF Research Database (Denmark)

    Mysling, Simon; Salbo, Rune; Ploug, Michael

    2014-01-01

    Characterization of disulfide bond-containing proteins by hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) requires reduction of the disulfide bonds under acidic and cold conditions, where the amide hydrogen exchange reaction is quenched (pH 2.5, 0 °C). The reduction typically...... of TCEP. In the present study, we explore the feasibility of using electrochemical reduction as a substitute for TCEP in HDX-MS analyses. Our results demonstrate that efficient disulfide bond reduction is readily achieved by implementing an electrochemical cell into the HDX-MS workflow. We also identify...... some challenges in using electrochemical reduction in HDX-MS analyses and provide possible conditions to attenuate these limitations. For example, high salt concentrations hamper disulfide bond reduction, necessitating additional dilution of the sample with aqueous acidic solution at quench conditions....

  8. Thiol-disulfide exchange in peptides derived from human growth hormone.

    Science.gov (United States)

    Chandrasekhar, Saradha; Epling, Daniel E; Sophocleous, Andreas M; Topp, Elizabeth M

    2014-04-01

    Disulfide bonds stabilize proteins by cross-linking distant regions into a compact three-dimensional structure. They can also participate in hydrolytic and oxidative pathways to form nonnative disulfide bonds and other reactive species. Such covalent modifications can contribute to protein aggregation. Here, we present experimental data for the mechanism of thiol-disulfide exchange in tryptic peptides derived from human growth hormone in aqueous solution. Reaction kinetics was monitored to investigate the effect of pH (6.0-10.0), temperature (4-50°C), oxidation suppressants [ethylenediaminetetraacetic acid (EDTA) and N2 sparging], and peptide secondary structure (amide cyclized vs. open form). The concentrations of free thiol containing peptides, scrambled disulfides, and native disulfide-linked peptides generated via thiol-disulfide exchange and oxidation reactions were determined using reverse-phase HPLC and liquid chromatography-mass spectrometry. Concentration versus time data were fitted to a mathematical model using nonlinear least squares regression analysis. At all pH values, the model was able to fit the data with R(2) ≥ 0.95. Excluding oxidation suppressants (EDTA and N2 sparging) resulted in an increase in the formation of scrambled disulfides via oxidative pathways but did not influence the intrinsic rate of thiol-disulfide exchange. In addition, peptide secondary structure was found to influence the rate of thiol-disulfide exchange. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

  9. Kinetic analysis of the mechanism and specificity of protein-disulfide isomerase using fluorescence-quenched peptides

    DEFF Research Database (Denmark)

    Westphal, V; Spetzler, J C; Meldal, M

    1998-01-01

    Protein-disulfide isomerase (PDI) is an abundant folding catalyst in the endoplasmic reticulum of eukaryotic cells. PDI introduces disulfide bonds into newly synthesized proteins and catalyzes disulfide bond isomerizations. We have synthesized a library of disulfide-linked fluorescence......-quenched peptides, individually linked to resin beads, for two purposes: 1) to probe PDI specificity, and 2) to identify simple, sensitive peptide substrates of PDI. Using this library, beads that became rapidly fluorescent by reduction by human PDI were selected. Amino acid sequencing of the bead-linked peptides...

  10. Disulfide Bridges: Bringing Together Frustrated Structure in a Bioactive Peptide.

    Science.gov (United States)

    Zhang, Yi; Schulten, Klaus; Gruebele, Martin; Bansal, Paramjit S; Wilson, David; Daly, Norelle L

    2016-04-26

    Disulfide bridges are commonly found covalent bonds that are usually believed to maintain structural stability of proteins. Here, we investigate the influence of disulfide bridges on protein dynamics through molecular dynamics simulations on the cysteine-rich trypsin inhibitor MCoTI-II with three disulfide bridges. Correlation analysis of the reduced cyclic peptide shows that two of the three disulfide distances (Cys(11)-Cys(23) and Cys(17)-Cys(29)) are anticorrelated within ∼1 μs of bridge formation or dissolution: when the peptide is in nativelike structures and one of the distances shortens to allow bond formation, the other tends to lengthen. Simulations over longer timescales, when the denatured state is less structured, do not show the anticorrelation. We propose that the native state contains structural elements that frustrate one another's folding, and that the two bridges are critical for snapping the frustrated native structure into place. In contrast, the Cys(4)-Cys(21) bridge is predicted to form together with either of the other two bridges. Indeed, experimental chromatography and nuclear magnetic resonance data show that an engineered peptide with the Cys(4)-Cys(21) bridge deleted can still fold into its near-native structure even in its noncyclic form, confirming the lesser role of the Cys(4)-Cys(21) bridge. The results highlight the importance of disulfide bridges in a small bioactive peptide to bring together frustrated structure in addition to maintaining protein structural stability. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  11. Determination of disulfide bridges of two spider toxins: hainantoxin-III and hainantoxin-IV

    Directory of Open Access Journals (Sweden)

    W Wang

    2009-01-01

    Full Text Available Peptide toxins are usually highly bridged proteins with multipairs of intrachain disulfide bonds. Analysis of disulfide connectivity is an important facet of protein structure determination. In this paper, we successfully assigned the disulfide linkage of two novel peptide toxins, called HNTX-III and HNTX-IV, isolated from the venom of Ornithoctonus hainana spider. Both peptides are useful inhibitors of TTX-sensitive voltage-gated sodium channels and are composed of six cysteine residues that form three disulfide bonds, respectively. Firstly, the peptides were partially reduced by tris(2-carboxyethyl-phosphine (TCEP in 0.1 M citrate buffer containing 6 M guanidine-HCl at 40° C for ten minutes. Subsequently, the partially reduced intermediates containing free thiols were separated by reversed-phase high-performance liquid chromatography (RP-HPLC and alkylated by rapid carboxamidomethylation. Then, the disulfide bonds of the intermediates were analyzed by Edman degradation. By using the strategy above, disulfide linkages of HNTX-III and HNTX-IV were determined as I-IV, II-V and III-VI pattern. In addition, this study also showed that this method may have a great potential for determining the disulfide bonds of spider peptide toxins.

  12. Multiple ways to make disulfides

    DEFF Research Database (Denmark)

    Bulleid, Neil J; Ellgaard, Lars

    2011-01-01

    Our concept of how disulfides form in proteins entering the secretory pathway has changed dramatically in recent years. The discovery of endoplasmic reticulum (ER) oxidoreductin 1 (ERO1) was followed by the demonstration that this enzyme couples oxygen reduction to de novo formation of disulfides...

  13. Intradomain Confinement of Disulfides in the Folding of Two Consecutive Modules of the LDL Receptor.

    Directory of Open Access Journals (Sweden)

    Juan Martínez-Oliván

    Full Text Available The LDL receptor internalizes circulating LDL and VLDL particles for degradation. Its extracellular binding domain contains ten (seven LA and three EGF cysteine-rich modules, each bearing three disulfide bonds. Despite the enormous number of disulfide combinations possible, LDLR oxidative folding leads to a single native species with 30 unique intradomain disulfides. Previous folding studies of the LDLR have shown that non native disulfides are initially formed that lead to compact species. Accordingly, the folding of the LDLR has been described as a "coordinated nonvectorial" reaction, and it has been proposed that early compaction funnels the reaction toward the native structure. Here we analyze the oxidative folding of LA4 and LA5, the modules critical for ApoE binding, isolated and in the LA45 tandem. Compared to LA5, LA4 folding is slow and inefficient, resembling that of LA5 disease-linked mutants. Without Ca++, it leads to a mixture of many two-disulfide scrambled species and, with Ca++, to the native form plus two three-disulfide intermediates. The folding of the LA45 tandem seems to recapitulate that of the individual repeats. Importantly, although the folding of the LA45 tandem takes place through formation of scrambled isomers, no interdomain disulfides are detected, i.e. the two adjacent modules fold independently without the assistance of interdomain covalent interactions. Reduction of incredibly large disulfide combinatorial spaces, such as that in the LDLR, by intradomain confinement of disulfide bond formation might be also essential for the efficient folding of other homologous disulfide-rich receptors.

  14. Conformational analysis and design of cross-strand disulfides in antiparallel β-sheets.

    Science.gov (United States)

    Indu, S; Kochat, V; Thakurela, S; Ramakrishnan, C; Varadarajan, Raghavan

    2011-01-01

    Cross-strand disulfides bridge two cysteines in a registered pair of antiparallel β-strands. A nonredundant data set comprising 5025 polypeptides containing 2311 disulfides was used to study cross-strand disulfides. Seventy-six cross-strand disulfides were found of which 75 and 1 occurred at non-hydrogen-bonded (NHB) and hydrogen-bonded (HB) registered pairs, respectively. Conformational analysis and modeling studies demonstrated that disulfide formation at HB pairs necessarily requires an extremely rare and positive χ¹ value for at least one of the cysteine residues. Disulfides at HB positions also have more unfavorable steric repulsion with the main chain. Thirteen pairs of disulfides were introduced in NHB and HB pairs in four model proteins: leucine binding protein (LBP), leucine, isoleucine, valine binding protein (LIVBP), maltose binding protein (MBP), and Top7. All mutants LIVBP T247C V331C showed disulfide formation either on purification, or on treatment with oxidants. Protein stability in both oxidized and reduced states of all mutants was measured. Relative to wild type, LBP and MBP mutants were destabilized with respect to chemical denaturation, although the sole exposed NHB LBP mutant showed an increase of 3.1°C in T(m). All Top7 mutants were characterized for stability through guanidinium thiocyanate chemical denaturation. Both exposed and two of the three buried NHB mutants were appreciably stabilized. All four HB Top7 mutants were destabilized (ΔΔG⁰ = -3.3 to -6.7 kcal/mol). The data demonstrate that introduction of cross-strand disulfides at exposed NHB pairs is a robust method of improving protein stability. All four exposed Top7 disulfide mutants showed mild redox activity. © 2010 Wiley-Liss, Inc.

  15. Structural basis for target protein recognition by the protein disulfide reductase thioredoxin

    DEFF Research Database (Denmark)

    Maeda, Kenji; Hägglund, Per; Finnie, Christine

    2006-01-01

    Thioredoxin is ubiquitous and regulates various target proteins through disulfide bond reduction. We report the structure of thioredoxin (HvTrxh2 from barley) in a reaction intermediate complex with a protein substrate, barley alpha-amylase/subtilisin inhibitor (BASI). The crystal structure...... of this mixed disulfide shows a conserved hydrophobic motif in thioredoxin interacting with a sequence of residues from BASI through van der Waals contacts and backbone-backbone hydrogen bonds. The observed structural complementarity suggests that the recognition of features around protein disulfides plays...... a major role in the specificity and protein disulfide reductase activity of thioredoxin. This novel insight into the function of thioredoxin constitutes a basis for comprehensive understanding of its biological role. Moreover, comparison with structurally related proteins shows that thioredoxin shares...

  16. Increasing the reactivity of an artificial dithiol-disulfide pair through modification of the electrostatic milieu

    DEFF Research Database (Denmark)

    Hansen, Rosa E; Østergaard, Henrik; Winther, Jakob R

    2005-01-01

    K(a) value of Cys149, as well as favorable electrostatic interactions with the negatively charged reagents. The results presented here show that the electrostatic milieu of cysteine thiols in proteins can have substantial effects on the rates of the thiol-disulfide exchange reactions.......The thiol-disulfide exchange reaction plays a central role in the formation of disulfide bonds in newly synthesized proteins and is involved in many aspects of cellular metabolism. Because the thiolate form of the cysteine residue is the key reactive species, its electrostatic milieu is thought...... surface. We have studied properties of vicinal cysteine residues in proteins using a model system based on redox-sensitive yellow fluorescent protein (rxYFP). In this system, the formation of a disulfide bond between two cysteines Cys149 and Cys202 is accompanied by a 2.2-fold decrease in fluorescence...

  17. Molecular Characterization and Analysis of a Novel Protein Disulfide Isomerase-Like Protein of Eimeria tenella

    OpenAIRE

    Han, Hongyu; Dong, Hui; Zhu, Shunhai; Zhao, Qiping; Jiang, Lianlian; Wang, Yange; Li, Liujia; Wu, Youlin; Huang, Bing

    2014-01-01

    Protein disulfide isomerase (PDI) and PDI-like proteins are members of the thioredoxin superfamily. They contain thioredoxin-like domains and catalyze the physiological oxidation, reduction and isomerization of protein disulfide bonds, which are involved in cell function and development in prokaryotes and eukaryotes. In this study, EtPDIL, a novel PDI-like gene of Eimeria tenella, was cloned using rapid amplification of cDNA ends (RACE) according to the expressed sequence tag (EST). The EtPDI...

  18. Characterization of Disulfide-Linked Peptides Using Tandem Mass Spectrometry Coupled with Automated Data Analysis Software

    Science.gov (United States)

    Liang, Zhidan; McGuinness, Kenneth N.; Crespo, Alejandro; Zhong, Wendy

    2018-05-01

    Disulfide bond formation is critical for maintaining structure stability and function of many peptides and proteins. Mass spectrometry has become an important tool for the elucidation of molecular connectivity. However, the interpretation of the tandem mass spectral data of disulfide-linked peptides has been a major challenge due to the lack of appropriate tools. Developing proper data analysis software is essential to quickly characterize disulfide-linked peptides. A thorough and in-depth understanding of how disulfide-linked peptides fragment in mass spectrometer is a key in developing software to interpret the tandem mass spectra of these peptides. Two model peptides with inter- and intra-chain disulfide linkages were used to study fragmentation behavior in both collisional-activated dissociation (CAD) and electron-based dissociation (ExD) experiments. Fragments generated from CAD and ExD can be categorized into three major types, which result from different S-S and C-S bond cleavage patterns. DiSulFinder is a computer algorithm that was newly developed based on the fragmentation observed in these peptides. The software is vendor neutral and capable of quickly and accurately identifying a variety of fragments generated from disulfide-linked peptides. DiSulFinder identifies peptide backbone fragments with S-S and C-S bond cleavages and, more importantly, can also identify fragments with the S-S bond still intact to aid disulfide linkage determination. With the assistance of this software, more comprehensive disulfide connectivity characterization can be achieved. [Figure not available: see fulltext.

  19. Characterization of Disulfide-Linked Peptides Using Tandem Mass Spectrometry Coupled with Automated Data Analysis Software.

    Science.gov (United States)

    Liang, Zhidan; McGuinness, Kenneth N; Crespo, Alejandro; Zhong, Wendy

    2018-01-25

    Disulfide bond formation is critical for maintaining structure stability and function of many peptides and proteins. Mass spectrometry has become an important tool for the elucidation of molecular connectivity. However, the interpretation of the tandem mass spectral data of disulfide-linked peptides has been a major challenge due to the lack of appropriate tools. Developing proper data analysis software is essential to quickly characterize disulfide-linked peptides. A thorough and in-depth understanding of how disulfide-linked peptides fragment in mass spectrometer is a key in developing software to interpret the tandem mass spectra of these peptides. Two model peptides with inter- and intra-chain disulfide linkages were used to study fragmentation behavior in both collisional-activated dissociation (CAD) and electron-based dissociation (ExD) experiments. Fragments generated from CAD and ExD can be categorized into three major types, which result from different S-S and C-S bond cleavage patterns. DiSulFinder is a computer algorithm that was newly developed based on the fragmentation observed in these peptides. The software is vendor neutral and capable of quickly and accurately identifying a variety of fragments generated from disulfide-linked peptides. DiSulFinder identifies peptide backbone fragments with S-S and C-S bond cleavages and, more importantly, can also identify fragments with the S-S bond still intact to aid disulfide linkage determination. With the assistance of this software, more comprehensive disulfide connectivity characterization can be achieved. Graphical Abstract ᅟ.

  20. Conformational analysis of large and highly disulfide-stabilized proteins by integrating online electrochemical reduction into an optimized H/D exchange mass spectrometry workflow

    DEFF Research Database (Denmark)

    Trabjerg, Esben; Jakobsen, Rasmus Uffe; Mysling, Simon

    2015-01-01

    Analysis of disulfide-bonded proteins by HDX-MS requires effective and rapid reduction of disulfide bonds before enzymatic digestion in order to increase sequence coverage. In a conventional HDX-MS workflow, disulfide bonds are reduced chemically by addition of a reducing agent to the quench......-antibody, respectively. The presented results demonstrate the successful electrochemical reduction during HDX-MS analysis of both a small exceptional tightly disulfide-bonded protein (NGF) as well as the largest protein attempted to date (IgG1-antibody). We envision that online electrochemical reduction...... the electrochemical reduction efficiency during HDX-MS analysis of two particularly challenging disulfide stabilized proteins: a therapeutic IgG1-antibody and Nerve Growth Factor-β (NGF). Several different parameters (flow rate, applied square wave potential as well as the type of labeling- and quench buffer) were...

  1. Identification of Thioredoxin Target Disulfides Using Isotope-Coded Affinity Tags

    DEFF Research Database (Denmark)

    Hägglund, Per; Bunkenborg, Jakob; Maeda, Kenji

    2014-01-01

    Thioredoxins (Trx) are small redox proteins that reduce disulfide bonds in various target proteins and maintain cellular thiol redox control. Here, a thiol-specific labeling and affinity enrichment approach for identification and relative quantification of Trx target disulfides in complex protein...... reduction is determined by LC-MS/MS-based quantification of tryptic peptides labeled with "light" (12C) and "heavy" (13C) ICAT reagents. The methodology can be adapted to monitor the effect of different reductants or oxidants on the redox status of thiol/disulfide proteomes in biological systems....... extracts is described. The procedure utilizes the isotope-coded affinity tag (ICAT) reagents containing a thiol reactive iodoacetamide group and a biotin affinity tag to target peptides containing reduced cysteine residues. The identification of substrates for Trx and the extent of target disulfide...

  2. Selective disulfide reduction for labeling and enhancement of Fab antibody fragments

    International Nuclear Information System (INIS)

    Kirley, Terence L.; Greis, Kenneth D.; Norman, Andrew B.

    2016-01-01

    Many methods have been developed for chemical labeling and enhancement of the properties of antibodies and their common fragments, including the Fab and F(ab’) 2 fragments. Somewhat selective reduction of some antibody disulfide bonds has been previously achieved, yielding antibodies and antibody fragments that can be labeled at defined sites, enhancing their utility and properties. Selective reduction of the two hinge disulfide bonds present in F(ab’) 2 fragments using mild reduction has been useful. However, such reduction is often not quantitative and results in the reduction of multiple disulfide bonds, and therefore subsequent multiple labeling or conjugation sites are neither homogenous nor stoichiometric. Here, a simple and efficient selective reduction of the single disulfide bond linking the partial heavy chain and the intact light chain which compose the Fab fragment is accomplished utilizing tris(2-carboxyethyl)phosphine (TCEP) immobilized on agarose beads. The resultant reduced cysteine residues were labeled with several cysteine-selective fluorescent reagents, as well as by cysteine-directed PEGylation. These two cysteine residues can also be re-ligated by means of a bifunctional cysteine cross-linking agent, dibromobimane, thereby both restoring a covalent linkage between the heavy and light chains at this site, far removed from the antigen binding site, and also introducing a fluorescent probe. There are many other research and clinical uses for these selectively partially reduced Fab fragments, including biotinylation, toxin and drug conjugation, and incorporation of radioisotopes, and this technique enables simple generation of very useful Fab fragment derivatives with many potential applications. - Highlights: • TCEP agarose is effective for selective reduction of a single Fab disulfide bond. • This disulfide is solvent accessible and distant from the antigen binding site. • A variety of buffers of varying pHs can be used, simplifying

  3. Mechanism of thioredoxin-catalyzed disulfide reduction. Activation of the buried thiol and role of the variable active-site residues

    NARCIS (Netherlands)

    Carvalho, A.P.; Swart, M.; van Stralen, J.N.P.; Fernandes, P.A.; Ramos, M.E.; Bickelhaupt, F.M.

    2008-01-01

    Thioredoxins (Trx) are enzymes with a characteristic CXYC active-site motif that catalyze the reduction of disulfide bonds in other proteins. We have theoretically explored this reaction mechanism, both in the gas phase and in water, using density functional theory. The mechanism of disulfide

  4. Role of protein disulfide isomerase and other thiol-reactive proteins in HIV-1 envelope protein-mediated fusion

    International Nuclear Information System (INIS)

    Ou Wu; Silver, Jonathan

    2006-01-01

    Cell-surface protein disulfide isomerase (PDI) has been proposed to promote disulfide bond rearrangements in HIV-1 envelope protein (Env) that accompany Env-mediated fusion. We evaluated the role of PDI in ways that have not been previously tested by downregulating PDI with siRNA and by overexpressing wild-type or variant forms of PDI in transiently and stably transfected cells. These manipulations, as well as treatment with anti-PDI antibodies, had only small effects on infection or cell fusion mediated by NL4-3 or AD8 strains of HIV-1. However, the cell-surface thiol-reactive reagent 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB) had a much stronger inhibitory effect in our system, suggesting that cell-surface thiol-containing molecules other than PDI, acting alone or in concert, have a greater effect than PDI on HIV-1 Env-mediated fusion. We evaluated one such candidate, thioredoxin, a PDI family member reported to reduce a labile disulfide bond in CD4. We found that the ability of thioredoxin to reduce the disulfide bond in CD4 is enhanced in the presence of HIV-1 Env gp120 and that thioredoxin also reduces disulfide bonds in gp120 directly in the absence of CD4. We discuss the implications of these observations for identification of molecules involved in disulfide rearrangements in Env during fusion

  5. Hexa-histidin tag position influences disulfide structure but not binding behavior of in vitro folded N-terminal domain of rat corticotropin-releasing factor receptor type 2a

    OpenAIRE

    Klose, Jana; Wendt, Norbert; Kubald, Sybille; Krause, Eberhard; Fechner, Klaus; Beyermann, Michael; Bienert, Michael; Rudolph, Rainer; Rothemund, Sven

    2004-01-01

    The oxidative folding, particularly the arrangement of disulfide bonds of recombinant extracellular N-terminal domains of the corticotropin-releasing factor receptor type 2a bearing five cysteines (C2 to C6), was investigated. Depending on the position of a His-tag, two types of disulfide patterns were found. In the case of an N-terminal His-tag, the disulfide bonds C2–C3 and C4–C6 were found, leaving C5 free, whereas the C-terminal position of the His-tag led to the disulfide pattern C2–C5 a...

  6. Conferring specificity in redox pathways by enzymatic thiol/disulfide exchange reactions.

    Science.gov (United States)

    Netto, Luis Eduardo S; de Oliveira, Marcos Antonio; Tairum, Carlos A; da Silva Neto, José Freire

    2016-01-01

    Thiol-disulfide exchange reactions are highly reversible, displaying nucleophilic substitutions mechanism (S(N)2 type). For aliphatic, low molecular thiols, these reactions are slow, but can attain million times faster rates in enzymatic processes. Thioredoxin (Trx) proteins were the first enzymes described to accelerate thiol-disulfide exchange reactions and their high reactivity is related to the high nucleophilicity of the attacking thiol. Substrate specificity in Trx is achieved by several factors, including polar, hydrophobic, and topological interactions through a groove in the active site. Glutaredoxin (Grx) enzymes also contain the Trx fold, but they do not share amino acid sequence similarity with Trx. A conserved glutathione binding site is a typical feature of Grx that can reduce substrates by two mechanisms (mono and dithiol). The high reactivity of Grx enzymes is related to the very acid pK(a) values of reactive Cys that plays roles as good leaving groups. Therefore, although distinct oxidoreductases catalyze similar thiol–disulfide exchange reactions, their enzymatic mechanisms vary. PDI and DsbA are two other oxidoreductases, but they are involved in disulfide bond formation, instead of disulfide reduction, which is related to the oxidative environment where they are found. PDI enzymes and DsbC are endowed with disulfide isomerase activity, which is related with their tetra-domain architecture. As illustrative description of specificity in thiol-disulfide exchange, redox aspects of transcription activation in bacteria, yeast, and mammals are presented in an evolutionary perspective. Therefore, thiol-disulfide exchange reactions play important roles in conferring specificity to pathways, a required feature for signaling.

  7. Rapid expansion of the protein disulfide isomerase gene family facilitates the folding of venom peptides

    DEFF Research Database (Denmark)

    Safavi-Hemami, Helena; Li, Qing; Jackson, Ronneshia L.

    2016-01-01

    Formation of correct disulfide bonds in the endoplasmic reticulum is a crucial step for folding proteins destined for secretion. Protein disulfide isomerases (PDIs) play a central role in this process. We report a previously unidentified, hypervariable family of PDIs that represents the most...... diverse gene family of oxidoreductases described in a single genus to date. These enzymes are highly expressed specifically in the venom glands of predatory cone snails, animals that synthesize a remarkably diverse set of cysteine-rich peptide toxins (conotoxins). Enzymes in this PDI family, termed...

  8. Inactivation of barley limit dextrinase inhibitor by thioredoxin-catalysed disulfide reduction

    DEFF Research Database (Denmark)

    Jensen, Johanne Mørch; Hägglund, Per; Christensen, Hans Erik Mølager

    2012-01-01

    and one glutathionylated cysteine. Here, thioredoxin is shown to progressively reduce disulfide bonds in LDI accompanied by loss of activity. A preferential reduction of the glutathionylated cysteine, as indicated by thiol quantification and molecular mass analysis using electrospray ionisation mass......Barley limit dextrinase (LD) that catalyses hydrolysis of α-1,6 glucosidic linkages in starch-derived dextrins is inhibited by limit dextrinase inhibitor (LDI) found in mature seeds. LDI belongs to the chloroform/methanol soluble protein family (CM-protein family) and has four disulfide bridges...... spectrometry, was not related to LDI inactivation. LDI reduction is proposed to cause conformational destabilisation leading to loss of function....

  9. Dissecting molecular interactions involved in recognition of target disulfides by the barley thioredoxin system

    DEFF Research Database (Denmark)

    Björnberg, Olof; Maeda, Kenji; Svensson, Birte

    2012-01-01

    Thioredoxin reduces disulfide bonds, thus regulating activities of target proteins in various biological systems, e.g., inactivation of inhibitors of starch hydrolases and proteases in germinating plant seeds. In the three-dimensional structure of a complex with barley α-amylase/subtilisin inhibi......Thioredoxin reduces disulfide bonds, thus regulating activities of target proteins in various biological systems, e.g., inactivation of inhibitors of starch hydrolases and proteases in germinating plant seeds. In the three-dimensional structure of a complex with barley α...... thioredoxin reductase. HvTrxh2 M88G and M88A adjacent to the invariant cis-proline lost efficiency in both BASI disulfide reduction and recycling by thioredoxin reductase. These effects were further pronounced in M88P lacking a backbone NH group. Remarkably, HvTrxh2 E86R in the same loop displayed overall...... retained catalytic properties, with the exception of a 3-fold increased activity toward BASI. From the 104VGA106 loop, a backbone hydrogen bond donated by A106 appears to be important for target disulfide recognition as A106P lost 90% activity toward BASI but was efficiently recycled by thioredoxin...

  10. Novel Roles of the Non-catalytic Elements of Yeast Protein-disulfide Isomerase in Its Interplay with Endoplasmic Reticulum Oxidoreductin 1*

    Science.gov (United States)

    Niu, Yingbo; Zhang, Lihui; Yu, Jiaojiao; Wang, Chih-chen; Wang, Lei

    2016-01-01

    The formation of disulfide bonds in the endoplasmic reticulum (ER) of eukaryotic cells is catalyzed by the sulfhydryl oxidase, ER oxidoreductin 1 (Ero1), and protein-disulfide isomerase (PDI). PDI is oxidized by Ero1 to continuously introduce disulfides into substrates, and feedback regulates Ero1 activity by manipulating the regulatory disulfides of Ero1. In this study we find that yeast Ero1p is enzymatically active even with its regulatory disulfides intact, and further activation of Ero1p by reduction of the regulatory disulfides requires the reduction of non-catalytic Cys90-Cys97 disulfide in Pdi1p. The principal client-binding site in the Pdi1p b′ domain is necessary not only for the functional Ero1p-Pdi1p disulfide relay but also for the activation of Ero1p. We also demonstrate by complementary activation assays that the regulatory disulfides in Ero1p are much more stable than those in human Ero1α. These new findings on yeast Ero1p-Pdi1p interplay reveal significant differences from our previously identified mode of human Ero1α-PDI interplay and provide insights into the evolution of the eukaryotic oxidative protein folding pathway. PMID:26846856

  11. Influence of Alternative Tubulin Inhibitors on the Potency of a Epirubicin-Immunochemotherapeutic Synthesized with an Ultra Violet Light-Activated Intermediate: Influence of incorporating an internal/integral disulfide bond structure and Alternative Tubulin/Microtubule Inhibitors on the Cytotoxic Anti-Neoplastic Potency of Epirubicin-(C3-amide)-Anti-HER2/neu Synthesized Utilizing a UV-Photoactivated Anthracycline Intermediate.

    Science.gov (United States)

    Coyne, C P; Jones, Toni; Bear, Ryan

    2012-11-01

    Immunochemotherapeutics, epirubicin-(C 3 - amide )-SS-[anti-HER2/ neu ] with an internal disulfide bond, and epirubicin-(C 3 - amide )-[anti-HER2/ neu ] were synthesized utilizing succinimidyl 2-[(4,4'-azipentanamido) ethyl]-1,3'-dithioproprionate or succinimidyl 4,4-azipentanoate respectively. Western blot analysis was used to determine the presence of any immunoglobulin fragmentation or IgG-IgG polymerization. Retained HER2/ neu binding characteristics of epirubicin-(C 3 - amide )-[anti-HER2/ neu ] and epirubicin-(C 3 - amide )-SS-[anti-HER2/ neu ] were validated by cell-ELISA using a mammary adenocarcinoma (SKBr-3) population that highly over-expresses trophic HER2/ neu receptor complexes. Cytotoxic anti-neoplastic potency of epirubicin-(C 3 - amide )-[anti-HER2/ neu ] and epirubicin-(C 3 - amide )-SS-[anti-HER2/ neu ] between epirubicin-equivalent concentrations of 10 -10 M and 10 -6 M was determined by measuring the vitality/proliferation of chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3 cell type). Cytotoxic anti-neoplastic potency of benzimidazoles (albendazole, flubendazole, membendazole) and griseofulvin were assessed between 0-to-2 μg/ml and 0-to-100 μg/ml respectively while mebendazole and griseofulvin were analyzed at fixed concentrations of 0.35 μg/ml and 35 g/ml respectively in dual combination with gradient concentrations of epirubicin-(C 3 - amide )-[anti-HER2/ neu ] and epirubicin-(C 3 - amide )-SS-[anti-HER2/ neu ]. Cytotoxic anti-neoplastic potency for epirubicin-(C 3 - amide )-[anti-HER2/ neu ] and epirubicin-(C 3 - amide )-SS-[anti-HER2/ neu ] against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) was nearly identical at epirubicin-equivalent concentrations of 10 -10 M and 10 -6 M. The benzimadazoles also possessed cytotoxic anti-neoplastic activity with flubendazole and albendazole being the most and least potent respectively. Similarly, griseofulvin had cytotoxic anti-neoplastic activity and was more potent than

  12. Antagonistic effect of disulfide-rich peptide aptamers selected by cDNA display on interleukin-6-dependent cell proliferation

    International Nuclear Information System (INIS)

    Nemoto, Naoto; Tsutsui, Chihiro; Yamaguchi, Junichi; Ueno, Shingo; Machida, Masayuki; Kobayashi, Toshikatsu; Sakai, Takafumi

    2012-01-01

    Highlights: ► Disulfide-rich peptide aptamer inhibits IL-6-dependent cell proliferation. ► Disulfide bond of peptide aptamer is essential for its affinity to IL-6R. ► Inhibitory effect of peptide depends on number and pattern of its disulfide bonds. -- Abstract: Several engineered protein scaffolds have been developed recently to circumvent particular disadvantages of antibodies such as their large size and complex composition, low stability, and high production costs. We previously identified peptide aptamers containing one or two disulfide-bonds as an alternative ligand to the interleukin-6 receptor (IL-6R). Peptide aptamers (32 amino acids in length) were screened from a random peptide library by in vitro peptide selection using the evolutionary molecular engineering method “cDNA display”. In this report, the antagonistic activity of the peptide aptamers were examined by an in vitro competition enzyme-linked immunosorbent assay (ELISA) and an IL-6-dependent cell proliferation assay. The results revealed that a disulfide-rich peptide aptamer inhibited IL-6-dependent cell proliferation with similar efficacy to an anti-IL-6R monoclonal antibody.

  13. PDILT, a divergent testis-specific protein disulfide isomerase with a non-classical SXXC motif that engages in disulfide-dependent interactions in the endoplasmic reticulum.

    Science.gov (United States)

    van Lith, Marcel; Hartigan, Nichola; Hatch, Jennifer; Benham, Adam M

    2005-01-14

    Protein disulfide isomerase (PDI) is the archetypal enzyme involved in the formation and reshuffling of disulfide bonds in the endoplasmic reticulum (ER). PDI achieves its redox function through two highly conserved thioredoxin domains, and PDI can also operate as an ER chaperone. The substrate specificities and the exact functions of most other PDI family proteins remain important unsolved questions in biology. Here, we characterize a new and striking member of the PDI family, which we have named protein disulfide isomerase-like protein of the testis (PDILT). PDILT is the first eukaryotic SXXC protein to be characterized in the ER. Our experiments have unveiled a novel, glycosylated PDI-like protein whose tissue-specific expression and unusual motifs have implications for the evolution, catalytic function, and substrate selection of thioredoxin family proteins. We show that PDILT is an ER resident glycoprotein that liaises with partner proteins in disulfide-dependent complexes within the testis. PDILT interacts with the oxidoreductase Ero1alpha, demonstrating that the N-terminal cysteine of the CXXC sequence is not required for binding of PDI family proteins to ER oxidoreductases. The expression of PDILT, in addition to PDI in the testis, suggests that PDILT performs a specialized chaperone function in testicular cells. PDILT is an unusual PDI relative that highlights the adaptability of chaperone and redox function in enzymes of the endoplasmic reticulum.

  14. Production of recombinant disulfide-rich venom peptides for structural and functional analysis via expression in the periplasm of E. coli.

    Directory of Open Access Journals (Sweden)

    Julie K Klint

    Full Text Available Disulfide-rich peptides are the dominant component of most animal venoms. These peptides have received much attention as leads for the development of novel therapeutic agents and bioinsecticides because they target a wide range of neuronal receptors and ion channels with a high degree of potency and selectivity. In addition, their rigid disulfide framework makes them particularly well suited for addressing the crucial issue of in vivo stability. Structural and functional characterization of these peptides necessitates the development of a robust, reliable expression system that maintains their native disulfide framework. The bacterium Escherichia coli has long been used for economical production of recombinant proteins. However, the expression of functional disulfide-rich proteins in the reducing environment of the E. coli cytoplasm presents a significant challenge. Thus, we present here an optimised protocol for the expression of disulfide-rich venom peptides in the periplasm of E. coli, which is where the endogenous machinery for production of disulfide-bonds is located. The parameters that have been investigated include choice of media, induction conditions, lysis methods, methods of fusion protein and peptide purification, and sample preparation for NMR studies. After each section a recommendation is made for conditions to use. We demonstrate the use of this method for the production of venom peptides ranging in size from 2 to 8 kDa and containing 2-6 disulfide bonds.

  15. Methods of measuring Protein Disulfide Isomerase activity: a critical overview

    Science.gov (United States)

    Watanabe, Monica; Laurindo, Francisco; Fernandes, Denise

    2014-09-01

    Protein disulfide isomerase is an essential redox chaperone from the endoplasmic reticulum (ER) and is responsible for correct disulfide bond formation in nascent proteins. PDI is also found in other cellular locations in the cell, particularly the cell surface. Overall, PDI contributes to ER and global cell redox homeostasis and signaling. The knowledge about PDI structure and function progressed substantially based on in vitro studies using recombinant PDI and chimeric proteins. In these experimental scenarios, PDI reductase and chaperone activities are readily approachable. In contrast, assays to measure PDI isomerase activity, the hallmark of PDI family, are more complex. Assessment of PDI roles in cells and tissues mainly relies on gain- or loss-of-function studies. However, there is limited information regarding correlation of experimental readouts with the distinct types of PDI activities. In this mini-review, we evaluate the main methods described for measuring the different kinds of PDI activity: thiol reductase, thiol oxidase, thiol isomerase and chaperone. We emphasize the need to use appropriate controls and the role of critical interferents (e.g., detergent, presence of reducing agents). We also discuss the translation of results from in vitro studies with purified recombinant PDI to cellular and tissue samples, with critical comments on the interpretation of results.

  16. Folding and activity of hybrid sequence, disulfide-stabilized peptides

    Energy Technology Data Exchange (ETDEWEB)

    Pease, J.H.B.; Storrs, R.W.; Wemmer, D.E. (Univ. of California, Berkeley (USA))

    1990-08-01

    Peptides have been synthesized that have hybrid sequences, partially derived from the bee venom peptide apamin and partially from the S peptide of ribonuclease A. The hybrid peptides were demonstrated by NMR spectroscopy to fold, forming the same disulfides and basic three-dimensional structure as native apamin, containing a {beta}-turn and an {alpha}-helix. These hybrids were active in complementing S protein, reactivating nuclease activity. In addition, the hybrid peptide was effective in inducing antibodies that cross-react with the RNase, without conjugation to a carrier protein. The stability of the folded structure of this peptide suggests that it should be possible to elicit antibodies that will react not only with a specific sequence, but also with a specific secondary structure. Hybrid sequence peptides also provide opportunities to study separately nucleation and propagation steps in formation of secondary structure. The authors show that in S peptide the {alpha}-helix does not end abruptly but rather terminates gradually over four or five residues. In general, these hybrid sequence peptides, which fold predictably because of disulfide bond formation, can provide opportunities for examining structure - function relationships for many biologically active sequences.

  17. Folding and activity of hybrid sequence, disulfide-stabilized peptides

    International Nuclear Information System (INIS)

    Pease, J.H.B.; Storrs, R.W.; Wemmer, D.E.

    1990-01-01

    Peptides have been synthesized that have hybrid sequences, partially derived from the bee venom peptide apamin and partially from the S peptide of ribonuclease A. The hybrid peptides were demonstrated by NMR spectroscopy to fold, forming the same disulfides and basic three-dimensional structure as native apamin, containing a β-turn and an α-helix. These hybrids were active in complementing S protein, reactivating nuclease activity. In addition, the hybrid peptide was effective in inducing antibodies that cross-react with the RNase, without conjugation to a carrier protein. The stability of the folded structure of this peptide suggests that it should be possible to elicit antibodies that will react not only with a specific sequence, but also with a specific secondary structure. Hybrid sequence peptides also provide opportunities to study separately nucleation and propagation steps in formation of secondary structure. The authors show that in S peptide the α-helix does not end abruptly but rather terminates gradually over four or five residues. In general, these hybrid sequence peptides, which fold predictably because of disulfide bond formation, can provide opportunities for examining structure - function relationships for many biologically active sequences

  18. Structural Basis of a Thiol-Disulfide Oxidoreductase in the Hedgehog-Forming Actinobacterium Corynebacterium matruchotii.

    Science.gov (United States)

    Luong, Truc Thanh; Tirgar, Reyhaneh; Reardon-Robinson, Melissa E; Joachimiak, Andrzej; Osipiuk, Jerzy; Ton-That, Hung

    2018-05-01

    The actinobacterium Corynebacterium matruchotii has been implicated in nucleation of oral microbial consortia leading to biofilm formation. Due to the lack of genetic tools, little is known about basic cellular processes, including protein secretion and folding, in this organism. We report here a survey of the C. matruchotii genome, which encodes a large number of exported proteins containing paired cysteine residues, and identified an oxidoreductase that is highly homologous to the Corynebacterium diphtheriae thiol-disulfide oxidoreductase MdbA (MdbA Cd ). Crystallization studies uncovered that the 1.2-Å resolution structure of C. matruchotii MdbA (MdbA Cm ) possesses two conserved features found in actinobacterial MdbA enzymes, a thioredoxin-like fold and an extended α-helical domain. By reconstituting the disulfide bond-forming machine in vitro , we demonstrated that MdbA Cm catalyzes disulfide bond formation within the actinobacterial pilin FimA. A new gene deletion method supported that mdbA is essential in C. matruchotii Remarkably, heterologous expression of MdbA Cm in the C. diphtheriae Δ mdbA mutant rescued its known defects in cell growth and morphology, toxin production, and pilus assembly, and this thiol-disulfide oxidoreductase activity required the catalytic motif CXXC. Altogether, the results suggest that MdbA Cm is a major thiol-disulfide oxidoreductase, which likely mediates posttranslocational protein folding in C. matruchotii by a mechanism that is conserved in Actinobacteria IMPORTANCE The actinobacterium Corynebacterium matruchotii has been implicated in the development of oral biofilms or dental plaque; however, little is known about the basic cellular processes in this organism. We report here a high-resolution structure of a C. matruchotii oxidoreductase that is highly homologous to the Corynebacterium diphtheriae thiol-disulfide oxidoreductase MdbA. By biochemical analysis, we demonstrated that C. matruchotii MdbA catalyzes disulfide

  19. Selective disulfide reduction for labeling and enhancement of Fab antibody fragments.

    Science.gov (United States)

    Kirley, Terence L; Greis, Kenneth D; Norman, Andrew B

    2016-11-25

    Many methods have been developed for chemical labeling and enhancement of the properties of antibodies and their common fragments, including the Fab and F(ab') 2 fragments. Somewhat selective reduction of some antibody disulfide bonds has been previously achieved, yielding antibodies and antibody fragments that can be labeled at defined sites, enhancing their utility and properties. Selective reduction of the two hinge disulfide bonds present in F(ab') 2 fragments using mild reduction has been useful. However, such reduction is often not quantitative and results in the reduction of multiple disulfide bonds, and therefore subsequent multiple labeling or conjugation sites are neither homogenous nor stoichiometric. Here, a simple and efficient selective reduction of the single disulfide bond linking the partial heavy chain and the intact light chain which compose the Fab fragment is accomplished utilizing tris(2-carboxyethyl)phosphine (TCEP) immobilized on agarose beads. The resultant reduced cysteine residues were labeled with several cysteine-selective fluorescent reagents, as well as by cysteine-directed PEGylation. These two cysteine residues can also be re-ligated by means of a bifunctional cysteine cross-linking agent, dibromobimane, thereby both restoring a covalent linkage between the heavy and light chains at this site, far removed from the antigen binding site, and also introducing a fluorescent probe. There are many other research and clinical uses for these selectively partially reduced Fab fragments, including biotinylation, toxin and drug conjugation, and incorporation of radioisotopes, and this technique enables simple generation of very useful Fab fragment derivatives with many potential applications. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. On the relevance of sophisticated structural annotations for disulfide connectivity pattern prediction.

    Directory of Open Access Journals (Sweden)

    Julien Becker

    Full Text Available Disulfide bridges strongly constrain the native structure of many proteins and predicting their formation is therefore a key sub-problem of protein structure and function inference. Most recently proposed approaches for this prediction problem adopt the following pipeline: first they enrich the primary sequence with structural annotations, second they apply a binary classifier to each candidate pair of cysteines to predict disulfide bonding probabilities and finally, they use a maximum weight graph matching algorithm to derive the predicted disulfide connectivity pattern of a protein. In this paper, we adopt this three step pipeline and propose an extensive study of the relevance of various structural annotations and feature encodings. In particular, we consider five kinds of structural annotations, among which three are novel in the context of disulfide bridge prediction. So as to be usable by machine learning algorithms, these annotations must be encoded into features. For this purpose, we propose four different feature encodings based on local windows and on different kinds of histograms. The combination of structural annotations with these possible encodings leads to a large number of possible feature functions. In order to identify a minimal subset of relevant feature functions among those, we propose an efficient and interpretable feature function selection scheme, designed so as to avoid any form of overfitting. We apply this scheme on top of three supervised learning algorithms: k-nearest neighbors, support vector machines and extremely randomized trees. Our results indicate that the use of only the PSSM (position-specific scoring matrix together with the CSP (cysteine separation profile are sufficient to construct a high performance disulfide pattern predictor and that extremely randomized trees reach a disulfide pattern prediction accuracy of [Formula: see text] on the benchmark dataset SPX[Formula: see text], which corresponds to

  1. Electrochemistry-assisted top-down characterization of disulfide-containing proteins.

    Science.gov (United States)

    Zhang, Yun; Cui, Weidong; Zhang, Hao; Dewald, Howard D; Chen, Hao

    2012-04-17

    Covalent disulfide bond linkage in a protein represents an important challenge for mass spectrometry (MS)-based top-down protein structure analysis as it reduces the backbone cleavage efficiency for MS/MS dissociation. This study presents a strategy for solving this critical issue via integrating electrochemistry (EC) online with a top-down MS approach. In this approach, proteins undergo electrolytic reduction in an electrochemical cell to break disulfide bonds and then undergo online ionization into gaseous ions for analysis by electron-capture dissociation (ECD) and collision-induced dissociation (CID). The electrochemical reduction of proteins allows one to remove disulfide bond constraints and also leads to increased charge numbers of the resulting protein ions. As a result, sequence coverage was significantly enhanced, as exemplified by β-lactoglobulin A (24 vs 75 backbone cleavages before and after electrolytic reduction, respectively) and lysozyme (5 vs 66 backbone cleavages before and after electrolytic reduction, respectively). This methodology is fast and does not need chemical reductants, which would have an important impact in high-throughput proteomics research.

  2. Measurement of glutathione-protein mixed disulfides

    International Nuclear Information System (INIS)

    Livesey, J.C.; Reed, D.J.

    1984-01-01

    The development of a sensitive and highly specific assay for the presence of mixed disulfides between protein thiol groups and endogenous thiols has been undertaken. Previous investigations on the concentrations of glutathione (GSH), glutathione disulfide (GSSG) and protein glutathione mixed disulfides (ProSSG) have been of limited usefulness because of the poor specificity of the assays used. Our assay for these forms of glutathione is based on high performance liquid chromatography (HPLC) and is an extension of an earlier method. After perchloric acid precipitation, the protein sample is washed with an organic solvent to fully denature the protein. Up to a 10-fold increase in GSH released from fetal bovine serum (FBS) protein has been found when the protein precipitate is washed with ethanol rather than ether, as earlier suggested. Similar effects have been observed with an as yet unidentified thiol which elutes in the chromatography system with a retention volume similar to cysteine

  3. Brain MRI findings of carbon disulfide poisoning

    International Nuclear Information System (INIS)

    Cha, Joo Hee; Kim, Mi Jung; Yim, Sang Hyuk; Kim, Sam Soo; Han, Heon; Kim, Rok Ho

    2002-01-01

    To evaluate the findings of brain MRI in patients with carbon disulfide poisoning. Ninety-one patients who had suffered carbon disulfide poisoning [male:female=87:4; age, 32-74 (mean 53.3) years] were included in this study. To determine the extent of white matter hyperintensity (Grade 0-V) and lacunar infarction, T2-weighted MR imaging of the brain was performed. T2-weighted images depicted white matter hyperintensity in 70 patients (76.9%) and lacunar infarcts in 27 (29.7%). In these patients, the prevalent findings at T2-weighted MR imaging of the brain were white matter hyperintensity and lacunar infarcts. Disturbance of the cardiovascular system by carbon disulfide might account for these results

  4. Disulphide bond formation in food protein aggregation and gelation

    NARCIS (Netherlands)

    Visschers, R.W.; Jongh, de H.H.J.

    2005-01-01

    In this short review we discuss the role of cysteine residues and cystine bridges for the functional aggregation of food proteins. We evaluate how formation and cleavage of disulphide bonds proceeds at a molecular level, and how inter- and intramolecular disulfide bonds can be detected and modified.

  5. Dynamic combinatorial chemistry with diselenides and disulfides in water

    DEFF Research Database (Denmark)

    Rasmussen, Brian; Sørensen, Anne; Gotfredsen, Henrik

    2014-01-01

    Diselenide exchange is introduced as a reversible reaction in dynamic combinatorial chemistry in water. At neutral pH, diselenides are found to mix with disulfides and form dynamic combinatorial libraries of diselenides, disulfides, and selenenylsulfides. This journal is......Diselenide exchange is introduced as a reversible reaction in dynamic combinatorial chemistry in water. At neutral pH, diselenides are found to mix with disulfides and form dynamic combinatorial libraries of diselenides, disulfides, and selenenylsulfides. This journal is...

  6. Functional differences in yeast protein disulfide isomerases

    DEFF Research Database (Denmark)

    Nørgaard, P; Westphal, V; Tachibana, C

    2001-01-01

    PDI1 is the essential gene encoding protein disulfide isomerase in yeast. The Saccharomyces cerevisiae genome, however, contains four other nonessential genes with homology to PDI1: MPD1, MPD2, EUG1, and EPS1. We have investigated the effects of simultaneous deletions of these genes. In several...

  7. An efficient algorithmic approach for mass spectrometry-based disulfide connectivity determination using multi-ion analysis

    Directory of Open Access Journals (Sweden)

    Yen Ten-Yang

    2011-02-01

    Full Text Available Abstract Background Determining the disulfide (S-S bond pattern in a protein is often crucial for understanding its structure and function. In recent research, mass spectrometry (MS based analysis has been applied to this problem following protein digestion under both partial reduction and non-reduction conditions. However, this paradigm still awaits solutions to certain algorithmic problems fundamental amongst which is the efficient matching of an exponentially growing set of putative S-S bonded structural alternatives to the large amounts of experimental spectrometric data. Current methods circumvent this challenge primarily through simplifications, such as by assuming only the occurrence of certain ion-types (b-ions and y-ions that predominate in the more popular dissociation methods, such as collision-induced dissociation (CID. Unfortunately, this can adversely impact the quality of results. Method We present an algorithmic approach to this problem that can, with high computational efficiency, analyze multiple ions types (a, b, bo, b*, c, x, y, yo, y*, and z and deal with complex bonding topologies, such as inter/intra bonding involving more than two peptides. The proposed approach combines an approximation algorithm-based search formulation with data driven parameter estimation. This formulation considers only those regions of the search space where the correct solution resides with a high likelihood. Putative disulfide bonds thus obtained are finally combined in a globally consistent pattern to yield the overall disulfide bonding topology of the molecule. Additionally, each bond is associated with a confidence score, which aids in interpretation and assimilation of the results. Results The method was tested on nine different eukaryotic Glycosyltransferases possessing disulfide bonding topologies of varying complexity. Its performance was found to be characterized by high efficiency (in terms of time and the fraction of search space

  8. Identification of a disulfide bridge important for transport function of SNAT4 neutral amino acid transporter.

    Directory of Open Access Journals (Sweden)

    Rugmani Padmanabhan Iyer

    Full Text Available SNAT4 is a member of system N/A amino acid transport family that primarily expresses in liver and muscles and mediates the transport of L-alanine. However, little is known about the structure and function of the SNAT family of transporters. In this study, we showed a dose-dependent inhibition in transporter activity of SNAT4 with the treatment of reducing agents, dithiothreitol (DTT and Tris(2-carboxyethylphosphine (TCEP, indicating the possible involvement of disulfide bridge(s. Mutation of residue Cys-232, and the two highly conserved residues Cys-249 and Cys-321, compromised the transport function of SNAT4. However, this reduction was not caused by the decrease of SNAT4 on the cell surface since the cysteine-null mutant generated by replacing all five cysteines with alanine was equally capable of being expressed on the cell surface as wild-type SNAT4. Interestingly, by retaining two cysteine residues, 249 and 321, a significant level of L-alanine uptake was restored, indicating the possible formation of disulfide bond between these two conserved residues. Biotinylation crosslinking of free thiol groups with MTSEA-biotin provided direct evidence for the existence of a disulfide bridge between Cys-249 and Cys-321. Moreover, in the presence of DTT or TCEP, transport activity of the mutant retaining Cys-249 and Cys-321 was reduced in a dose-dependent manner and this reduction is gradually recovered with increased concentration of H2O2. Disruption of the disulfide bridge also decreased the transport of L-arginine, but to a lesser degree than that of L-alanine. Together, these results suggest that cysteine residues 249 and 321 form a disulfide bridge, which plays an important role in substrate transport but has no effect on trafficking of SNAT4 to the cell surface.

  9. Loss of metal ions, disulfide reduction and mutations related to familial ALS promote formation of amyloid-like aggregates from superoxide dismutase.

    Directory of Open Access Journals (Sweden)

    Zeynep A Oztug Durer

    Full Text Available Mutations in the gene encoding Cu-Zn superoxide dismutase (SOD1 are one of the causes of familial amyotrophic lateral sclerosis (FALS. Fibrillar inclusions containing SOD1 and SOD1 inclusions that bind the amyloid-specific dye thioflavin S have been found in neurons of transgenic mice expressing mutant SOD1. Therefore, the formation of amyloid fibrils from human SOD1 was investigated. When agitated at acidic pH in the presence of low concentrations of guanidine or acetonitrile, metalated SOD1 formed fibrillar material which bound both thioflavin T and Congo red and had circular dichroism and infrared spectra characteristic of amyloid. While metalated SOD1 did not form amyloid-like aggregates at neutral pH, either removing metals from SOD1 with its intramolecular disulfide bond intact or reducing the intramolecular disulfide bond of metalated SOD1 was sufficient to promote formation of these aggregates. SOD1 formed amyloid-like aggregates both with and without intermolecular disulfide bonds, depending on the incubation conditions, and a mutant SOD1 lacking free sulfhydryl groups (AS-SOD1 formed amyloid-like aggregates at neutral pH under reducing conditions. ALS mutations enhanced the ability of disulfide-reduced SOD1 to form amyloid-like aggregates, and apo-AS-SOD1 formed amyloid-like aggregates at pH 7 only when an ALS mutation was also present. These results indicate that some mutations related to ALS promote formation of amyloid-like aggregates by facilitating the loss of metals and/or by making the intramolecular disulfide bond more susceptible to reduction, thus allowing the conversion of SOD1 to a form that aggregates to form resembling amyloid. Furthermore, the occurrence of amyloid-like aggregates per se does not depend on forming intermolecular disulfide bonds, and multiple forms of such aggregates can be produced from SOD1.

  10. 1.42 A crystal structure of mini-IGF-1(2): an analysis of the disulfide isomerization property and receptor binding property of IGF-1 based on the three-dimensional structure

    International Nuclear Information System (INIS)

    Yun Caihong; Tang Yuehua; Feng Youmin; An Xiaomin; Chang Wenrui; Liang Dongcai

    2004-01-01

    Insulin and insulin-like growth factor 1 (IGF-1) share a homologous sequence, a similar three-dimensional structure and weakly overlapping biological activity, but IGF-1 folds into two thermodynamically stable disulfide isomers, while insulin folds into one unique stable tertiary structure. This is a very interesting phenomenon in which one amino acid sequence encodes two three-dimensional structures, and its molecular mechanism has remained unclear for a long time. In this study, the crystal structure of mini-IGF-1(2), a disulfide isomer of an artificial analog of IGF-1, was solved by the SAD/SIRAS method using our in-house X-ray source. Evidence was found in the structure showing that the intra-A-chain/domain disulfide bond of some molecules was broken; thus, it was proposed that disulfide isomerization begins with the breakdown of this disulfide bond. Furthermore, based on the structural comparison of IGF-1 and insulin, a new assumption was made that in insulin the several hydrogen bonds formed between the N-terminal region of the B-chain and the intra-A-chain disulfide region of the A-chain are the main reason for the stability of the intra-A-chain disulfide bond and for the prevention of disulfide isomerization, while Phe B1 and His B5 are very important for the formation of these hydrogen bonds. Moreover, the receptor binding property of IGF-1 was analyzed in detail based on the structural comparison of mini-IGF-1(2), native IGF-1, and small mini-IGF-1

  11. Investigation of protein FTT1103 electroactivity using carbon and mercury electrodes. Surface-inhibition approach for disulfide oxidoreductases using silver amalgam powder

    Czech Academy of Sciences Publication Activity Database

    Večerková, R.; Hernychová, L.; Dobeš, P.; Vrba, J.; Josypčuk, Bohdan; Bartošík, M.; Vacek, J.

    2014-01-01

    Roč. 830, JUN 2014 (2014), s. 23-32 ISSN 0003-2670 Institutional support: RVO:61388955 Keywords : Disulfide bond forming protein * Electrochemical sensing * Membrane proteins Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 4.513, year: 2014

  12. Thiol-Disulfide Exchange between Glutaredoxin and Glutathione

    DEFF Research Database (Denmark)

    Iversen, Rasmus; Andersen, Peter Anders; Jensen, Kristine Steen

    2010-01-01

    Glutaredoxins are ubiquitous thiol-disulfide oxidoreductases which catalyze the reduction of glutathione-protein mixed disulfides. Belonging to the thioredoxin family, they contain a conserved active site CXXC motif. The N-proximal active site cysteine can form a mixed disulfide with glutathione ...... has been replaced with serine. The exchange reaction between the reduced protein and oxidized glutathione leading to formation of the mixed disulfide could readily be monitored by isothermal titration calorimetry (ITC) due to the enthalpic contributions from the noncovalent interactions...

  13. Photoinduced Cross-Linking of Dynamic Poly(disulfide) Films via Thiol Oxidative Coupling.

    Science.gov (United States)

    Feillée, Noémi; Chemtob, Abraham; Ley, Christian; Croutxé-Barghorn, Céline; Allonas, Xavier; Ponche, Arnaud; Le Nouen, Didier; Majjad, Hicham; Jacomine, Léandro

    2016-01-01

    Initially developed as an elastomer with an excellent record of barrier and chemical resistance properties, poly(disulfide) has experienced a revival linked to the dynamic nature of the S-S covalent bond. A novel photobase-catalyzed oxidative polymerization of multifunctional thiols to poly(disulfide) network is reported. Based solely on air oxidation, the single-step process is triggered by the photodecarboxylation of a xanthone acetic acid liberating a strong bicyclic guanidine base. Starting with a 1 μm thick film based on trithiol poly(ethylene oxide) oligomer, the UV-mediated oxidation of thiols to disulfides occurs in a matter of minutes both selectively, i.e., without overoxidation, and quantitatively as assessed by a range of spectroscopic techniques. Thiolate formation and film thickness determine the reaction rates and yield. Spatial control of the photopolymerization serves to generate robust micropatterns, while the reductive cleavage of S-S bridges allows the recycling of 40% of the initial thiol groups. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Structure of conkunitzin-S1, a neurotoxin and Kunitz-fold disulfide variant from cone snail

    International Nuclear Information System (INIS)

    Dy, Catherine Y.; Buczek, Pawel; Imperial, Julita S.; Bulaj, Grzegorz; Horvath, Martin P.

    2006-01-01

    Most Kunitz proteins like BPTI and α-dendrotoxin are stabilized by three disulfide bonds. The crystal structure shows how subtle repacking of non-covalent interactions may compensate for disulfide bond loss in a naturally occurring two-disulfide variant, conkunitzin-S1, the first discovered member of a new conotoxin family. Cone snails (Conus) are predatory marine mollusks that immobilize prey with venom containing 50–200 neurotoxic polypeptides. Most of these polypeptides are small disulfide-rich conotoxins that can be classified into families according to their respective ion-channel targets and patterns of cysteine–cysteine disulfides. Conkunitzin-S1, a potassium-channel pore-blocking toxin isolated from C. striatus venom, is a member of a newly defined conotoxin family with sequence homology to Kunitz-fold proteins such as α-dendrotoxin and bovine pancreatic trypsin inhibitor (BPTI). While conkunitzin-S1 and α-dendrotoxin are 42% identical in amino-acid sequence, conkunitzin-S1 has only four of the six cysteines normally found in Kunitz proteins. Here, the crystal structure of conkunitzin-S1 is reported. Conkunitzin-S1 adopts the canonical 3 10 –β–β–α Kunitz fold complete with additional distinguishing structural features including two completely buried water molecules. The crystal structure, although completely consistent with previously reported NMR distance restraints, provides a greater degree of precision for atomic coordinates, especially for S atoms and buried solvent molecules. The region normally cross-linked by cysteines II and IV in other Kunitz proteins retains a network of hydrogen bonds and van der Waals interactions comparable to those found in α-dendrotoxin and BPTI. In conkunitzin-S1, glycine occupies the sequence position normally reserved for cysteine II and the special steric properties of glycine allow additional van der Waals contacts with the glutamine residue substituting for cysteine IV. Evolution has thus defrayed the

  15. Compact conformations of human protein disulfide isomerase.

    Directory of Open Access Journals (Sweden)

    Shang Yang

    Full Text Available Protein disulfide isomerase (PDI composed of four thioredoxin-like domains a, b, b', and a', is a key enzyme catalyzing oxidative protein folding in the endoplasmic reticulum. Large scale molecular dynamics simulations starting from the crystal structures of human PDI (hPDI in the oxidized and reduced states were performed. The results indicate that hPDI adopts more compact conformations in solution than in the crystal structures, which are stabilized primarily by inter-domain interactions, including the salt bridges between domains a and b' observed for the first time. A prominent feature of the compact conformations is that the two catalytic domains a and a' can locate close enough for intra-molecular electron transfer, which was confirmed by the characterization of an intermediate with a disulfide between the two domains. Mutations, which disrupt the inter-domain interactions, lead to decreased reductase activity of hPDI. Our molecular dynamics simulations and biochemical experiments reveal the intrinsic conformational dynamics of hPDI and its biological impact.

  16. Protein binding of N-2-mercaptoethyl-1,3-diaminopropane via mixed disulfide formation after oral administration of WR 2721

    Energy Technology Data Exchange (ETDEWEB)

    Tabachnik, N.F.; Blackburn, P.; Peterson, C.M.; Cerami, A.

    1982-02-01

    Earlier studies have shown that WR 2721 (H2N-(CH2)3-NH(CH2)2SPO3H2) is converted to its free thiol form, N-2-mercaptoethyl-1,3-diaminopropane (MDP), at the acidic pH of the stomach. MDP is a radioprotective compound and a mucolytic agent capable of decreasing sputum viscosity in the lungs of patients with cystic fibrosis. Conversion of WR 2721 and MDP to the corresponding sulfonic acid (MDP-SO3H) permits quantitative determination of these compounds in physiological fluids by use of an automatic amino acid analyzer. After oral administration of WR 2721 to human patients and rabbits it is converted to MDP and the free thiol form of the drug associates with plasma proteins by mixed disulfide linkage. The plasma proteins serve as a depot and reservoir of MDP for potential exchange at the tissues. When incubated with whole sputum or with purified mucin solutions in vitro, MDP decreased the viscosity of these solutions by reduction of the accessible disulfide bonds of the mucin molecule and was subsequently found in mixed disulfide association with the mucin molecule. The association of MDP with proteins via mixed disulfide linkage has important implications for the development of optimal dose regimens for administration of WR 2721 to patients.

  17. Protein binding of N-2-mercaptoethyl-1,3-diaminopropane via mixed disulfide formation after oral administration of WR 2721

    International Nuclear Information System (INIS)

    Tabachnik, N.F.; Blackburn, P.; Peterson, C.M.; Cerami, A.

    1982-01-01

    Earlier studies have shown that WR 2721 [H2N-(CH2)3-NH(CH2)2SPO3H2] is converted to its free thiol form, N-2-mercaptoethyl-1,3-diaminopropane (MDP), at the acidic pH of the stomach. MDP is a radioprotective compound and a mucolytic agent capable of decreasing sputum viscosity in the lungs of patients with cystic fibrosis. Conversion of WR 2721 and MDP to the corresponding sulfonic acid (MDP-SO3H) permits quantitative determination of these compounds in physiological fluids by use of an automatic amino acid analyzer. After oral administration of WR 2721 to human patients and rabbits it is converted to MDP and the free thiol form of the drug associates with plasma proteins by mixed disulfide linkage. The plasma proteins serve as a depot and reservoir of MDP for potential exchange at the tissues. When incubated with whole sputum or with purified mucin solutions in vitro, MDP decreased the viscosity of these solutions by reduction of the accessible disulfide bonds of the mucin molecule and was subsequently found in mixed disulfide association with the mucin molecule. The association of MDP with proteins via mixed disulfide linkage has important implications for the development of optimal dose regimens for administration of WR 2721 to patients

  18. CuI-Catalyzed: One-Pot Synthesis of Diaryl Disulfides from Aryl Halides and Carbon Disulfide

    Directory of Open Access Journals (Sweden)

    Mohammad Soleiman-Beigi

    2013-01-01

    Full Text Available A new application of carbon disulfide in the presence of KF/Al2O3 is reported for the synthesis of organic symmetrical diaryl disulfides. These products were synthesized by one-pot reaction of aryl halides with the in situ generated trithiocarbonate ion in the presence of copper under air atmosphere.

  19. Studying Chemical Reactions, One Bond at a Time, with Single Molecule AFM Techniques

    Science.gov (United States)

    Fernandez, Julio M.

    2008-03-01

    The mechanisms by which mechanical forces regulate the kinetics of a chemical reaction are unknown. In my lecture I will demonstrate how we use single molecule force-clamp spectroscopy and protein engineering to study the effect of force on the kinetics of thiol/disulfide exchange. Reduction of disulfide bond via the thiol/disulfide exchange chemical reaction is crucial in regulating protein function and is of common occurrence in mechanically stressed proteins. While reduction is thought to proceed through a substitution nucleophilic bimolecular (SN2) reaction, the role of a mechanical force in modulating this chemical reaction is unknown. We apply a constant stretching force to single engineered disulfide bonds and measure their rate of reduction by dithiothreitol (DTT). We find that while the reduction rate is linearly dependent on the concentration of DTT, it is exponentially dependent on the applied force, increasing 10-fold over a 300 pN range. This result predicts that the disulfide bond lengthens by 0.34 å at the transition state of the thiol/disulfide exchange reaction. In addition to DTT, we also study the reduction of the engineered disulfide bond by the E. coli enzyme thioredoxin (Trx). Thioredoxins are enzymes that catalyze disulfide bond reduction in all organisms. As before, we apply a mechanical force in the range of 25-450 pN to the engineered disulfide bond substrate and monitor the reduction of these bonds by individual enzymes. In sharp contrast with the data obtained with DTT, we now observe two alternative forms of the catalytic reaction, the first requiring a reorientation of the substrate disulfide bond, causing a shortening of the substrate polypeptide by 0.76±0.07 å, and the second elongating the substrate disulfide bond by 0.21±0.01 å. These results support the view that the Trx active site regulates the geometry of the participating sulfur atoms, with sub-ångström precision, in order to achieve efficient catalysis. Single molecule

  20. Cysteine-Rich Peptide Family with Unusual Disulfide Connectivity from Jasminum sambac.

    Science.gov (United States)

    Kumari, Geeta; Serra, Aida; Shin, Joon; Nguyen, Phuong Q T; Sze, Siu Kwan; Yoon, Ho Sup; Tam, James P

    2015-11-25

    Cysteine-rich peptides (CRPs) are natural products with privileged peptidyl structures that represent a potentially rich source of bioactive compounds. Here, the discovery and characterization of a novel plant CRP family, jasmintides from Jasminum sambac of the Oleaceae family, are described. Two 27-amino acid jasmintides (jS1 and jS2) were identified at the gene and protein levels. Disulfide bond mapping of jS1 by mass spectrometry and its confirmation by NMR spectroscopy revealed disulfide bond connectivity of C-1-C-5, C-2-C-4, and C-3-C-6, a cystine motif that has not been reported in plant CRPs. Structural determination showed that jS1 displays a well-defined structure framed by three short antiparallel β-sheets. Genomic analysis showed that jasmintides share a three-domain precursor arrangement with a C-terminal mature domain preceded by a long pro-domain of 46 residues and an intron cleavage site between the signal sequence and pro-domain. The compact cysteine-rich structure together with an N-terminal pyroglutamic acid residue confers jasmintides high resistance to heat and enzymatic degradation, including exopeptidase treatment. Collectively, these results reveal a new plant CRP structure with an unusual cystine connectivity, which could be useful as a scaffold for designing peptide drugs.

  1. Thermal ripples in model molybdenum disulfide monolayers

    Energy Technology Data Exchange (ETDEWEB)

    Remsing, Richard C.; Klein, Michael L. [Institute for Computational Molecular Science, Center for the Computational, Design of Functional Layered Materials, and Department of Chemistry, Temple University, 1925 N. 12th St., 19122, Philadelphia, PA (United States); Waghmare, Umesh V. [Theoretical Sciences Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, 560 064, Jakkur, Bangalore (India)

    2017-01-15

    Molybdenum disulfide (MoS{sub 2}) monolayers have the potential to revolutionize nanotechnology. To reach this potential, it will be necessary to understand the behavior of this two-dimensional (2D) material on large length scales and under thermal conditions. Herein, we use molecular dynamics (MD) simulations to investigate the nature of the rippling induced by thermal fluctuations in monolayers of the 2H and 1T phases of MoS{sub 2}. The 1T phase is found to be more rigid than the 2H phase. Both monolayer phases are predicted to follow long wavelength scaling behavior typical of systems with anharmonic coupling between vibrational modes as predicted by classic theories of membrane-like systems. (copyright 2017 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  2. Preparation and Photoluminescence of Tungsten Disulfide Monolayer

    Directory of Open Access Journals (Sweden)

    Yanfei Lv

    2018-05-01

    Full Text Available Tungsten disulfide (WS2 monolayer is a direct band gap semiconductor. The growth of WS2 monolayer hinders the progress of its investigation. In this paper, we prepared the WS2 monolayer through chemical vapor transport deposition. This method makes it easier for the growth of WS2 monolayer through the heterogeneous nucleation-and-growth process. The crystal defects introduced by the heterogeneous nucleation could promote the photoluminescence (PL emission. We observed the strong photoluminescence emission in the WS2 monolayer, as well as thermal quenching, and the PL energy redshift as the temperature increases. We attribute the thermal quenching to the energy or charge transfer of the excitons. The redshift is related to the dipole moment of WS2.

  3. Towards a Molecular Movie: Real Time Observation of Hydrogen Bond Breaking by Transient 2D-IR Spectroscopy in a Cyclic Peptide

    Science.gov (United States)

    Kolano, Christoph; Helbing, Jan; Sander, Wolfram; Hamm, Peter

    Transient two-dimensional infrared spectroscopy (T2D-IR) has been used to observe in real time the non-equilibrium structural dynamics of intramolecular hydrogen bond breaking in a small cyclic disulfide-bridged peptide.

  4. Chemoreactomic analysis of thiamine disulfide, thiamine hydrochloride, and benfotiamine molecules

    Directory of Open Access Journals (Sweden)

    O. A. Gromova

    2017-01-01

    Full Text Available Objective: to analyze the interactions that could indicate the potential pharmacological properties of the molecules of thiamin, thiamine disulfide, and others.Material and methods. The investigators simulated the properties of thiamine disulfide (bistiamin versus those of the reference molecules of thiamin hydrochloride and benfotiamine. The study was performed using chemoreactomic simulation that is the newest area in post-genome pharmacology.Results and discussion. Chemoreactomic analysis has shown that thiamine disulfide can inhibit the molecular receptors involved in blood pressure regulation: adrenoceptors, vasopressin receptor, and angiotensin receptor. Thiamine disulfide can inhibit the reuptake of serotonin, increase its levels, inhibit benzodiazepine receptor and dopamine reuptake, and enhance neuronal acetylcholine release to a large extent than benfotiamine. These molecular effects are consistent with the sedative and anticonvulsant action profile of thiamine disulfide. Simulation has indicated that thiamine disulfide has neuroprotective, anti-inflammatory, normolipidemic, and antitumor activities.Conclusion. The simulation results are confirmed by the available clinical and experimental findings and indicate the virtually unstudied molecular mechanisms of action of thiamine disulfide, benfotiamine, and thiamin hydrochloride. 

  5. Steric effects in peptide and protein exchange with activated disulfides.

    Science.gov (United States)

    Kerr, Jason; Schlosser, Jessica L; Griffin, Donald R; Wong, Darice Y; Kasko, Andrea M

    2013-08-12

    Disulfide exchange is an important bioconjugation tool, enabling chemical modification of peptides and proteins containing free cysteines. We previously reported the synthesis of a macromer bearing an activated disulfide and its incorporation into hydrogels. Despite their ability to diffuse freely into hydrogels, larger proteins were unable to undergo in-gel disulfide exchange. In order to understand this phenomenon, we synthesized four different activated disulfide-bearing model compounds (Mn = 300 Da to 10 kDa) and quantified their rate of disulfide exchange with a small peptide (glutathione), a moderate-sized protein (β-lactoglobulin), and a large protein (bovine serum albumin) in four different pH solutions (6.0, 7.0, 7.4, and 8.0) to mimic biological systems. Rate constants of exchange depend significantly on the size and accessibility of the thiolate. pH also significantly affects the rate of reaction, with the faster reactions occurring at higher pH. Surprisingly, little difference in exchange rates is seen between macromolecular disulfides of varying size (Mn = 2 kDa - 10 kDa), although all undergo exchange more slowly than their small molecule analogue (MW = 300 g/mol). The maximum exchange efficiencies (% disulfides exchanged after 24 h) are not siginificantly affected by thiol size or pH, but somewhat affected by disulfide size. Therefore, while all three factors investigated (pH, disulfide size, and thiolate size) can influence the exchange kinetics and extent of reaction, the size of the thiolate and its accessibility plays the most significant role.

  6. Bond Issues.

    Science.gov (United States)

    Pollack, Rachel H.

    2000-01-01

    Notes trends toward increased borrowing by colleges and universities and offers guidelines for institutions that are considering issuing bonds to raise money for capital projects. Discussion covers advantages of using bond financing, how use of bonds impacts on traditional fund raising, other cautions and concerns, and some troubling aspects of…

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

    International Nuclear Information System (INIS)

    Meinke, Gretchen; Phelan, Paul; Fradet-Turcotte, Amélie; Archambault, Jacques; Bullock, Peter A.

    2011-01-01

    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

  8. Chemoreactomic analysis of thiamine disulfide, thiamine hydrochloride, and benfotiamine molecules

    OpenAIRE

    O. A. Gromova; I. Yu. Torshin; L. V. Stakhovskaya; L. E. Fedotova

    2017-01-01

    Objective: to analyze the interactions that could indicate the potential pharmacological properties of the molecules of thiamin, thiamine disulfide, and others.Material and methods. The investigators simulated the properties of thiamine disulfide (bistiamin) versus those of the reference molecules of thiamin hydrochloride and benfotiamine. The study was performed using chemoreactomic simulation that is the newest area in post-genome pharmacology.Results and discussion. Chemoreactomic analysis...

  9. Lithium/disulfide battery R and D

    Science.gov (United States)

    Kaun, T. D.; Deluca, W.; Lee, J.; Redey, L.; Nelson, P. A.

    The focus of molten-salt cell R and D in the past year at Argonne National Laboratory has been on developing an understanding of the excellent performance and stability of a lithium/disulfide cell using LiCl-LiBr-KBr electrolyte. For further improvement, we have initiated development of a rod-electrode cell design and design of cells which can tolerate overdischarge and overcharge abuse. Earlier Li/FeS2 cells offered performance quite below expectations and had high capacity decline rates: 0.10 to 0.25 percent per cycle. Approaches for reducing the capacity decline rates of the earlier cells also reduced cell performance. However, our improved Li/FeS2 cell tests indicate good prospects for attaining cell development goals of specific energy of 200 Wh/kg at a 4-h discharge rate, a specific power of 200 W/kg at 80 percent depth of discharge, and a cycle life of 1000 cycles.

  10. Raman Signatures of Polytypism in Molybdenum Disulfide.

    Science.gov (United States)

    Lee, Jae-Ung; Kim, Kangwon; Han, Songhee; Ryu, Gyeong Hee; Lee, Zonghoon; Cheong, Hyeonsik

    2016-02-23

    Since the stacking order sensitively affects various physical properties of layered materials, accurate determination of the stacking order is important for studying the basic properties of these materials as well as for device applications. Because 2H-molybdenum disulfide (MoS2) is most common in nature, most studies so far have focused on 2H-MoS2. However, we found that the 2H, 3R, and mixed stacking sequences exist in few-layer MoS2 exfoliated from natural molybdenite crystals. The crystal structures are confirmed by HR-TEM measurements. The Raman signatures of different polytypes are investigated by using three different excitation energies that are nonresonant and resonant with A and C excitons, respectively. The low-frequency breathing and shear modes show distinct differences for each polytype, whereas the high-frequency intralayer modes show little difference. For resonant excitations at 1.96 and 2.81 eV, distinct features are observed that enable determination of the stacking order.

  11. Parental Bonding

    Directory of Open Access Journals (Sweden)

    T. Paul de Cock

    2014-08-01

    Full Text Available Estimating the early parent–child bonding relationship can be valuable in research and practice. Retrospective dimensional measures of parental bonding provide a means for assessing the experience of the early parent–child relationship. However, combinations of dimensional scores may provide information that is not readily captured with a dimensional approach. This study was designed to assess the presence of homogeneous groups in the population with similar profiles on parental bonding dimensions. Using a short version of the Parental Bonding Instrument (PBI, three parental bonding dimensions (care, authoritarianism, and overprotection were used to assess the presence of unobserved groups in the population using latent profile analysis. The class solutions were regressed on 23 covariates (demographics, parental psychopathology, loss events, and childhood contextual factors to assess the validity of the class solution. The results indicated four distinct profiles of parental bonding for fathers as well as mothers. Parental bonding profiles were significantly associated with a broad range of covariates. This person-centered approach to parental bonding has broad utility in future research which takes into account the effect of parent–child bonding, especially with regard to “affectionless control” style parenting.

  12. Purification of correctly oxidized MHC class I heavy-chain molecules under denaturing conditions: a novel strategy exploiting disulfide assisted protein folding

    DEFF Research Database (Denmark)

    Ferré, Henrik; Ruffet, Emmanuel; Blicher, Thomas

    2003-01-01

    The aim of this study has been to develop a strategy for purifying correctly oxidized denatured major histocompability complex class I (MHC-I) heavy-chain molecules, which on dilution, fold efficiently and become functional. Expression of heavy-chain molecules in bacteria results in the formation...... of insoluble cellular inclusion bodies, which must be solubilized under denaturing conditions. Their subsequent purification and refolding is complicated by the fact that (1). correct folding can only take place in combined presence of beta(2)-microglobulin and a binding peptide; and (2). optimal in vitro...... conditions for disulfide bond formation ( approximately pH 8) and peptide binding ( approximately pH 6.6) are far from complementary. Here we present a two-step strategy, which relies on uncoupling the events of disulfide bond formation and peptide binding. In the first phase, heavy-chain molecules...

  13. Purification of correctly oxidized MHC class I heavy-chain molecules under denaturing conditions: A novel strategy exploiting disulfide assisted protein folding

    DEFF Research Database (Denmark)

    Ferré, Henrik; Ruffet, E.; Blicher, T.

    2003-01-01

    The aim of this study has been to develop a strategy for purifying correctly oxidized denatured major histocompability complex class I (MHC-I) heavy-chain molecules, which on dilution, fold efficiently and become functional. Expression of heavy-chain molecules in bacteria results in the formation...... of insoluble cellular inclusion bodies, which must be solubilized under denaturing conditions. Their subsequent purification and refolding is complicated by the fact that (1) correct folding can only take place in combined presence of beta(2)-microglobulin and a binding peptide; and (2) optimal in vitro...... conditions for disulfide bond formation (similar topH 8) and peptide binding (similar topH 6.6) are far from complementary. Here we present a two-step strategy, which relies on uncoupling the events of disulfide bond formation and peptide binding. In the first phase, heavy-chain molecules with correct...

  14. 11-Hydroxyundecyl octadecyl disulfide self-assembled monolayers on Au(1 1 1)

    Energy Technology Data Exchange (ETDEWEB)

    Albayrak, Erol [Department of Materials and Metallurgical Engineering, Ahi Evran University, Kırşehir 40000 (Turkey); Karabuga, Semistan [Department of Chemistry, Kahramanmaraş Sütçü İmam University, Kahramanmaraş 46030 (Turkey); Bracco, Gianangelo [CNR-IMEM and Department of Physics, University of Genoa, via Dodecaneso 33, Genoa 16146 (Italy); Danışman, M. Fatih, E-mail: danisman@metu.edu.tr [Department of Chemistry, Middle East Technical University, Ankara 06800 (Turkey)

    2014-08-30

    Highlights: • 11-Hydroxyundecyl octadecyl disulfide self-assembled monolayers on Au(1 1 1) surface were grown by supersonic molecular beam deposition. • Two different lying down monolayer phases were observed depending on the substrate temperature. • High temperature monolayer phase has a diffraction pattern similar to that of mercaptoundecanol SAMs. • Desorption from several different chemisorbed and physisorbed states were observed. - Abstract: Here, we report a helium atom diffraction study of 11-hydroxyundecyl octadecyl disulfide (CH{sub 3}-(CH{sub 2}){sub 17}-S-S-(CH{sub 2}){sub 11}-OH, HOD) self-assembled monolayers (SAMs) produced by supersonic molecular beam deposition (SMBD). Two different lying down monolayer phases were observed depending on the substrate temperature. At low temperatures a poorly ordered phase was observed, while the diffraction patterns of the film grown at high temperatures were similar to that of mercaptoundecanol (MUD) SAMs reported previously in the literature. The transition from the low temperature phase to the high temperature phase is due to S-S bond cleavage at the surface. Desorption from several different chemisorbed and physisorbed states were observed with energies in the same range as observed for MUD and octadecanelthiol (ODT) SAMs.

  15. Functional Role of the Disulfide Isomerase ERp57 in Axonal Regeneration.

    Directory of Open Access Journals (Sweden)

    Valentina Castillo

    Full Text Available ERp57 (also known as grp58 and PDIA3 is a protein disulfide isomerase that catalyzes disulfide bonds formation of glycoproteins as part of the calnexin and calreticulin cycle. ERp57 is markedly upregulated in most common neurodegenerative diseases downstream of the endoplasmic reticulum (ER stress response. Despite accumulating correlative evidence supporting a neuroprotective role of ERp57, the contribution of this foldase to the physiology of the nervous system remains unknown. Here we developed a transgenic mouse model that overexpresses ERp57 in the nervous system under the control of the prion promoter. We analyzed the susceptibility of ERp57 transgenic mice to undergo neurodegeneration. Unexpectedly, ERp57 overexpression did not affect dopaminergic neuron loss and striatal denervation after injection of a Parkinson's disease-inducing neurotoxin. In sharp contrast, ERp57 transgenic animals presented enhanced locomotor recovery after mechanical injury to the sciatic nerve. These protective effects were associated with enhanced myelin removal, macrophage infiltration and axonal regeneration. Our results suggest that ERp57 specifically contributes to peripheral nerve regeneration, whereas its activity is dispensable for the survival of a specific neuronal population of the central nervous system. These results demonstrate for the first time a functional role of a component of the ER proteostasis network in peripheral nerve regeneration.

  16. Multimolecular Salivary Mucin Complex Is Altered in Saliva of Cigarette Smokers: Detection of Disulfide Bridges by Raman Spectroscopy

    Directory of Open Access Journals (Sweden)

    Motoe Taniguchi

    2013-01-01

    Full Text Available Saliva contains mucins, which protect epithelial cells. We showed a smaller amount of salivary mucin, both MG1 and MG2, in the premenopausal female smokers than in their nonsmoking counterparts. Smokers' MG1, which contains almost 2% cysteine/half cystine in its amino acid residues, turned out to be chemically altered in the nonsmoker’s saliva. The smaller acidic glycoprotein bands were detectable only in smoker’s saliva in the range of 20–25 kDa and at 45 kDa, suggesting that degradation, at least in part, caused the reduction of MG1 mucin. This is in agreement with the previous finding that free radicals in cigarette smoke modify mucins in both sugar and protein moieties. Moreover, proteins such as amylase and albumin are bound to other proteins through disulfide bonds and are identifiable only after reduction with DTT. Confocal laser Raman microspectroscopy identified a disulfide stretch band of significantly stronger intensity per protein in the stimulated saliva of smokers alone. We conclude that the saliva of smokers, especially stimulated saliva, contains significantly more oxidized form of proteins with increased disulfide bridges, that reduces protection for oral epithelium. Raman microspectroscopy can be used for an easy detection of the damaged salivary proteins.

  17. Redox Reactivity of Cerium Oxide Nanoparticles Induces the Formation of Disulfide Bridges in Thiol-Containing Biomolecules.

    Science.gov (United States)

    Rollin-Genetet, Françoise; Seidel, Caroline; Artells, Ester; Auffan, Mélanie; Thiéry, Alain; Vidaud, Claude

    2015-12-21

    The redox state of disulfide bonds is implicated in many redox control systems, such as the cysteine-cystine couple. Among proteins, ubiquitous cysteine-rich metallothioneins possess thiolate metal binding groups susceptible to metal exchange in detoxification processes. CeO2 NPs are commonly used in various industrial applications due to their redox properties. These redox properties that enable dual oxidation states (Ce(IV)/Ce(III)) to exist at their surface may act as oxidants for biomolecules. The interaction among metallothioneins, cysteine, and CeO2 NPs was investigated through various biophysical approaches to shed light on the potential effects of the Ce(4+)/Ce(3+) redox system on the thiol groups of these biomolecules. The possible reaction mechanisms include the formation of a disulfide bridge/Ce(III) complex resulting from the interaction between Ce(IV) and the thiol groups, leading to metal unloading from the MTs, depending on their metal content and cluster type. The formation of stable Ce(3+) disulfide complexes has been demonstrated via their fluorescence properties. This work provides the first evidence of thiol concentration-dependent catalytic oxidation mechanisms between pristine CeO2 NPs and thiol-containing biomolecules.

  18. Thiol/disulfide redox states in signaling and sensing

    Science.gov (United States)

    Go, Young-Mi; Jones, Dean P.

    2015-01-01

    Rapid advances in redox systems biology are creating new opportunities to understand complexities of human disease and contributions of environmental exposures. New understanding of thiol-disulfide systems have occurred during the past decade as a consequence of the discoveries that thiol and disulfide systems are maintained in kinetically controlled steady-states displaced from thermodynamic equilibrium, that a widely distributed family of NADPH oxidases produces oxidants that function in cell signaling, and that a family of peroxiredoxins utilize thioredoxin as a reductant to complement the well-studied glutathione antioxidant system for peroxide elimination and redox regulation. This review focuses on thiol/disulfide redox state in biologic systems and the knowledge base available to support development of integrated redox systems biology models to better understand the function and dysfunction of thiol-disulfide redox systems. In particular, central principles have emerged concerning redox compartmentalization and utility of thiol/disulfide redox measures as indicators of physiologic function. Advances in redox proteomics show that, in addition to functioning in protein active sites and cell signaling, cysteine residues also serve as redox sensors to integrate biologic functions. These advances provide a framework for translation of redox systems biology concepts to practical use in understanding and treating human disease. Biological responses to cadmium, a widespread environmental agent, are used to illustrate the utility of these advances to the understanding of complex pleiotropic toxicities. PMID:23356510

  19. A molybdenum disulfide/carbon nanotube heterogeneous complementary inverter.

    Science.gov (United States)

    Huang, Jun; Somu, Sivasubramanian; Busnaina, Ahmed

    2012-08-24

    We report a simple, bottom-up/top-down approach for integrating drastically different nanoscale building blocks to form a heterogeneous complementary inverter circuit based on layered molybdenum disulfide and carbon nanotube (CNT) bundles. The fabricated CNT/MoS(2) inverter is composed of n-type molybdenum disulfide (MOS(2)) and p-type CNT transistors, with a high voltage gain of 1.3. The CNT channels are fabricated using directed assembly while the layered molybdenum disulfide channels are fabricated by mechanical exfoliation. This bottom-up fabrication approach for integrating various nanoscale elements with unique characteristics provides an alternative cost-effective methodology to complementary metal-oxide-semiconductors, laying the foundation for the realization of high performance logic circuits.

  20. Quantifying the global cellular thiol-disulfide status

    DEFF Research Database (Denmark)

    Hansen, Rosa E; Roth, Doris; Winther, Jakob R

    2009-01-01

    It is widely accepted that the redox status of protein thiols is of central importance to protein structure and folding and that glutathione is an important low-molecular-mass redox regulator. However, the total cellular pools of thiols and disulfides and their relative abundance have never been...... determined. In this study, we have assembled a global picture of the cellular thiol-disulfide status in cultured mammalian cells. We have quantified the absolute levels of protein thiols, protein disulfides, and glutathionylated protein (PSSG) in all cellular protein, including membrane proteins. These data...... cell types. However, when cells are exposed to a sublethal dose of the thiol-specific oxidant diamide, PSSG levels increase to >15% of all protein cysteine. Glutathione is typically characterized as the "cellular redox buffer"; nevertheless, our data show that protein thiols represent a larger active...

  1. Electrostatic influence of local cysteine environments on disulfide exchange kinetics.

    Science.gov (United States)

    Snyder, G H; Cennerazzo, M J; Karalis, A J; Field, D

    1981-11-10

    The ionic strength dependence of the bimolecular rate constant for reaction of the negative disulfide 5,5'-dithiobis (2-nitrobenzoic acid) with cysteines in fragments of naturally occurring proteins was determined by stopped-flow spectroscopy. The Debye-Hückel relationship was applied to determine the effective charge at the cysteine and thereby determine the extent to which nearby neighbors in the primary sequence influence the kinetics. Corrections for the secondary salt effect on cysteine pKs were determined by direct spectrometric pH titration of sulfhydryl groups or by observation of the ionic strength dependence of kinetics of cysteine reaction with the neutral disulfide 2,2'-dithiodipyridine. Quantitative expressions was verified by model studies with N-acetyl-cystein. At ionic strengths equal to or greater than 20 mM, the net charge at the polypeptide cysteine site is the sum of the single negative charge of the thiolate anion and the charges of the amino acids immediately preceding and following the cysteine in the primary sequence. At lower ionic strengths, more distant residues influence kinetics. At pH 7.0, 23 degree C, and an ionic strength of 20 mM, rate constants for reaction of the negative disulfide with a cysteine having two positive neighbors, one positive and one neutral neighbor, or two neutral neighbors are 132000, 3350, and 367 s-1 M-1, respectively. This corresponds to a contribution to the activation energy of 0.65- 1.1 kcal/mol per ion pair involved in collision between the cysteine and disulfide regions. The results permit the estimation that cysteine local environments may provide a means of achieving a 10(6)-fold range in rate constants in disulfide exchange reactions in random-coil proteins. This range may prove useful in developing strategies for directing disulfide pairing in synthetic proteins.

  2. Structures and related properties of helical, disulfide-stabilized peptides

    Energy Technology Data Exchange (ETDEWEB)

    Pagel, Mark D. [Univ. of California, Berkeley, CA (United States). Dept. of Chemistry

    1993-11-01

    The three dimensional structure of several peptides were determined by NMR spectroscopy and distance geometry calculations. Each peptide formed a predictable, rigid structure, consisting of an α-helix, a "scaffold" region which packed along one face of the helix, and two disulfide bridges which covalently connect the helix and scaffold regions. The peptide Apa-M5 was designed to constrain the M5 peptide from MLCK in a helical geometry using the apamin disulfide scaffold. This scaffold constrains the N- terminal end of the helix with two disulfide bridges and a reverse turn. Like the M5 peptide, Apa-M5 was found to bind calmodulin in a Ca2+-dependent 1:1 stoichiometry. However, the dissociation constant of the (Apa-M5)-calmodulin complex, 107 nM, was 100-fold higher than the dissociation constant of the M5-calmodulin complex. This difference was due to a putative steric overlap between the Apa-M5 scaffold and calmodulin. The peptide Apa-Cro was designed to replace the large structural protein matrix of λ Cro with the apamin disulfide scaffold. However, Apa-Cro did not bind the consensus DNA operator half-site of λ Cro, probably due to a steric overlap between the Apa-Cro disulfide framework and the DNA. The amino acid sequence of the scaffold-disulfide bridge arrangement of the peptide Max was derived from the core sequence of scyllatoxin, which contains an α-helix constrained at the C-terminal end by two disulfide bridges and a two-stranded βsheet scaffold. Max was shown to fold with >84% yield to form a predictable, stable structure that is similar to scyllatoxin. The folding and stability properties of Max make this scaffold and disulfide bridge arrangement an ideal candidate for the development of hybrid sequence peptides. The dynamics of a fraying C-terminal end of the helix of the peptide Apa-AlaN was determined by analysis of 15N NMR relaxation properties.

  3. Structural differences between glycosylated, disulfide-linked heterodimeric Knob-into-Hole Fc fragment and its homodimeric Knob-Knob and Hole-Hole side products.

    Science.gov (United States)

    Kuglstatter, A; Stihle, M; Neumann, C; Müller, C; Schaefer, W; Klein, C; Benz, J

    2017-09-01

    An increasing number of bispecific therapeutic antibodies are progressing through clinical development. The Knob-into-Hole (KiH) technology uses complementary mutations in the CH3 region of the antibody Fc fragment to achieve heavy chain heterodimerization. Here we describe the X-ray crystal structures of glycosylated and disulfide-engineered heterodimeric KiH Fc fragment and its homodimeric Knob-Knob and Hole-Hole side products. The heterodimer structure confirms the KiH design principle and supports the hypothesis that glycosylation stabilizes a closed Fc conformation. Both homodimer structures show parallel Fc fragment architectures, in contrast to recently reported crystal structures of the corresponding aglycosylated Fc fragments which in the absence of disulfide mutations show an unexpected antiparallel arrangement. The glycosylated Knob-Knob Fc fragment is destabilized as indicated by variability in the relative orientation of its CH3 domains. The glycosylated Hole-Hole Fc fragment shows an unexpected intermolecular disulfide bond via the introduced Y349C Hole mutation which results in a large CH3 domain shift and a new CH3-CH3 interface. The crystal structures of glycosylated, disulfide-linked KiH Fc fragment and its Knob-Knob and Hole-Hole side products reported here will facilitate further design of highly efficient antibody heterodimerization strategies. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  4. Radioiodine-labeled disulfide: a novel radiotracer for evaluation of tumor uptake

    Energy Technology Data Exchange (ETDEWEB)

    Ryu, E. K.; Choi, Y. S.; Byun, S. S.; Baek, J. Y.; Lee, K. H.; Kim, S. E.; Choi, Y.; Kim, B. T. [Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of)

    2002-07-01

    Diallyl disulfide found in garlic has been known to inhibit the growth of various cancer cells. In this study, iodine-substituted disulfides were synthesized and their growth inhibitory effects on cancer cells (SUN C5 and MCF-7) were investigated. Dibenzyl disulfide was labeled with {sup 123}I/{sup 125}I for evaluation of tumor uptake. Halogen-substituted disulfides were synthesized using 2,2'-dithiobis(benzothiazole) and one equivalent each of the corresponding thiols. Growth inhibition studies were performed on cancer cells that were grown at 37 .deg. C for 48 hr prior to exposure to the disulfides. Radioiodine-labeled disulfide was prepared by halogen exchange reaction on the 4-bromodibenzyl disulfide in the presence of Na{sup 123}I/{sup 125}I and CuCl at 150 .deg. C for 60 min, followed by HPLC purification. Uptake of the radioactivity to SUN C5 cells was measured as a function of time, and inhibition studies were performed in the presence of either S-methyl methanethiosulfonate (MMTS) or diallyl disulfide. Disulfides were synthesized in the high yields (90%). Tumor growth inhibition studies by the 3 iododisulfides showed the inhibition (>95%) comparable to diallyl disulfide (100%). Cu(I)-assisted radioiodination gave 4-{sup 123}I/{sup 125}I-iododibenzyl disulfide in overall 30-40% radiochemical yield and with high specific activity. Cell uptake studies of the radiolabeled disulfide showed a time-dependent increase of the uptake (4-fold increase from 15 min to 2 hr). Both MMTS, a glutathione depleting agent, and diallyl disulfide reduced the uptake of the radioactivity in a dose-dependent manner. Inhibition studies suggest that uptake of disulfide to the tumor cells could be mediated by thiol-disulfide exchange. This study demonstrates that radioiodine-labeled dibenzyl disulfide may be useful for evaluation of tumor uptake.

  5. Electrical Transport Properties of Polycrystalline Monolayer Molybdenum Disulfide

    Science.gov (United States)

    2014-07-14

    Lou, Sina Najmaei, Matin Amani, Matthew L. Chin, Zheng Se. TASK NUMBER Liu Sf. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAMES AND ADDRESSES 8...Transport Properties of Polycrystalline Monolayer Molybdenum Disulfide Sina Najmaei,t.§ Matin Ama ni,M Matthew L. Chin,* Zhe ng liu/ ·"·v: A. Gle n

  6. Impaired Thiol-Disulfide Balance in Acute Brucellosis.

    Science.gov (United States)

    Kolgelier, Servet; Ergin, Merve; Demir, Lutfi Saltuk; Inkaya, Ahmet Cagkan; Aktug Demir, Nazlim; Alisik, Murat; Erel, Ozcan

    2017-05-24

    The objective of this study was to examine a novel profile: thiol-disulfide homeostasis in acute brucellosis. The study included 90 patients with acute brucellosis, and 27 healthy controls. Thiol-disulfide profile tests were analyzed by a recently developed method, and ceruloplasmin levels were determined. Native thiol levels were 256.72 ± 48.20 μmol/L in the acute brucellosis group and 461.13 ± 45.37 μmol/L in the healthy group, and total thiol levels were 298.58 ± 51.78 μmol/L in the acute brucellosis group and 504.83 ± 51.05 μmol/L in the healthy group (p brucellosis than in the healthy controls (p brucellosis. The strong associations between thiol-disulfide parameters and a positive acute-phase reactant reflected the disruption of the balance between the antioxidant and oxidant systems. Since thiol groups act as anti-inflammatory mediators, the alteration in the thiol-disulfide homeostasis may be involved in brucellosis.

  7. Band engineering in twisted molybdenum disulfide bilayers

    Science.gov (United States)

    Zhao, Yipeng; Liao, Chengwei; Ouyang, Gang

    2018-05-01

    In order to explore the theoretical relationship between interlayer spacing, interaction and band offset at the atomic level in vertically stacked two-dimensional (2D) van der Waals (vdW) structures, we propose an analytical model to address the evolution of interlayer vdW coupling with random stacking configurations in MoS2 bilayers based on the atomic-bond-relaxation correlation mechanism. We found that interlayer spacing changes substantially with respect to the orientations, and the bandgap increases from 1.53 eV (AB stacking) to 1.68 eV (AA stacking). Our results reveal that the evolution of interlayer vdW coupling originates from the interlayer interaction, leading to interlayer separations and electronic properties changing with stacking configurations. Our predictions constitute a demonstration of twist engineering the band shift in the emergent class of 2D crystals, transition-metal dichalcogenides.

  8. Transpeptidase activity of penicillin-binding protein SpoVD in peptidoglycan synthesis conditionally depends on the disulfide reductase StoA.

    Science.gov (United States)

    Bukowska-Faniband, Ewa; Hederstedt, Lars

    2017-07-01

    Endospore cortex peptidoglycan synthesis is not required for bacterial growth but essential for endospore heat resistance. It therefore constitutes an amenable system for research on peptidoglycan biogenesis. The Bacillus subtilis sporulation-specific class B penicillin-binding protein (PBP) SpoVD and many homologous PBPs contain two conserved cysteine residues of unknown function in the transpeptidase domain - one as residue x in the SxN catalytic site motif and the other in a flexible loop near the catalytic site. A disulfide bond between these residues blocks the function of SpoVD in cortex synthesis. With a combination of experiments with purified proteins and B. subtilis mutant cells, it was shown that in active SpoVD the two cysteine residues most probably interact by hydrogen bonding and that this is important for peptidoglycan synthesis in vivo. It was furthermore demonstrated that the sporulation-specific thiol-disulfide oxidoreductase StoA reduces SpoVD and that requirement of StoA for cortex synthesis can be suppressed by two completely different types of structural alterations in SpoVD. It is concluded that StoA plays a critical role mainly during maturation of SpoVD in the forespore outer membrane. The findings advance our understanding of essential PBPs and redox control of extra-cytoplasmic protein disulfides in bacterial cells. © 2017 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.

  9. Bonds Boom.

    Science.gov (United States)

    Reynolds, Cathryn

    1989-01-01

    The combined effect of the "Serrano" decision and Proposition 13 left California school districts with aging, overcrowded facilities. Chico schools won a $18.5 million general obligation bond election for facilities construction. With $11 billion needed for new school construction, California will need to tap local sources. A sidebar…

  10. Disulfide mapping the voltage-sensing mechanism of a voltage-dependent potassium channel.

    Science.gov (United States)

    Nozaki, Tomohiro; Ozawa, Shin-Ichiro; Harada, Hitomi; Kimura, Tomomi; Osawa, Masanori; Shimada, Ichio

    2016-11-17

    Voltage-dependent potassium (Kv) channels allow for the selective permeability of potassium ions in a membrane potential dependent manner, playing crucial roles in neurotransmission and muscle contraction. Kv channel is a tetramer, in which each subunit possesses a voltage-sensing domain (VSD) and a pore domain (PD). Although several lines of evidence indicated that membrane depolarization is sensed as the movement of helix S4 of the VSD, the detailed voltage-sensing mechanism remained elusive, due to the difficulty of structural analyses at resting potential. In this study, we conducted a comprehensive disulfide locking analysis of the VSD using 36 double Cys mutants, in order to identify the proximal residue pairs of the VSD in the presence or absence of a membrane potential. An intramolecular SS-bond was formed between 6 Cys pairs under both polarized and depolarized environment, and one pair only under depolarized environment. The multiple conformations captured by the SS-bond can be divided by two states, up and down, where S4 lies on the extracellular and intracellular sides of the membrane, respectively, with axial rotation of 180°. The transition between these two states is caused by the S4 translocation of 12 Å, enabling allosteric regulation of the gating at the PD.

  11. Identification, activity and disulfide connectivity of C-di-GMP regulating proteins in Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Kajal Gupta

    2010-11-01

    Full Text Available C-di-GMP, a bacterial second messenger plays a key role in survival and adaptation of bacteria under different environmental conditions. The level of c-di-GMP is regulated by two opposing activities, namely diguanylate cyclase (DGC and phosphodiesterase (PDE-A exhibited by GGDEF and EAL domain, respectively in the same protein. Previously, we reported a bifunctional GGDEF-EAL domain protein, MSDGC-1 from Mycobacterium smegmatis showing both these activities (Kumar and Chatterji, 2008. In this current report, we have identified and characterized the homologous protein from Mycobacterium tuberculosis (Rv 1354c named as MtbDGC. MtbDGC is also a bifunctional protein, which can synthesize and degrade c-di-GMP in vitro. Further we expressed Mtbdgc in M. smegmatis and it was able to complement the MSDGC-1 knock out strain by restoring the long term survival of M. smegmatis. Another protein Rv 1357c, named as MtbPDE, is an EAL domain protein and degrades c-di-GMP to pGpG in vitro. Rv1354c and 1357c have seven cysteine amino acids in their sequence, distributed along the full length of the protein. Disulfide bonds play an important role in stabilizing protein structure and regulating protein function. By proteolytic digestion and mass spectrometric analysis of MtbDGC, connectivity between cysteine pairs Cys94-Cys584, Cys2-Cys479 and Cys429-Cys614 was determined, whereas the third cysteine (Cys406 from N terminal was found to be free in MtbDGC protein, which was further confirmed by alkylation with iodoacetamide labeling. Bioinformatics modeling investigations also supported the pattern of disulfide connectivity obtained by Mass spectrometric analysis. Cys406 was mutated to serine by site directed mutagenesis and the mutant MtbC406S was not found to be active and was not able to synthesize or degrade c-di-GMP. The disulfide connectivity established here would help further in understanding the structure - function relationship in MtbDGC.

  12. Disulfide Bond-Containing Ajoene Analogues As Novel Quorum Sensing Inhibitors of Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Fong, July; Yuan, Mingjun; Jakobsen, Tim Holm

    2017-01-01

    Since its discovery 22 years ago, the bacterial cell-to-cell communication system, termed quorum sensing (QS), has shown potential as antipathogenic target. Previous studies reported that ajoene from garlic inhibits QS in opportunistic human pathogen Pseudomonas aeruginosa. In this study, screening...

  13. Radiolytic Reduction Characteristics of Artificial Oligodeoxynucleotides Possessing 2-Oxoalkyl Group or Disulfide Bonds

    Directory of Open Access Journals (Sweden)

    Kazuhito Tanabe

    2011-01-01

    Full Text Available A number of advances have been made in the development of modified oligodeoxynucleotides (ODNs, and chemical or physical properties of which are controlled by external stimuli. These intelligent ODNs are promising for the next generation of gene diagnostics and therapy. This paper focuses on the molecular design of artificial ODNs that are activated by X-irradiation and their applications to regulation of hybridization properties, conformation change, radiation-activated DNAzyme, and decoy molecules.

  14. Diffusion bonding

    International Nuclear Information System (INIS)

    Anderson, R.C.

    1976-01-01

    A method is described for joining beryllium to beryllium by diffusion bonding. At least one surface portion of at least two beryllium pieces is coated with nickel. A coated surface portion is positioned in a contiguous relationship with another surface portion and subjected to an environment having an atmosphere at a pressure lower than ambient pressure. A force is applied on the beryllium pieces for causing the contiguous surface portions to abut against each other. The contiguous surface portions are heated to a maximum temperature less than the melting temperature of the beryllium, and the applied force is decreased while increasing the temperature after attaining a temperature substantially above room temperature. A portion of the applied force is maintained at a temperature corresponding to about maximum temperature for a duration sufficient to effect the diffusion bond between the contiguous surface portions

  15. Photodegradable, Photoadaptable Hydrogels via Radical-Mediated Disulfide Fragmentation Reaction.

    Science.gov (United States)

    Fairbanks, Benjamin D; Singh, Samir P; Bowman, Christopher N; Anseth, Kristi S

    2011-04-26

    Various techniques have been adopted to impart a biological responsiveness to synthetic hydrogels for the delivery of therapeutic agents as well as the study and manipulation of biological processes and tissue development. Such techniques and materials include polyelectrolyte gels that swell and deswell with changes in pH, thermosensitive gels that contract at physiological temperatures, and peptide cross-linked hydrogels that degrade upon peptidolysis by cell-secreted enzymes. Herein we report a unique approach to photochemically deform and degrade disulfide cross-linked hydrogels, mitigating the challenges of light attenuation and low quantum yield, permitting the degradation of hydrogels up to 2 mm thick within 120 s at low light intensities (10 mW/cm(2) at 365 nm). Hydrogels were formed by the oxidation of thiol-functionalized 4-armed poly(ethylene glycol) macromolecules. These disulfide cross-linked hydrogels were then swollen in a lithium acylphosphinate photoinitiator solution. Upon exposure to light, photogenerated radicals initiate multiple fragmentation and disulfide exchange reactions, permitting and promoting photodeformation, photowelding, and photodegradation. This novel, but simple, approach to generate photoadaptable hydrogels portends the study of cellular response to mechanically and topographically dynamic substrates as well as novel encapsulations by the welding of solid substrates. The principles and techniques described herein hold implications for more than hydrogel materials but also for photoadaptable polymers more generally.

  16. Preliminary crystallographic data of the three homologues of the thiol–disulfide oxidoreductase DsbA in Neisseria meningitidis

    Energy Technology Data Exchange (ETDEWEB)

    Lafaye, Céline [Laboratoire des Protéines Membranaires, Institut de Biologie Structurale, CEA/CNRS/Université Joseph Fourier, 41 Rue Jules Horowitz, 38027 Grenoble CEDEX 01 (France); Iwena, Thomas; Ferrer, Jean-Luc [Laboratoire de Cristallogénèse et Cristallisation des Protéines, Institut de Biologie Structurale, CEA/CNRS/Université Joseph Fourier, 41 Rue Jules Horowitz, 38027 Grenoble CEDEX 01 (France); Kroll, J. Simon [Department of Paediatrics, Imperial College London, St Mary’s Hospital Campus, Norfolk Place, London W2 1PG (United Kingdom); Griat, Mickael; Serre, Laurence, E-mail: laurence.serre@ibs.fr [Laboratoire des Protéines Membranaires, Institut de Biologie Structurale, CEA/CNRS/Université Joseph Fourier, 41 Rue Jules Horowitz, 38027 Grenoble CEDEX 01 (France)

    2008-02-01

    The Neisseria meningitidis genome possesses three genes encoding active DsbAs. To throw light on the reason for this genetic multiplicity, the three enzymes have been purified and crystallized. Bacterial virulence depends on the correct folding of surface-exposed proteins, a process that is catalyzed by the thiol-disulfide oxidoreductase DsbA, which facilitates the synthesis of disulfide bonds in Gram-negative bacteria. Uniquely among bacteria, the Neisseria meningitidis genome possesses three genes encoding active DsbAs: DsbA1, DsbA2 and DsbA3. DsbA1 and DsbA2 have been characterized as lipoproteins involved in natural competence and in host-interactive biology, while the function of DsbA3 remains unknown. In an attempt to shed light on the reason for this multiplicity of dsbA genes, the three enzymes from N. meningitidis have been purified and crystallized in the presence of high concentrations of ammonium sulfate. The best crystals were obtained using DsbA1 and DsbA3; they belong to the orthorhombic and tetragonal systems and diffract to 1.5 and 2.7 Å resolution, respectively.

  17. Modification of molybdenum disulfide in methanol solvent for hydrogen evolution reaction

    Science.gov (United States)

    Niyitanga, Theophile; Jeong, Hae Kyung

    2018-05-01

    Molybdenum disulfide is a promising catalyst to replace the expensive platinum as an electrocatalyst but needs to be modified to present excellent electrocatalytic properties. Herein, we successfully modify molybdenum disulfide in methanol solvent for hydrogen evolution reaction by using a simple hydrothermal method. Overpotential reduced to -0.6 V from -1.5 V, and energy band gap decreased from 1.73 eV to 1.58 eV after the modification. The modified molybdenum disulfide also demonstrated lower resistance (42 Ω) at high frequency (1000 kHz) compared with that (240 Ω) of the precursor, showing that conductivity of the modified molybdenum disulfide has improved.

  18. Functional and structural studies of the disulfide isomerase DsbC from the plant pathogen Xylella fastidiosa reveals a redox-dependent oligomeric modulation in vitro.

    Science.gov (United States)

    Santos, Clelton A; Toledo, Marcelo A S; Trivella, Daniela B B; Beloti, Lilian L; Schneider, Dilaine R S; Saraiva, Antonio M; Crucello, Aline; Azzoni, Adriano R; Souza, Alessandra A; Aparicio, Ricardo; Souza, Anete P

    2012-10-01

    Xylella fastidiosa is a Gram-negative bacterium that grows as a biofilm inside the xylem vessels of susceptible plants and causes several economically relevant crop diseases. In the present study, we report the functional and low-resolution structural characterization of the X. fastidiosa disulfide isomerase DsbC (XfDsbC). DsbC is part of the disulfide bond reduction/isomerization pathway in the bacterial periplasm and plays an important role in oxidative protein folding. In the present study, we demonstrate the presence of XfDsbC during different stages of X. fastidiosa biofilm development. XfDsbC was not detected during X. fastidiosa planktonic growth; however, after administering a sublethal copper shock, we observed an overexpression of XfDsbC that also occurred during planktonic growth. These results suggest that X. fastidiosa can use XfDsbC in vivo under oxidative stress conditions similar to those induced by copper. In addition, using dynamic light scattering and small-angle X-ray scattering, we observed that the oligomeric state of XfDsbC in vitro may be dependent on the redox environment. Under reducing conditions, XfDsbC is present as a dimer, whereas a putative tetrameric form was observed under nonreducing conditions. Taken together, our findings demonstrate the overexpression of XfDsbC during biofilm formation and provide the first structural model of a bacterial disulfide isomerase in solution. © 2012 The Authors Journal compilation © 2012 FEBS.

  19. An Internal Disulfide Locks a Misfolded Aggregation-prone Intermediate in Cataract-linked Mutants of Human γD-Crystallin.

    Science.gov (United States)

    Serebryany, Eugene; Woodard, Jaie C; Adkar, Bharat V; Shabab, Mohammed; King, Jonathan A; Shakhnovich, Eugene I

    2016-09-02

    Considerable mechanistic insight has been gained into amyloid aggregation; however, a large number of non-amyloid protein aggregates are considered "amorphous," and in most cases, little is known about their mechanisms. Amorphous aggregation of γ-crystallins in the eye lens causes cataract, a widespread disease of aging. We combined simulations and experiments to study the mechanism of aggregation of two γD-crystallin mutants, W42R and W42Q: the former a congenital cataract mutation, and the latter a mimic of age-related oxidative damage. We found that formation of an internal disulfide was necessary and sufficient for aggregation under physiological conditions. Two-chain all-atom simulations predicted that one non-native disulfide in particular, between Cys(32) and Cys(41), was likely to stabilize an unfolding intermediate prone to intermolecular interactions. Mass spectrometry and mutagenesis experiments confirmed the presence of this bond in the aggregates and its necessity for oxidative aggregation under physiological conditions in vitro Mining the simulation data linked formation of this disulfide to extrusion of the N-terminal β-hairpin and rearrangement of the native β-sheet topology. Specific binding between the extruded hairpin and a distal β-sheet, in an intermolecular chain reaction similar to domain swapping, is the most probable mechanism of aggregate propagation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. An Internal Disulfide Locks a Misfolded Aggregation-prone Intermediate in Cataract-linked Mutants of Human γD-Crystallin*

    Science.gov (United States)

    Serebryany, Eugene; Woodard, Jaie C.; Adkar, Bharat V.; Shabab, Mohammed; King, Jonathan A.; Shakhnovich, Eugene I.

    2016-01-01

    Considerable mechanistic insight has been gained into amyloid aggregation; however, a large number of non-amyloid protein aggregates are considered “amorphous,” and in most cases, little is known about their mechanisms. Amorphous aggregation of γ-crystallins in the eye lens causes cataract, a widespread disease of aging. We combined simulations and experiments to study the mechanism of aggregation of two γD-crystallin mutants, W42R and W42Q: the former a congenital cataract mutation, and the latter a mimic of age-related oxidative damage. We found that formation of an internal disulfide was necessary and sufficient for aggregation under physiological conditions. Two-chain all-atom simulations predicted that one non-native disulfide in particular, between Cys32 and Cys41, was likely to stabilize an unfolding intermediate prone to intermolecular interactions. Mass spectrometry and mutagenesis experiments confirmed the presence of this bond in the aggregates and its necessity for oxidative aggregation under physiological conditions in vitro. Mining the simulation data linked formation of this disulfide to extrusion of the N-terminal β-hairpin and rearrangement of the native β-sheet topology. Specific binding between the extruded hairpin and a distal β-sheet, in an intermolecular chain reaction similar to domain swapping, is the most probable mechanism of aggregate propagation. PMID:27417136

  1. Molecular Docking Simulation of Neuraminidase Influenza A Subtype H1N1 with Potential Inhibitor of Disulfide Cyclic Peptide (DNY, NNY, LRL)

    Science.gov (United States)

    Putra, R. P.; Imaniastuti, R.; Nasution, M. A. F.; Kerami, Djati; Tambunan, U. S. F.

    2018-04-01

    Oseltamivir resistance as an inhibitor of neuraminidase influenza A virus subtype H1N1 has been reported lately. Therefore, to solve this problem, several kinds of research has been conducted to design and discover disulfide cyclic peptide ligands through molecular docking method, to find the potential inhibitors for neuraminidase H1N1 which then can disturb the virus replication. This research was studied and evaluated the interaction of ligands toward enzyme using molecular docking simulation, which was performed on three disulfide cyclic peptide inhibitors (DNY, LRL, and NNT), along with oseltamivir and zanamivir as the standard ligands using MOE 2008.10 software. The docking simulation shows that all disulfide cyclic peptide ligands have lower Gibbs free binding energies (ΔGbinding) than the standard ligands, with DNY ligand has the lowest ΔGbinding at -7.8544 kcal/mol. Furthermore, these ligands were also had better molecular interactions with neuraminidase than the standards, owing by the hydrogen bonds that were formed during the docking simulation. In the end, we concluded that DNY, LRL and NNT ligands have the potential to be developed as the inhibitor of neuraminidase H1N1.

  2. Oxidation of the N-terminal domain of the wheat metallothionein Ec -1 leads to the formation of three distinct disulfide bridges.

    Science.gov (United States)

    Tarasava, Katsiaryna; Chesnov, Serge; Freisinger, Eva

    2016-05-01

    Metallothioneins (MTs) are low molecular weight proteins, characterized by a high cysteine content and the ability to coordinate large amounts of d(10) metal ions, for example, Zn(II), Cd(II), and Cu(I), in form of metal-thiolate clusters. Depending on intracellular conditions such as redox potential or metal ion concentrations, MTs can occur in various states ranging from the fully metal-loaded holo- to the metal-free apo-form. The Cys thiolate groups in the apo-form can be either reduced or be involved in disulfide bridges. Although oxidation-mediated Zn(II) release might be a possible mechanism for the regulation of Zn(II) availability by MTs, no concise information regarding the associated pathways and the structure of oxidized apo-MT forms is available. Using the well-studied Zn2 γ-Ec -1 domain of the wheat Zn6 Ec -1 MT we attempt here to answer several question regarding the structure and biophysical properties of oxidized MT forms, such as: (1) does disulfide bond formation increase the stability against proteolysis, (2) is the overall peptide backbone fold similar for the holo- and the oxidized apo-MT form, and (3) are disulfide bridges specifically or randomly formed? Our investigations show that oxidation leads to three distinct disulfide bridges independently of the applied oxidation conditions and of the initial species used for oxidation, that is, the apo- or the holo-form. In addition, the oxidized apo-form is as stable against proteolysis as Zn2 γ-Ec -1, rendering the currently assumed degradation of oxidized MTs unlikely and suggesting a role of the oxidation process for the extension of protein lifetime in absence of sufficient amounts of metal ions. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 295-308, 2016. © 2016 Wiley Periodicals, Inc.

  3. Bond strength of masonry

    NARCIS (Netherlands)

    Pluijm, van der R.; Vermeltfoort, A.Th.

    1992-01-01

    Bond strength is not a well defined property of masonry. Normally three types of bond strength can be distinguished: - tensile bond strength, - shear (and torsional) bond strength, - flexural bond strength. In this contribution the behaviour and strength of masonry in deformation controlled uniaxial

  4. Selenocysteine in thiol/disulfide-like exchange reactions.

    Science.gov (United States)

    Hondal, Robert J; Marino, Stefano M; Gladyshev, Vadim N

    2013-05-01

    Among trace elements used as cofactors in enzymes, selenium is unique in that it is incorporated into proteins co-translationally in the form of an amino acid, selenocysteine (Sec). Sec differs from cysteine (Cys) by only one atom (selenium versus sulfur), yet this switch dramatically influences important aspects of enzyme reactivity. The main focus of this review is an updated and critical discussion on how Sec might be used to accelerate thiol/disulfide-like exchange reactions in natural selenoenzymes, compared with their Cys-containing homologs. We discuss in detail three major aspects associated with thiol/disulfide exchange reactions: (i) nucleophilicity of the attacking thiolate (or selenolate); (ii) electrophilicity of the center sulfur (or selenium) atom; and (iii) stability of the leaving group (sulfur or selenium). In all these cases, we analyze the benefits that selenium might provide in these types of reactions. It is the biological thiol oxidoreductase-like function that benefits from the use of Sec, since Sec functions to chemically accelerate the rate of these reactions. We review various hypotheses that could help explain why Sec is used in enzymes, particularly with regard to competitive chemical advantages provided by the presence of the selenium atom in enzymes. Ultimately, these chemical advantages must be connected to biological functions of Sec.

  5. The Chemistry of Alk-1-yn-1-yl DisulfidesA Review

    DEFF Research Database (Denmark)

    Senning, Alexander Erich Eugen

    2009-01-01

    The preparation and the properties of the elusive alk-1-yn-1-yl disulfides are reviewed, including the most recent quantum chemical findings with regard to their reactivity.......The preparation and the properties of the elusive alk-1-yn-1-yl disulfides are reviewed, including the most recent quantum chemical findings with regard to their reactivity....

  6. Regulation of interleukin-4 signaling by extracellular reduction of intramolecular disulfides

    International Nuclear Information System (INIS)

    Curbo, Sophie; Gaudin, Raphael; Carlsten, Mattias; Malmberg, Karl-Johan; Troye-Blomberg, Marita; Ahlborg, Niklas; Karlsson, Anna; Johansson, Magnus; Lundberg, Mathias

    2009-01-01

    Interleukin-4 (IL-4) contains three structurally important intramolecular disulfides that are required for the bioactivity of the cytokine. We show that the cell surface of HeLa cells and endotoxin-activated monocytes can reduce IL-4 intramolecular disulfides in the extracellular space and inhibit binding of IL-4 to the IL-4Rα receptor. IL-4 disulfides were in vitro reduced by thioredoxin 1 (Trx1) and protein disulfide isomerase (PDI). Reduction of IL-4 disulfides by the cell surface of HeLa cells was inhibited by auranofin, an inhibitor of thioredoxin reductase that is an electron donor to both Trx1 and PDI. Both Trx1 and PDI have been shown to be located at the cell surface and our data suggests that these enzymes are involved in catalyzing reduction of IL-4 disulfides. The pro-drug N-acetylcysteine (NAC) that promotes T-helper type 1 responses was also shown to mediate the reduction of IL-4 disulfides. Our data provides evidence for a novel redox dependent pathway for regulation of cytokine activity by extracellular reduction of intramolecular disulfides at the cell surface by members of the thioredoxin enzyme family.

  7. Inhibition of carbon disulfide on bio-desulfurization in the process of ...

    African Journals Online (AJOL)

    Biological desulfurization is a novel technology for the removal of hydrogen sulfide from some biogas or sour gas, in which there are always a certain amounts of carbon disulfide together with much hydrogen sulfide. Nowadays, carbon disulfide is found to have negative effect on the biological desulfurization, but seldom ...

  8. Molecular characterization and analysis of a novel protein disulfide isomerase-like protein of Eimeria tenella.

    Science.gov (United States)

    Han, Hongyu; Dong, Hui; Zhu, Shunhai; Zhao, Qiping; Jiang, Lianlian; Wang, Yange; Li, Liujia; Wu, Youlin; Huang, Bing

    2014-01-01

    Protein disulfide isomerase (PDI) and PDI-like proteins are members of the thioredoxin superfamily. They contain thioredoxin-like domains and catalyze the physiological oxidation, reduction and isomerization of protein disulfide bonds, which are involved in cell function and development in prokaryotes and eukaryotes. In this study, EtPDIL, a novel PDI-like gene of Eimeria tenella, was cloned using rapid amplification of cDNA ends (RACE) according to the expressed sequence tag (EST). The EtPDIL cDNA contained 1129 nucleotides encoding 216 amino acids. The deduced EtPDIL protein belonged to thioredoxin-like superfamily and had a single predicted thioredoxin domain with a non-classical thioredoxin-like motif (SXXC). BLAST analysis showed that the EtPDIL protein was 55-59% identical to PDI-like proteins of other apicomplexan parasites. The transcript and protein levels of EtPDIL at different development stages were investigated by real-time quantitative PCR and western blot. The messenger RNA and protein levels of EtPDIL were higher in sporulated oocysts than in unsporulated oocysts, sporozoites or merozoites. Protein expression was barely detectable in unsporulated oocysts. Western blots showed that rabbit antiserum against recombinant EtPDIL recognized only a native 24 kDa protein from parasites. Immunolocalization with EtPDIL antibody showed that EtPDIL had a disperse distribution in the cytoplasm of whole sporozoites and merozoites. After sporozoites were incubated in complete medium, EtPDIL protein concentrated at the anterior of the sporozoites and appeared on the surface of parasites. Specific staining was more intense and mainly located on the parasite surface after merozoites released from mature schizonts invaded DF-1 cells. After development of parasites in DF-1 cells, staining intensified in trophozoites, immature schizonts and mature schizonts. Antibody inhibition of EtPDIL function reduced the ability of E. tenella to invade DF-1 cells. These results

  9. Hydrothermal Synthesis of Disulfide-Containing Uranyl Compounds. In Situ Ligand Synthesis versus Direct Assembly

    Energy Technology Data Exchange (ETDEWEB)

    Rowland, Clare E. [George Washington Univ., Washington, DC (United States); Belai, Nebebech [George Washington Univ., Washington, DC (United States); Knope, Karah E. [George Washington Univ., Washington, DC (United States); Cahill, Christopher L. [George Washington Univ., Washington, DC (United States)

    2010-01-29

    Three disulfide-containing uranyl compounds, [UO2(C7H4O2S)3]·H2O (1), [UO2(C7H4O2S)2(C7H5O2S)] (2), and [UO2(C7H4O2S)4] (3) have been hydrothermally synthesized. Both in situ disulfide bond formation from 3- and 4-mercaptobenzoic acid (C7H5O2S, MBA) to yield 3,3'- and 4,4'-dithiobisbenzoic acid (C14H8O4S2, DTBA) and direct assembly with the presynthesized dimeric ligands have been explored. While the starting materials 4-MBA and 4,4'-DTBA both yield 2 via in situ ligand synthesis and direct assembly, respectively, we observe the formation of 1 from the starting material 3-MBA via in situ ligand synthesis and of 3 from the direct assembly of the uranyl cation with 3,3'-DTBA. Concurrently with the synthesis of 1 and 2, we have observed the in situ formation of the crystalline dimeric organic species, 3,3'-DTBA, [(C7H5O2S)2] (4) and 4,4'-DTBA, [(C7H5O2S)2] (5). Herein we report the synthesis and crystallographic characterization of 1-5, as well as observations regarding the utility of product formation via direct assembly and in situ ligand synthesis.

  10. Molecular characterization and analysis of a novel protein disulfide isomerase-like protein of Eimeria tenella.

    Directory of Open Access Journals (Sweden)

    Hongyu Han

    Full Text Available Protein disulfide isomerase (PDI and PDI-like proteins are members of the thioredoxin superfamily. They contain thioredoxin-like domains and catalyze the physiological oxidation, reduction and isomerization of protein disulfide bonds, which are involved in cell function and development in prokaryotes and eukaryotes. In this study, EtPDIL, a novel PDI-like gene of Eimeria tenella, was cloned using rapid amplification of cDNA ends (RACE according to the expressed sequence tag (EST. The EtPDIL cDNA contained 1129 nucleotides encoding 216 amino acids. The deduced EtPDIL protein belonged to thioredoxin-like superfamily and had a single predicted thioredoxin domain with a non-classical thioredoxin-like motif (SXXC. BLAST analysis showed that the EtPDIL protein was 55-59% identical to PDI-like proteins of other apicomplexan parasites. The transcript and protein levels of EtPDIL at different development stages were investigated by real-time quantitative PCR and western blot. The messenger RNA and protein levels of EtPDIL were higher in sporulated oocysts than in unsporulated oocysts, sporozoites or merozoites. Protein expression was barely detectable in unsporulated oocysts. Western blots showed that rabbit antiserum against recombinant EtPDIL recognized only a native 24 kDa protein from parasites. Immunolocalization with EtPDIL antibody showed that EtPDIL had a disperse distribution in the cytoplasm of whole sporozoites and merozoites. After sporozoites were incubated in complete medium, EtPDIL protein concentrated at the anterior of the sporozoites and appeared on the surface of parasites. Specific staining was more intense and mainly located on the parasite surface after merozoites released from mature schizonts invaded DF-1 cells. After development of parasites in DF-1 cells, staining intensified in trophozoites, immature schizonts and mature schizonts. Antibody inhibition of EtPDIL function reduced the ability of E. tenella to invade DF-1 cells

  11. A novel disulfide-rich protein motif from avian eggshell membranes.

    Directory of Open Access Journals (Sweden)

    Vamsi K Kodali

    2011-03-01

    Full Text Available Under the shell of a chicken egg are two opposed proteinaceous disulfide-rich membranes. They are fabricated in the avian oviduct using fibers formed from proteins that are extensively coupled by irreversible lysine-derived crosslinks. The intractability of these eggshell membranes (ESM has slowed their characterization and their protein composition remains uncertain. In this work, reductive alkylation of ESM followed by proteolytic digestion led to the identification of a cysteine rich ESM protein (abbreviated CREMP that was similar to spore coat protein SP75 from cellular slime molds. Analysis of the cysteine repeats in partial sequences of CREMP reveals runs of remarkably repetitive patterns. Module a contains a C-X(4-C-X(5-C-X(8-C-X(6 pattern (where X represents intervening non-cysteine residues. These inter-cysteine amino acid residues are also strikingly conserved. The evolutionarily-related module b has the same cysteine spacing as a, but has 11 amino acid residues at its C-terminus. Different stretches of CREMP sequences in chicken genomic DNA fragments show diverse repeat patterns: e.g. all a modules; an alternation of a-b modules; or an a-b-b arrangement. Comparable CREMP proteins are found in contigs of the zebra finch (Taeniopygia guttata and in the oviparous green anole lizard (Anolis carolinensis. In all these cases the long runs of highly conserved modular repeats have evidently led to difficulties in the assembly of full length DNA sequences. Hence the number, and the amino acid lengths, of CREMP proteins are currently unknown. A 118 amino acid fragment (representing an a-b-a-b pattern from a chicken oviduct EST library expressed in Escherichia coli is a well folded, highly anisotropic, protein with a large chemical shift dispersion in 2D solution NMR spectra. Structure is completely lost on reduction of the 8 disulfide bonds of this protein fragment. Finally, solid state NMR spectra suggest a surprising degree of order in intact

  12. Understanding Bonds - Denmark

    DEFF Research Database (Denmark)

    Rimmer, Nina Røhr

    2016-01-01

    Undervisningsmateriale. A bond is a debt security, similar to an ”I Owe You document” (IOU). When you purchase a bond, you are lending money to a government, municipality, corporation, federal agency or other entity known as the issuer. In return for the loan, the issuer promises to pay you...... a specified rate of interest during the life of the bond and to repay the face value of the bond (the principal) when it “matures,” or comes due. Among the types of bonds you can choose from are: Government securities, municipal bonds, corporate bonds, mortgage and asset-backed securities, federal agency...... securities and foreign government bonds....

  13. Wire bonding in microelectronics

    CERN Document Server

    Harman, George G

    2010-01-01

    Wire Bonding in Microelectronics, Third Edition, has been thoroughly revised to help you meet the challenges of today's small-scale and fine-pitch microelectronics. This authoritative guide covers every aspect of designing, manufacturing, and evaluating wire bonds engineered with cutting-edge techniques. In addition to gaining a full grasp of bonding technology, you'll learn how to create reliable bonds at exceedingly high yields, test wire bonds, solve common bonding problems, implement molecular cleaning methods, and much more. Coverage includes: Ultrasonic bonding systems and technologies, including high-frequency systems Bonding wire metallurgy and characteristics, including copper wire Wire bond testing Gold-aluminum intermetallic compounds and other interface reactions Gold and nickel-based bond pad plating materials and problems Cleaning to improve bondability and reliability Mechanical problems in wire bonding High-yield, fine-pitch, specialized-looping, soft-substrate, and extreme-temperature wire bo...

  14. Piezoelectricity in two dimensions: Graphene vs. molybdenum disulfide

    Science.gov (United States)

    Song, Xiaoxue; Hui, Fei; Knobloch, Theresia; Wang, Bingru; Fan, Zhongchao; Grasser, Tibor; Jing, Xu; Shi, Yuanyuan; Lanza, Mario

    2017-08-01

    The synthesis of piezoelectric two-dimensional (2D) materials is very attractive for implementing advanced energy harvesters and transducers, as these materials provide enormously large areas for the exploitation of the piezoelectric effect. Among all 2D materials, molybdenum disulfide (MoS2) has shown the largest piezoelectric activity. However, all research papers in this field studied just a single material, and this may raise concerns because different setups could provide different values depending on experimental parameters (e.g., probes used and areas analyzed). By using conductive atomic force microscopy, here we in situ demonstrate that the piezoelectric currents generated in MoS2 are gigantic (65 mA/cm2), while the same experiments in graphene just showed noise currents. These results provide the most reliable comparison yet reported on the piezoelectric effect in graphene and MoS2.

  15. Raman investigation of molybdenum disulfide with different polytypes

    Science.gov (United States)

    Lee, Jae-Ung; Kim, Kangwon; Han, Songhee; Ryu, Gyeong Hee; Lee, Zonghoon; Cheong, Hyeonsik

    The Raman spectra of molybdenum disulfide (MoS2) with different polytypes are investigated. Although 2H-MoS2 is most common in nature, the 3R phase can exist due to a small difference in the formation energy. However, only a few studies are reported for the 3R phase, and most studies have focused on the 2H phase. We found the 2H, 3R and mixed phases of exfoliated few-layer MoS2 from natural molybdenite crystals. The crystal structures of 2H- and 3R-MoS2 are confirmed by the HR-TEM measurements. By using 3 different excitation energies, we compared the Raman spectra of different polytypes in detail. We show that the Raman spectroscopy can be used to identify not only the number of layers but also the polytypes of MoS2.

  16. Graphite oxide and molybdenum disulfide composite for hydrogen evolution reaction

    Science.gov (United States)

    Niyitanga, Theophile; Jeong, Hae Kyung

    2017-10-01

    Graphite oxide and molybdenum disulfide (GO-MoS2) composite is prepared through a wet process by using hydrolysis of ammonium tetrathiomolybdate, and it exhibits excellent catalytic activity of the hydrogen evolution reaction (HER) with a low overpotential of -0.47 V, which is almost two and three times lower than those of precursor MoS2 and GO. The high performance of HER of the composite attributes to the reduced GO supporting MoS2, providing a conducting network for fast electron transport from MoS2 to electrodes. The composite also shows high stability after 500 cycles, demonstrating a synergistic effect of MoS2 and GO for efficient HER.

  17. Plasmon modes of bilayer molybdenum disulfide: a density functional study

    Science.gov (United States)

    Torbatian, Z.; Asgari, R.

    2017-11-01

    We explore the collective electronic excitations of bilayer molybdenum disulfide (MoS2) using density functional theory together with random phase approximation. The many-body dielectric function and electron energy-loss spectra are calculated using an ab initio based model involving material-realistic physical properties. The electron energy-loss function of the bilayer MoS2 system is found to be sensitive to either electron or hole doping and this is due to the fact that the Kohn-Sham band dispersions are not symmetric for energies above and below the zero Fermi level. Three plasmon modes are predicted, a damped high-energy mode, one optical mode (in-phase mode) for which the plasmon dispersion exhibits \\sqrt q in the long wavelength limit originating from low-energy electron scattering and finally a highly damped acoustic mode (out-of-phase mode).

  18. Tuning thermal conductivity in molybdenum disulfide by electrochemical intercalation

    Science.gov (United States)

    Zhu, Gaohua; Liu, Jun; Zheng, Qiye; Zhang, Ruigang; Li, Dongyao; Banerjee, Debasish; Cahill, David G.

    2016-01-01

    Thermal conductivity of two-dimensional (2D) materials is of interest for energy storage, nanoelectronics and optoelectronics. Here, we report that the thermal conductivity of molybdenum disulfide can be modified by electrochemical intercalation. We observe distinct behaviour for thin films with vertically aligned basal planes and natural bulk crystals with basal planes aligned parallel to the surface. The thermal conductivity is measured as a function of the degree of lithiation, using time-domain thermoreflectance. The change of thermal conductivity correlates with the lithiation-induced structural and compositional disorder. We further show that the ratio of the in-plane to through-plane thermal conductivity of bulk crystal is enhanced by the disorder. These results suggest that stacking disorder and mixture of phases is an effective mechanism to modify the anisotropic thermal conductivity of 2D materials. PMID:27767030

  19. Large area synthesis, characterization, and anisotropic etching of two dimensional tungsten disulfide films

    International Nuclear Information System (INIS)

    Mutlu, Zafer; Ozkan, Mihrimah; Ozkan, Cengiz S.

    2016-01-01

    Emergent properties of tungsten disulfide at the quantum confinement limit hold promise for electronic and optoelectronic applications. Here we report on the large area synthesis of atomically thin tungsten disulfide films with strong photoluminescence properties via sulfurization of the pre-deposited tungsten films. Detailed characterization of the pre-deposited tungsten films and tungsten disulfide films are performed using microscopy and spectroscopy methods. By directly heating tungsten disulfide films in air, we have shown that the films tend to be etched into a series of triangular shaped pits with the same orientations, revealing the anisotropic etching behavior of tungsten disulfide edges. Moreover, the dimensions of the triangular pits increase with the number of layers, suggesting a thickness dependent behavior of etching in tungsten disulfide films. This method offers a promising new avenue for engineering the edge structures of tungsten disulfide films. - Highlights: • Large-scale synthesis of WS_2 films is achieved via sulfurization of W films. • Annealing of W films leads to a substantial improvement in the quality of WS_2 films. • WS_2 films show laser power dependent photoluminescence characteristics. • WS_2 films are etched with well-oriented triangular pits upon annealing in air. • Anisotropic oxidative etching is greatly affected by the thickness of WS_2 films.

  20. Variation in the Subcellular Localization and Protein Folding Activity among Arabidopsis thaliana Homologs of Protein Disulfide Isomerase

    Directory of Open Access Journals (Sweden)

    Christen Y. L. Yuen

    2013-10-01

    Full Text Available Protein disulfide isomerases (PDIs catalyze the formation, breakage, and rearrangement of disulfide bonds to properly fold nascent polypeptides within the endoplasmic reticulum (ER. Classical animal and yeast PDIs possess two catalytic thioredoxin-like domains (a, a′ and two non-catalytic domains (b, b′, in the order a-b-b′-a′. The model plant, Arabidopsis thaliana, encodes 12 PDI-like proteins, six of which possess the classical PDI domain arrangement (AtPDI1 through AtPDI6. Three additional AtPDIs (AtPDI9, AtPDI10, AtPDI11 possess two thioredoxin domains, but without intervening b-b′ domains. C-terminal green fluorescent protein (GFP fusions to each of the nine dual-thioredoxin PDI homologs localized predominantly to the ER lumen when transiently expressed in protoplasts. Additionally, expression of AtPDI9:GFP-KDEL and AtPDI10: GFP-KDDL was associated with the formation of ER bodies. AtPDI9, AtPDI10, and AtPDI11 mediated the oxidative folding of alkaline phosphatase when heterologously expressed in the Escherichia coli protein folding mutant, dsbA−. However, only three classical AtPDIs (AtPDI2, AtPDI5, AtPDI6 functionally complemented dsbA−. Interestingly, chemical inducers of the ER unfolded protein response were previously shown to upregulate most of the AtPDIs that complemented dsbA−. The results indicate that Arabidopsis PDIs differ in their localization and protein folding activities to fulfill distinct molecular functions in the ER.

  1. Disulfide-mediated stabilization of the IκB kinase binding domain of NF-κB essential modulator (NEMO).

    Science.gov (United States)

    Zhou, Li; Yeo, Alan T; Ballarano, Carmine; Weber, Urs; Allen, Karen N; Gilmore, Thomas D; Whitty, Adrian

    2014-12-23

    Human NEMO (NF-κB essential modulator) is a 419 residue scaffolding protein that, together with catalytic subunits IKKα and IKKβ, forms the IκB kinase (IKK) complex, a key regulator of NF-κB pathway signaling. NEMO is an elongated homodimer comprising mostly α-helix. It has been shown that a NEMO fragment spanning residues 44-111, which contains the IKKα/β binding site, is structurally disordered in the absence of bound IKKβ. Herein we show that enforcing dimerization of NEMO1-120 or NEMO44-111 constructs through introduction of one or two interchain disulfide bonds, through oxidation of the native Cys54 residue and/or at position 107 through a Leu107Cys mutation, induces a stable α-helical coiled-coil structure that is preorganized to bind IKKβ with high affinity. Chemical and thermal denaturation studies showed that, in the context of a covalent dimer, the ordered structure was stabilized relative to the denatured state by up to 3 kcal/mol. A full-length NEMO-L107C protein formed covalent dimers upon treatment of mammalian cells with H2O2. Furthermore, NEMO-L107C bound endogenous IKKβ in A293T cells, reconstituted TNF-induced NF-κB signaling in NEMO-deficient cells, and interacted with TRAF6. Our results indicate that the IKKβ binding domain of NEMO possesses an ordered structure in the unbound state, provided that it is constrained within a dimer as is the case in the constitutively dimeric full-length NEMO protein. The stability of the NEMO coiled coil is maintained by strong interhelix interactions in the region centered on residue 54. The disulfide-linked constructs we describe herein may be useful for crystallization of NEMO's IKKβ binding domain in the absence of bound IKKβ, thereby facilitating the structural characterization of small-molecule inhibitors.

  2. Rv2969c, essential for optimal growth in Mycobacterium tuberculosis, is a DsbA-like enzyme that interacts with VKOR-derived peptides and has atypical features of DsbA-like disulfide oxidases

    International Nuclear Information System (INIS)

    Premkumar, Lakshmanane; Heras, Begoña; Duprez, Wilko; Walden, Patricia; Halili, Maria; Kurth, Fabian; Fairlie, David P.; Martin, Jennifer L.

    2013-01-01

    The gene product of M. tuberculosis Rv2969c is shown to be a disulfide oxidase enzyme that has a canonical DsbA-like fold with novel structural and functional characteristics. The bacterial disulfide machinery is an attractive molecular target for developing new antibacterials because it is required for the production of multiple virulence factors. The archetypal disulfide oxidase proteins in Escherichia coli (Ec) are DsbA and DsbB, which together form a functional unit: DsbA introduces disulfides into folding proteins and DsbB reoxidizes DsbA to maintain it in the active form. In Mycobacterium tuberculosis (Mtb), no DsbB homologue is encoded but a functionally similar but structurally divergent protein, MtbVKOR, has been identified. Here, the Mtb protein Rv2969c is investigated and it is shown that it is the DsbA-like partner protein of MtbVKOR. It is found that it has the characteristic redox features of a DsbA-like protein: a highly acidic catalytic cysteine, a highly oxidizing potential and a destabilizing active-site disulfide bond. Rv2969c also has peptide-oxidizing activity and recognizes peptide segments derived from the periplasmic loops of MtbVKOR. Unlike the archetypal EcDsbA enzyme, Rv2969c has little or no activity in disulfide-reducing and disulfide-isomerase assays. The crystal structure of Rv2969c reveals a canonical DsbA fold comprising a thioredoxin domain with an embedded helical domain. However, Rv2969c diverges considerably from other DsbAs, including having an additional C-terminal helix (H8) that may restrain the mobility of the catalytic helix H1. The enzyme is also characterized by a very shallow hydrophobic binding surface and a negative electrostatic surface potential surrounding the catalytic cysteine. The structure of Rv2969c was also used to model the structure of a paralogous DsbA-like domain of the Ser/Thr protein kinase PknE. Together, these results show that Rv2969c is a DsbA-like protein with unique properties and a limited

  3. Rv2969c, essential for optimal growth in Mycobacterium tuberculosis, is a DsbA-like enzyme that interacts with VKOR-derived peptides and has atypical features of DsbA-like disulfide oxidases

    Energy Technology Data Exchange (ETDEWEB)

    Premkumar, Lakshmanane, E-mail: p.lakshmanane@imb.uq.edu.au; Heras, Begoña; Duprez, Wilko; Walden, Patricia; Halili, Maria; Kurth, Fabian; Fairlie, David P.; Martin, Jennifer L., E-mail: p.lakshmanane@imb.uq.edu.au [University of Queensland, St Lucia, QLD 4067 (Australia)

    2013-10-01

    The gene product of M. tuberculosis Rv2969c is shown to be a disulfide oxidase enzyme that has a canonical DsbA-like fold with novel structural and functional characteristics. The bacterial disulfide machinery is an attractive molecular target for developing new antibacterials because it is required for the production of multiple virulence factors. The archetypal disulfide oxidase proteins in Escherichia coli (Ec) are DsbA and DsbB, which together form a functional unit: DsbA introduces disulfides into folding proteins and DsbB reoxidizes DsbA to maintain it in the active form. In Mycobacterium tuberculosis (Mtb), no DsbB homologue is encoded but a functionally similar but structurally divergent protein, MtbVKOR, has been identified. Here, the Mtb protein Rv2969c is investigated and it is shown that it is the DsbA-like partner protein of MtbVKOR. It is found that it has the characteristic redox features of a DsbA-like protein: a highly acidic catalytic cysteine, a highly oxidizing potential and a destabilizing active-site disulfide bond. Rv2969c also has peptide-oxidizing activity and recognizes peptide segments derived from the periplasmic loops of MtbVKOR. Unlike the archetypal EcDsbA enzyme, Rv2969c has little or no activity in disulfide-reducing and disulfide-isomerase assays. The crystal structure of Rv2969c reveals a canonical DsbA fold comprising a thioredoxin domain with an embedded helical domain. However, Rv2969c diverges considerably from other DsbAs, including having an additional C-terminal helix (H8) that may restrain the mobility of the catalytic helix H1. The enzyme is also characterized by a very shallow hydrophobic binding surface and a negative electrostatic surface potential surrounding the catalytic cysteine. The structure of Rv2969c was also used to model the structure of a paralogous DsbA-like domain of the Ser/Thr protein kinase PknE. Together, these results show that Rv2969c is a DsbA-like protein with unique properties and a limited

  4. Edge eigen-stress and eigen-displacement of armchair molybdenum disulfide nanoribbons

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Quan; Li, Xi [Corrosion and Protection Center, Key Laboratory for Environmental Fracture (MOE), University of Science and Technology Beijing, Beijing 100083 (China); Volinsky, Alex A., E-mail: volinsky@usf.edu [Department of Mechanical Engineering, University of South Florida, Tampa, FL 33620 (United States); Su, Yanjing, E-mail: yjsu@ustb.edu.cn [Corrosion and Protection Center, Key Laboratory for Environmental Fracture (MOE), University of Science and Technology Beijing, Beijing 100083 (China)

    2017-05-10

    Edge effects on mechanical properties of armchair molybdenum disulfide nanoribbons were investigated using first principles calculations. The edge eigen-stress model was applied to explain the relaxation process of forming molybdenum disulfide nanoribbon. Edge effects on surface atoms fluctuation degree were obtained from each fully relaxed nanoribbon with different width. Changes of the relaxed armchair molybdenum disulfide nanoribbons structure can be expressed using hexagonal perimeters pattern. Based on the thickness change, relaxed armchair molybdenum disulfide nanoribbons tensile/compression tests were simulated, providing intrinsic edge elastic parameters, such as eigen-stress, Young's modulus and Poisson's ratio. - Highlights: • Edge effects on mechanical properties of armchair MoS{sub 2} nanoribbons were investigated. • Structure changes of different width armchair MoS{sub 2} nanoribbons were obtained. • Tensile/compressive tests were conducted to determine elastic constants. • Mechanical properties are compared for two and three dimensional conditions.

  5. Disulfide-functional poly(amido amine)s with tunable degradability for gene delivery

    NARCIS (Netherlands)

    Elzes, M. Rachel; Akeroyd, Niels; Engbersen, Johan F. J.; Paulusse, Jos M. J.

    2016-01-01

    Controlled degradability in response to the local environment is one of the most effective strategies to achieve spatiotemporal release of genes from a polymeric carrier. Exploiting the differences in reduction potential between the extracellular and intracellular environment, disulfides are

  6. Generation of a Multicomponent Library of Disulfide Donor-Acceptor Architectures Using Dynamic Combinatorial Chemistry.

    Science.gov (United States)

    Drożdż, Wojciech; Kołodziejski, Michał; Markiewicz, Grzegorz; Jenczak, Anna; Stefankiewicz, Artur R

    2015-07-17

    We describe here the generation of new donor-acceptor disulfide architectures obtained in aqueous solution at physiological pH. The application of a dynamic combinatorial chemistry approach allowed us to generate a large number of new disulfide macrocyclic architectures together with a new type of [2]catenanes consisting of four distinct components. Up to fifteen types of structurally-distinct dynamic architectures have been generated through one-pot disulfide exchange reactions between four thiol-functionalized aqueous components. The distribution of disulfide products formed was found to be strongly dependent on the structural features of the thiol components employed. This work not only constitutes a success in the synthesis of topologically- and morphologically-complex targets, but it may also open new horizons for the use of this methodology in the construction of molecular machines.

  7. Stability of a metabolizable ester bond in radioimmunoconjugates

    International Nuclear Information System (INIS)

    Arano, Yasushi; Wakisaka, Kouji; Mukai, Takahiro; Uezono, Takashi; Motonari, Hiroshi; Akizawa, Hiromichi; Kairiyama, Claudia; Ohmomo, Yoshiro; Tanaka, Chiaki; Ishiyama, Munetaka; Sakahara, Harumi; Konishi, Junji; Yokoyama, Akira

    1996-01-01

    Ester bonds have been used as metabolizable linkages to reduce radioactivity levels in non-target tissues following the administration of antibodies labeled with metallic radionuclides. In this radiochemical design of antibodies, while the ester bonds should be cleaved rapidly in non-target tissues, high stability of the ester bonds in plasma is also required to preserve target radioactivity levels. To assess the structural requirements to stabilize the ester bond, a new benzyl-EDTA-derived bifunctional chelating agent with an ester bond, (1-[4-[4-(2-maleimidoethoxy)succinamido]benzyl]ethylenediamine-N,N,N',N'- tetraacetic acid; MESS-Bz-EDTA), was developed. MESS-Bz-EDTA was coupled with a thiolated monoclonal antibody (OST7, IgG 1 ) prepared by reducing its disulfide bonds to introduce the ester bond close and proximal to the antibody molecule. For comparison, 1-[4-(5-maleimidopentyl)aminobenzyl]ethylenediamine-N,N,N',N'-tetraacetic acid (EMCS-Bz-EDTA) and meleimidoethyl 3-[ 131 I]iodohippurate (MIH) was coupled to OST7 under the same conjunction chemistry. When incubated in 50% murine plasma or a buffered-solution of neutral pH, OST7-MESS-Bz-EDTA- 111 In rapidly released the radioactivity, and more than 95% of the initial radioactivity was liberated after a 24 h incubation in both solutions, due to a cleavage of the ester bond. On the other hand, only about 20% of the radioactivity was released from OST7-MIH- 131 I in both solutions during the same incubation period. In mice biodistribution studies, while a slightly faster radioactivity clearance from the blood with less radioactivity levels in the liver and kidneys was observed with OST7-MIH- 131 I than with OST7-EMCS-Bz-EDTA- 111 In, OST7-MESS-Bz-EDTA- 111 In indicated radioactivity clearance from the blood much faster than and almost comparable to that of OST7-MIH- 131 I and succinamidobenzyl-EDTA- 111 In, respectively. These findings as well as previous findings on radiolabeled antibodies with ester bonds

  8. The dehydrogenase region of the NADPH oxidase component Nox2 acts as a protein disulfide isomerase (PDI) resembling PDIA3 with a role in the binding of the activator protein p67phox

    Science.gov (United States)

    Bechor, Edna; Dahan, Iris; Fradin, Tanya; Berdichevsky, Yevgeny; Zahavi, Anat; Rafalowski, Meirav; Federman-Gross, Aya; Pick, Edgar

    2015-02-01

    The superoxide (O2.-)-generating NADPH oxidase of phagocytes consists of a membrane component, cytochrome b558 (a heterodimer of Nox2 and p22phox), and four cytosolic components, p47phox, p67phox, p40phox, and Rac. The catalytic component, responsible for O2.- generation, is Nox2. It is activated by the interaction of the dehydrogenase region (DHR) of Nox2 with the cytosolic components, principally with p67phox. Using a peptide-protein binding assay, we found that Nox2 peptides containing a 369CysGlyCys371 triad (CGC) bound p67phox with high affinity, dependent upon the establishment of a disulfide bond between the two cysteines. Serially truncated recombinant Nox2 DHR proteins bound p67phox only when they comprised the CGC triad. CGC resembles the catalytic motif (CGHC) of protein disulfide isomerases (PDIs). This led to the hypothesis that Nox2 establishes disulfide bonds with p67phox via a thiol-dilsulfide exchange reaction and, thus, functions as a PDI. Evidence for this was provided by the following: 1. Recombinant Nox2 protein, which contained the CGC triad, exhibited PDI-like disulfide reductase activity; 2. Truncation of Nox2 C-terminal to the CGC triad or mutating C369 and C371 to R, resulted in loss of PDI activity; 3. Comparison of the sequence of the DHR of Nox2 with PDI family members revealed three small regions of homology with PDIA3; 4. Two monoclonal anti-Nox2 antibodies, with epitopes corresponding to regions of Nox2/PDIA3 homology, reacted with PDIA3 but not with PDIA1; 5. A polyclonal anti-PDIA3 (but not an anti-PDIA1) antibody reacted with Nox2; 6. p67phox, in which all cysteines were mutated to serines, lost its ability to bind to a Nox2 peptide containing the CGC triad and had an impaired capacity to support oxidase activity in vitro. We propose a model of oxidase assembly in which binding of p67phox to Nox2 via disulfide bonds, by virtue of the intrinsic PDI activity of Nox2, stabilizes the primary interaction between the two components.

  9. Bonding with Your Baby

    Science.gov (United States)

    ... the future bonding of the child and parent. Adoptive parents may be concerned about bonding with their ... general emotional support. And it's OK to ask family members and friends for help in the days — ...

  10. Live-cell imaging of biothiols via thiol/disulfide exchange to trigger the photoinduced electron transfer of gold-nanodot sensor

    International Nuclear Information System (INIS)

    Liu, Ching-Ping; Wu, Te-Haw; Liu, Chia-Yeh; Lin, Shu-Yi

    2014-01-01

    Highlights: • The ultrasmall size, PAMAM dendrimer-entrapped Au 8 -clusters were synthesized. • Thiol/disulfide exchange with biothiols to release 2-PyT resulted in quenching. • The sensing platform can detect both low and high molecular weight thiols. • Capable of imaging biothiols including protein thiols in living cells. - Abstract: Biothiols have been reported to involve in intracellular redox-homeostasis against oxidative stress. In this study, a highly selective and sensitive fluorescent probe for sensing biothiols is explored by using an ultrasmall gold nanodot (AuND), the dendrimer-entrapped Au 8 -cluster. This strategy relies upon a thiol/disulfide exchange to trigger the fluorescence change through a photoinduced electron transfer (PET) process between the Au 8 -cluster (as an electron donor) and 2-pyridinethiol (2-PyT) (as an electron acceptor) for sensing biothiols. When 2-PyT is released via the cleavage of disulfide bonds by biothiols, the PET process from the Au 8 -cluster to 2-PyT is initiated, resulting in fluorescence quenching. The fluorescence intensity was found to decrease linearly with glutathione (GSH) concentration (0–1500 μM) at physiological relevant levels and the limit of detection for GSH was 15.4 μM. Compared to most nanoparticle-based fluorescent probes that are limited to detect low molecular weight thiols (LMWTs; i.e., GSH and cysteine), the ultrasmall Au 8 -cluster-based probe exhibited less steric hindrance and can be directly applied in selectively and sensitively detecting both LMWTs and high molecular weight thiols (HMWTs; i.e., protein thiols). Based on such sensing platform, the surface-functionalized Au 8 -cluster has significant promise for use as an efficient nanoprobe for intracellular fluorescence imaging of biothiols including protein thiols in living cells whereas other nanoparticle-based fluorescent probes cannot

  11. Live-cell imaging of biothiols via thiol/disulfide exchange to trigger the photoinduced electron transfer of gold-nanodot sensor

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Ching-Ping; Wu, Te-Haw; Liu, Chia-Yeh; Lin, Shu-Yi, E-mail: shuyi@nhri.org.tw

    2014-11-07

    Highlights: • The ultrasmall size, PAMAM dendrimer-entrapped Au{sub 8}-clusters were synthesized. • Thiol/disulfide exchange with biothiols to release 2-PyT resulted in quenching. • The sensing platform can detect both low and high molecular weight thiols. • Capable of imaging biothiols including protein thiols in living cells. - Abstract: Biothiols have been reported to involve in intracellular redox-homeostasis against oxidative stress. In this study, a highly selective and sensitive fluorescent probe for sensing biothiols is explored by using an ultrasmall gold nanodot (AuND), the dendrimer-entrapped Au{sub 8}-cluster. This strategy relies upon a thiol/disulfide exchange to trigger the fluorescence change through a photoinduced electron transfer (PET) process between the Au{sub 8}-cluster (as an electron donor) and 2-pyridinethiol (2-PyT) (as an electron acceptor) for sensing biothiols. When 2-PyT is released via the cleavage of disulfide bonds by biothiols, the PET process from the Au{sub 8}-cluster to 2-PyT is initiated, resulting in fluorescence quenching. The fluorescence intensity was found to decrease linearly with glutathione (GSH) concentration (0–1500 μM) at physiological relevant levels and the limit of detection for GSH was 15.4 μM. Compared to most nanoparticle-based fluorescent probes that are limited to detect low molecular weight thiols (LMWTs; i.e., GSH and cysteine), the ultrasmall Au{sub 8}-cluster-based probe exhibited less steric hindrance and can be directly applied in selectively and sensitively detecting both LMWTs and high molecular weight thiols (HMWTs; i.e., protein thiols). Based on such sensing platform, the surface-functionalized Au{sub 8}-cluster has significant promise for use as an efficient nanoprobe for intracellular fluorescence imaging of biothiols including protein thiols in living cells whereas other nanoparticle-based fluorescent probes cannot.

  12. Approach to characterization of the higher order structure of disulfide-containing proteins using hydrogen/deuterium exchange and top-down mass spectrometry.

    Science.gov (United States)

    Wang, Guanbo; Kaltashov, Igor A

    2014-08-05

    Top-down hydrogen/deuterium exchange (HDX) with mass spectrometric (MS) detection has recently matured to become a potent biophysical tool capable of providing valuable information on higher order structure and conformational dynamics of proteins at an unprecedented level of structural detail. However, the scope of the proteins amenable to the analysis by top-down HDX MS still remains limited, with the protein size and the presence of disulfide bonds being the two most important limiting factors. While the limitations imposed by the physical size of the proteins gradually become more relaxed as the sensitivity, resolution and dynamic range of modern MS instrumentation continue to improve at an ever accelerating pace, the presence of the disulfide linkages remains a much less forgiving limitation even for the proteins of relatively modest size. To circumvent this problem, we introduce an online chemical reduction step following completion and quenching of the HDX reactions and prior to the top-down MS measurements of deuterium occupancy of individual backbone amides. Application of the new methodology to the top-down HDX MS characterization of a small (99 residue long) disulfide-containing protein β2-microglobulin allowed the backbone amide protection to be probed with nearly a single-residue resolution across the entire sequence. The high-resolution backbone protection pattern deduced from the top-down HDX MS measurements carried out under native conditions is in excellent agreement with the crystal structure of the protein and high-resolution NMR data, suggesting that introduction of the chemical reduction step to the top-down routine does not trigger hydrogen scrambling either during the electrospray ionization process or in the gas phase prior to the protein ion dissociation.

  13. Graphene oxide – molybdenum disulfide hybrid membranes for hydrogen separation

    KAUST Repository

    Ostwal, Mayur

    2017-12-24

    Graphene oxide – molybdenum disulfide hybrid membranes were prepared using vacuum filtration technique. The thickness and the MoS2 content in the membranes were varied and their H2 permeance and H2/CO2 selectivity are reported. A 60nm hybrid membrane containing ~75% by weight of MoS2 exhibited the highest H2 permeance of 804×10−9mol/m2·s·Pa with corresponding H2/CO2 selectivity of 26.7; while a 150nm hybrid membrane with ~29% MoS2 showed the highest H2/CO2 selectivity of 44.2 with corresponding H2 permeance of 287×10−9mol/m2·s·Pa. The hybrid membranes exhibited much higher H2 permeance compared to graphene oxide membranes and higher selectivity compared to MoS2 membranes, which fully demonstrated the synergistic effect of both nanomaterials. The membranes also displayed excellent operational long-term stability.

  14. DNA origami deposition on native and passivated molybdenum disulfide substrates

    Directory of Open Access Journals (Sweden)

    Xiaoning Zhang

    2014-04-01

    Full Text Available Maintaining the structural fidelity of DNA origami structures on substrates is a prerequisite for the successful fabrication of hybrid DNA origami/semiconductor-based biomedical sensor devices. Molybdenum disulfide (MoS2 is an ideal substrate for such future sensors due to its exceptional electrical, mechanical and structural properties. In this work, we performed the first investigations into the interaction of DNA origami with the MoS2 surface. In contrast to the structure-preserving interaction of DNA origami with mica, another atomically flat surface, it was observed that DNA origami structures rapidly lose their structural integrity upon interaction with MoS2. In a further series of studies, pyrene and 1-pyrenemethylamine, were evaluated as surface modifications which might mitigate this effect. While both species were found to form adsorption layers on MoS2 via physisorption, 1-pyrenemethylamine serves as a better protective agent and preserves the structures for significantly longer times. These findings will be beneficial for the fabrication of future DNA origami/MoS2 hybrid electronic structures.

  15. Graphene oxide – molybdenum disulfide hybrid membranes for hydrogen separation

    KAUST Repository

    Ostwal, Mayur; Shinde, Digambar B.; Wang, Xinbo; Gadwal, Ikhlas; Lai, Zhiping

    2017-01-01

    Graphene oxide – molybdenum disulfide hybrid membranes were prepared using vacuum filtration technique. The thickness and the MoS2 content in the membranes were varied and their H2 permeance and H2/CO2 selectivity are reported. A 60nm hybrid membrane containing ~75% by weight of MoS2 exhibited the highest H2 permeance of 804×10−9mol/m2·s·Pa with corresponding H2/CO2 selectivity of 26.7; while a 150nm hybrid membrane with ~29% MoS2 showed the highest H2/CO2 selectivity of 44.2 with corresponding H2 permeance of 287×10−9mol/m2·s·Pa. The hybrid membranes exhibited much higher H2 permeance compared to graphene oxide membranes and higher selectivity compared to MoS2 membranes, which fully demonstrated the synergistic effect of both nanomaterials. The membranes also displayed excellent operational long-term stability.

  16. Prediction of dimethyl disulfide levels from biosolids using statistical modeling.

    Science.gov (United States)

    Gabriel, Steven A; Vilalai, Sirapong; Arispe, Susanna; Kim, Hyunook; McConnell, Laura L; Torrents, Alba; Peot, Christopher; Ramirez, Mark

    2005-01-01

    Two statistical models were used to predict the concentration of dimethyl disulfide (DMDS) released from biosolids produced by an advanced wastewater treatment plant (WWTP) located in Washington, DC, USA. The plant concentrates sludge from primary sedimentation basins in gravity thickeners (GT) and sludge from secondary sedimentation basins in dissolved air flotation (DAF) thickeners. The thickened sludge is pumped into blending tanks and then fed into centrifuges for dewatering. The dewatered sludge is then conditioned with lime before trucking out from the plant. DMDS, along with other volatile sulfur and nitrogen-containing chemicals, is known to contribute to biosolids odors. These models identified oxidation/reduction potential (ORP) values of a GT and DAF, the amount of sludge dewatered by centrifuges, and the blend ratio between GT thickened sludge and DAF thickened sludge in blending tanks as control variables. The accuracy of the developed regression models was evaluated by checking the adjusted R2 of the regression as well as the signs of coefficients associated with each variable. In general, both models explained observed DMDS levels in sludge headspace samples. The adjusted R2 value of the regression models 1 and 2 were 0.79 and 0.77, respectively. Coefficients for each regression model also had the correct sign. Using the developed models, plant operators can adjust the controllable variables to proactively decrease this odorant. Therefore, these models are a useful tool in biosolids management at WWTPs.

  17. Protein Disulfide Isomerase and Host-Pathogen Interaction

    Directory of Open Access Journals (Sweden)

    Beatriz S. Stolf

    2011-01-01

    Full Text Available Reactive oxygen species (ROS production by immunological cells is known to cause damage to pathogens. Increasing evidence accumulated in the last decade has shown, however, that ROS (and redox signals functionally regulate different cellular pathways in the host-pathogen interaction. These especially affect (i pathogen entry through protein redox switches and redox modification (i.e., intra- and interdisulfide and cysteine oxidation and (ii phagocytic ROS production via Nox family NADPH oxidase enzyme and the control of phagolysosome function with key implications for antigen processing. The protein disulfide isomerase (PDI family of redox chaperones is closely involved in both processes and is also implicated in protein unfolding and trafficking across the endoplasmic reticulum (ER and towards the cytosol, a thiol-based redox locus for antigen processing. Here, we summarise examples of the cellular association of host PDI with different pathogens and explore the possible roles of pathogen PDIs in infection. A better understanding of these complex regulatory steps will provide insightful information on the redox role and coevolutional biological process, and assist the development of more specific therapeutic strategies in pathogen-mediated infections.

  18. 9-Fluorenylmethyl (Fm) Disulfides: Biomimetic Precursors for Persulfides

    Energy Technology Data Exchange (ETDEWEB)

    Park, Chung-Min; Johnson, Brett A.; Duan, Jicheng; Park, Jeong-Jin; Day, Jacob J.; Gang, David; Qian, Wei-Jun; Xian, Ming

    2016-03-04

    Protein S-sulfhydration has been recognized as an important post-translational modification that regulates H2S signals. However, the reactivity and biological implications of the products of S-sulfhydration, i.e. persulfides, are still unclear. This is mainly due to the instability of persulfides and difficulty to access these molecules. Under physiological conditions persulfides mainly exist in anionic forms because of their low pKa values. However, current methods do not allow for the direct generation of persulfide anions under biomimetic and non-H2S conditions. Herein we report the development of a functional disulfide, FmSSPy-A (Fm =9-fluorenylmethyl; Py = pyridinyl). This reagent can effectively convert both small molecule and protein thiols (-SH) to form –S-SFm adducts under mild conditions. It allows for a H2S-free and biomimetic protocol to generate highly reactive persulfides (in their anionic forms). We also demonstrated the high nucleophilicity of persulfides toward a number of thiol-blocking reagents. This method holds promise for further understanding the chemical biology of persulfides and S-sulfhydration.

  19. Metallic molybdenum disulfide nanosheet-based electrochemical actuators

    Science.gov (United States)

    Acerce, Muharrem; Akdoğan, E. Koray; Chhowalla, Manish

    2017-09-01

    Actuators that convert electrical energy to mechanical energy are useful in a wide variety of electromechanical systems and in robotics, with applications such as steerable catheters, adaptive wings for aircraft and drag-reducing wind turbines. Actuation systems can be based on various stimuli, such as heat, solvent adsorption/desorption, or electrochemical action (in systems such as carbon nanotube electrodes, graphite electrodes, polymer electrodes and metals). Here we demonstrate that the dynamic expansion and contraction of electrode films formed by restacking chemically exfoliated nanosheets of two-dimensional metallic molybdenum disulfide (MoS2) on thin plastic substrates can generate substantial mechanical forces. These films are capable of lifting masses that are more than 150 times that of the electrode over several millimetres and for hundreds of cycles. Specifically, the MoS2 films are able to generate mechanical stresses of about 17 megapascals—higher than mammalian muscle (about 0.3 megapascals) and comparable to ceramic piezoelectric actuators (about 40 megapascals)—and strains of about 0.6 per cent, operating at frequencies up to 1 hertz. The actuation performance is attributed to the high electrical conductivity of the metallic 1T phase of MoS2 nanosheets, the elastic modulus of restacked MoS2 layers (2 to 4 gigapascals) and fast proton diffusion between the nanosheets. These results could lead to new electrochemical actuators for high-strain and high-frequency applications.

  20. Australia's Bond Home Bias

    OpenAIRE

    Anil V. Mishra; Umaru B. Conteh

    2014-01-01

    This paper constructs the float adjusted measure of home bias and explores the determinants of bond home bias by employing the International Monetary Fund's high quality dataset (2001 to 2009) on cross-border bond investment. The paper finds that Australian investors' prefer investing in countries with higher economic development and more developed bond markets. Exchange rate volatility appears to be an impediment for cross-border bond investment. Investors prefer investing in countries with ...

  1. Phenylacetylene and H bond

    Indian Academy of Sciences (India)

    ... all resembling H bonds. Non-linear H bonds due to secondary interactions. C-H stretching frequency shows blue shift. Heavy atom distances are longer than the sum of van der Waals radii. Formed a task group through IUPAC to come up with a modern definition of H bond. 15 international experts including Desiraju.

  2. Chemical bond fundamental aspects of chemical bonding

    CERN Document Server

    Frenking, Gernot

    2014-01-01

    This is the perfect complement to ""Chemical Bonding - Across the Periodic Table"" by the same editors, who are two of the top scientists working on this topic, each with extensive experience and important connections within the community. The resulting book is a unique overview of the different approaches used for describing a chemical bond, including molecular-orbital based, valence-bond based, ELF, AIM and density-functional based methods. It takes into account the many developments that have taken place in the field over the past few decades due to the rapid advances in quantum chemica

  3. Ionic properties of non-aqueous liquid and PVDF-based gel electrolytes containing a cesium thiolate/disulfide redox couple

    International Nuclear Information System (INIS)

    Renard, Ingrid; Li Hongmei; Marsan, Benoit

    2003-01-01

    Liquid electrolytes containing a cesium thiolate/disulfide redox couple, prepared from 5-mercapto-1-methyltetrazole cesium salt (CsT) and di-5-(1-methyltetrazole)disulfide (T 2 ) dissolved in several aprotic solvents and solvent mixtures, have been studied using various techniques. FTIR spectroscopy reveals that relatively strong interactions occur between the reduced species T - and DMSO or DMF while Cs + ions are very weakly coordinated to the S=O or C=O bond. It is shown that the electrolyte consisting of 1.55 mol kg -1 CsT in the solvent mixture DMSO/DMF (40/60%) exhibits the highest conductivity (1.1x10 -2 and 2.3x10 -2 S cm -1 at 23 and 80 deg. C, respectively), and that the presence of the oxidized species T 2 does not affect significantly its electrical properties up to a CsT:T 2 molar ratio of 5:1. Conductivity values as a function of salt concentration are discussed in terms of the effective number of charge carriers, taking into account the level of ionic association, and of the ionic mobility. Optically transparent gel electrolytes have been prepared by incorporation of the optimal liquid electrolyte into various amounts of poly(vinylidene fluoride) (PVDF). It is shown that ionic mobility is not much affected by the polymer concentration, suggesting that migration of ions occurs mainly through the solvent mixture surrounded by the PVDF matrix

  4. A thiol-disulfide oxidoreductase of the Gram-positive pathogen Corynebacterium diphtheriae is essential for viability, pilus assembly, toxin production and virulence.

    Science.gov (United States)

    Reardon-Robinson, Melissa E; Osipiuk, Jerzy; Jooya, Neda; Chang, Chungyu; Joachimiak, Andrzej; Das, Asis; Ton-That, Hung

    2015-12-01

    The Gram-positive pathogen Corynebacterium diphtheriae exports through the Sec apparatus many extracellular proteins that include the key virulence factors diphtheria toxin and the adhesive pili. How these proteins attain their native conformations after translocation as unfolded precursors remains elusive. The fact that the majority of these exported proteins contain multiple cysteine residues and that several membrane-bound oxidoreductases are encoded in the corynebacterial genome suggests the existence of an oxidative protein-folding pathway in this organism. Here we show that the shaft pilin SpaA harbors a disulfide bond in vivo and alanine substitution of these cysteines abrogates SpaA polymerization and leads to the secretion of degraded SpaA peptides. We then identified a thiol-disulfide oxidoreductase (MdbA), whose structure exhibits a conserved thioredoxin-like domain with a CPHC active site. Remarkably, deletion of mdbA results in a severe temperature-sensitive cell division phenotype. This mutant also fails to assemble pilus structures and is greatly defective in toxin production. Consistent with these defects, the ΔmdbA mutant is attenuated in a guinea pig model of diphtheritic toxemia. Given its diverse cellular functions in cell division, pilus assembly and toxin production, we propose that MdbA is a component of the general oxidative folding machine in C. diphtheriae. © 2015 John Wiley & Sons Ltd.

  5. Intermolecular-directed reactivity in solid media. Radiogenic formation of phosphorus-centered radicals in chiral diphosphine disulfides studied by ESR

    Energy Technology Data Exchange (ETDEWEB)

    Aagaard, O.M.; Janssen, R.A.J.; de Waal, B.F.M.; Buck, H.M. (Eindhoven Univ. of Technology (Netherlands)); Kanters, J.A.; Schouten, A. (State Univ. of Utrecht (Netherlands))

    1990-07-04

    Single-crystal, powder, and frozen-matrix ESR experiments have been performed to study the radiogenic electron-capture properties of several diastereoisomeric and asymmetric diphosphine disulfides (R{sub 1}R{sub 2}P(S)P(S)R{sub 3}R{sub 4}). The principal values of the hyperfine couplings of several phosphorus-centered radical configurations are determined and related to the spin density distribution. Attention is focused on the strong differences in radiogenic properties, observed between the meso and racemic forms of phenyl- and tolyl-substituted diphosphine disulfides. The most striking result is that X irradiation of the crystalline meso compounds MePhP(S)P(S)MePh, Me(p-Tol)P(S)P(S)Me(p-Tol), and Ph(PhCH{sub 2})P(S)P(S)Ph(CH{sub 2}Ph) does not lead to the formation of a three-electron bond P-P {sigma}* radical but invariably results in configurations in which the unpaired electron is primarily localized on one half of the molecule. X irradiation of the corresponding racemic forms, on the other hand, gives rise to P-P {sigma}* configurations.

  6. Mutation of the Streptococcus gordonii Thiol-Disulfide Oxidoreductase SdbA Leads to Enhanced Biofilm Formation Mediated by the CiaRH Two-Component Signaling System.

    Directory of Open Access Journals (Sweden)

    Lauren Davey

    Full Text Available Streptococcus gordonii is a commensal inhabitant of human oral biofilms. Previously, we identified an enzyme called SdbA that played an important role in biofilm formation by S. gordonii. SdbA is thiol-disulfide oxidoreductase that catalyzes disulfide bonds in secreted proteins. Surprisingly, inactivation of SdbA results in enhanced biofilm formation. In this study we investigated the basis for biofilm formation by the ΔsdbA mutant. The results revealed that biofilm formation was mediated by the interaction between the CiaRH and ComDE two-component signalling systems. Although it did not affect biofilm formation by the S. gordonii parent strain, CiaRH was upregulated in the ΔsdbA mutant and it was essential for the enhanced biofilm phenotype. The biofilm phenotype was reversed by inactivation of CiaRH or by the addition of competence stimulating peptide, the production of which is blocked by CiaRH activity. Competition assays showed that the enhanced biofilm phenotype also corresponded to increased oral colonization in mice. Thus, the interaction between SdbA, CiaRH and ComDE affects biofilm formation both in vitro and in vivo.

  7. Atypical protein disulfide isomerases (PDI: Comparison of the molecular and catalytic properties of poplar PDI-A and PDI-M with PDI-L1A.

    Directory of Open Access Journals (Sweden)

    Benjamin Selles

    Full Text Available Protein disulfide isomerases are overwhelmingly multi-modular redox catalysts able to perform the formation, reduction or isomerisation of disulfide bonds. We present here the biochemical characterization of three different poplar PDI isoforms. PDI-A is characterized by a single catalytic Trx module, the so-called a domain, whereas PDI-L1a and PDI-M display an a-b-b'-a' and a°-a-b organisation respectively. Their activities have been tested in vitro using purified recombinant proteins and a series of model substrates as insulin, NADPH thioredoxin reductase, NADP malate dehydrogenase (NADP-MDH, peroxiredoxins or RNase A. We demonstrated that PDI-A exhibited none of the usually reported activities, although the cysteines of the WCKHC active site signature are able to form a disulfide with a redox midpoint potential of -170 mV at pH 7.0. The fact that it is able to bind a [Fe2S2] cluster upon Escherichia coli expression and anaerobic purification might indicate that it does not have a function in dithiol-disulfide exchange reactions. The two other proteins were able to catalyze oxidation or reduction reactions, PDI-L1a being more efficient in most cases, except that it was unable to activate the non-physiological substrate NADP-MDH, in contrast to PDI-M. To further evaluate the contribution of the catalytic domains of PDI-M, the dicysteinic motifs have been independently mutated in each a domain. The results indicated that the two a domains seem interconnected and that the a° module preferentially catalyzed oxidation reactions whereas the a module catalyzed reduction reactions, in line with the respective redox potentials of -170 mV and -190 mV at pH 7.0. Overall, these in vitro results illustrate that the number and position of a and b domains influence the redox properties and substrate recognition (both electron donors and acceptors of PDI which contributes to understand why this protein family expanded along evolution.

  8. Crystal structure of Aquifex aeolicus gene product Aq1627: a putative phosphoglucosamine mutase reveals a unique C-terminal end-to-end disulfide linkage.

    Science.gov (United States)

    Sridharan, Upasana; Kuramitsu, Seiki; Yokoyama, Shigeyuki; Kumarevel, Thirumananseri; Ponnuraj, Karthe

    2017-06-27

    The Aq1627 gene from Aquifex aeolicus, a hyperthermophilic bacterium has been cloned and overexpressed in Escherichia coli. The protein was purified to homogeneity and its X-ray crystal structure was determined to 1.3 Å resolution using multiple wavelength anomalous dispersion phasing. The structural and sequence analysis of Aq1627 is suggestive of a putative phosphoglucosamine mutase. The structural features of Aq1627 further indicate that it could belong to a new subclass of the phosphoglucosamine mutase family. Aq1627 structure contains a unique C-terminal end-to-end disulfide bond, which links two monomers and this structural information can be used in protein engineering to make proteins more stable in different applications.

  9. Changes in Thiol-Disulfide Homeostasis of the Body to Surgical Trauma in Laparoscopic Cholecystectomy Patients.

    Science.gov (United States)

    Polat, Murat; Ozcan, Onder; Sahan, Leyla; Üstündag-Budak, Yasemin; Alisik, Murat; Yilmaz, Nigar; Erel, Özcan

    2016-12-01

    We aimed to investigate the short-term effect of laparoscopic surgery on serum thiol-disulfide homeostasis levels as a marker of oxidant stress of surgical trauma in elective laparoscopic cholecystectomy patients. Venous blood samples were collected, and levels of native thiols, total thiols, and disulfides were determined with a novel automated assay. Total antioxidant capacity (measured as the ferric-reducing ability of plasma) and serum ischemia modified albumin, expressed as absorbance units assayed by the albumin cobalt binding test, were determined. The major findings of the present study were that native thiol (283 ± 45 versus 241 ± 61 μmol/L), total thiol (313 ± 49 versus 263 ± 67 μmol/L), and disulfide (14.9 ± 4.6 versus 11.0 ± 6.1 μmol/L) levels were decreased significantly during operation and although they increased, they did not return to preoperation levels 24 hours after laparoscopic surgery compared to the levels at baseline. Disulfide/native thiol and disulfide/total thiol levels did not change during laparoscopic surgery. The decrease in plasma level of native and total thiol groups suggests impairment of the antioxidant capacity of plasma; however, the delicate balance between the different redox forms of thiols was maintained during surgery.

  10. Enhancement of anti-tumor activity of hybrid peptide in conjugation with carboxymethyl dextran via disulfide linkers.

    Science.gov (United States)

    Gaowa, Arong; Horibe, Tomohisa; Kohno, Masayuki; Tabata, Yasuhiko; Harada, Hiroshi; Hiraoka, Masahiro; Kawakami, Koji

    2015-05-01

    To improve the anti-tumor activity of EGFR2R-lytic hybrid peptide, we prepared peptide-modified dextran conjugates with the disulfide bonds between thiolated carboxymethyl dextran (CMD-Cys) and cysteine-conjugated peptide (EGFR2R-lytic-Cys). In vitro release studies showed that the peptide was released from the CMD-s-s-peptide conjugate in a concentration-dependent manner in the presence of glutathione (GSH, 2μM-2mM). The CMD-s-s-peptide conjugate exhibited a similar cytotoxic activity with free peptide alone against human pancreatic cancer BxPC-3 cells in vitro. Furthermore, it was shown that the CMD-s-s-peptide conjugates were highly accumulated in tumor tissue in a mouse xenograft model using BxPC-3 cells, and the anti-tumor activity of the conjugate was more effective than that of the free peptide. In addition, the plasma concentrations of peptide were moderately increased and the elimination half-life of the peptide was prolonged after intravenous injection of CMD-s-s-peptide conjugates. These results demonstrated that the conjugate based on thiolated CMD polymer would be potentially useful carriers for the sustained release of the hybrid peptide in vivo. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Redox and pH Responsive Poly (Amidoamine Dendrimer-Heparin Conjugates via Disulfide Linkages for Letrozole Delivery

    Directory of Open Access Journals (Sweden)

    Thanh Luan Nguyen

    2017-01-01

    Full Text Available Heparin (Hep conjugated to poly (amidoamine dendrimer G3.5 (P via redox-sensitive disulfide bond (P-SS-Hep was studied. The redox and pH dual-responsive nanocarriers were prepared by a simple method that minimized many complex steps as previous studies. The functional characterization of G3.5 coated Hep was investigated by the proton nuclear magnetic resonance spectroscopy. The size and formation were characterized by the dynamic light scattering, zeta potential, and transmission electron microscopy. P-SS-Hep was spherical in shape with average diameter about 11 nm loaded with more than 20% letrozole. This drug carrier could not only eliminate toxicity to cells and improve the drugs solubility but also increase biocompatibility of the system under reductive environment of glutathione. In particular, P-SS-Hep could enhance the effectiveness of cancer therapy after removing Hep from the surface. These results demonstrated that the P-SS-Hep conjugates could be a promising candidate as redox and pH responsive nanocarriers for cancer chemotherapy.

  12. Interface passivation and trap reduction via hydrogen fluoride for molybdenum disulfide on silicon oxide back-gate transistors

    Science.gov (United States)

    Hu, Yaoqiao; San Yip, Pak; Tang, Chak Wah; Lau, Kei May; Li, Qiang

    2018-04-01

    Layered semiconductor molybdenum disulfide (MoS2) has recently emerged as a promising material for flexible electronic and optoelectronic devices because of its finite bandgap and high degree of gate control. Here, we report a hydrogen fluoride (HF) passivation technique for improving the carrier mobility and interface quality of chemical vapor deposited monolayer MoS2 on a SiO2/Si substrate. After passivation, the fabricated MoS2 back-gate transistors demonstrate a more than double improvement in average electron mobility, a reduced gate hysteresis gap of 3 V, and a low interface trapped charge density of ˜5.8 × 1011 cm-2. The improvements are attributed to the satisfied interface dangling bonds, thus a reduction of interface trap states and trapped charges. Surface x-ray photoelectron spectroscopy analysis and first-principles simulation were performed to verify the HF passivation effect. The results here highlight the necessity of a MoS2/dielectric passivation strategy and provides a viable route for enhancing the performance of MoS2 nano-electronic devices.

  13. Prokaryotic soluble overexpression and purification of bioactive human growth hormone by fusion to thioredoxin, maltose binding protein, and protein disulfide isomerase.

    Directory of Open Access Journals (Sweden)

    Minh Tan Nguyen

    Full Text Available Human growth hormone (hGH is synthesized by somatotroph cells of the anterior pituitary gland and induces cell proliferation and growth. This protein has been approved for the treatment of various conditions, including hGH deficiency, chronic renal failure, and Turner syndrome. Efficient production of hGH in Escherichia coli (E. coli has proven difficult because the E. coli-expressed hormone tends to aggregate and form inclusion bodies, resulting in poor solubility. In this study, seven N-terminal fusion partners, hexahistidine (His6, thioredoxin (Trx, glutathione S-transferase (GST, maltose-binding protein (MBP, N-utilization substance protein A (NusA, protein disulfide bond isomerase (PDI, and the b'a' domain of PDI (PDIb'a', were tested for soluble overexpression of codon-optimized hGH in E. coli. We found that MBP and hPDI tags significantly increased the solubility of the hormone. In addition, lowering the expression temperature to 18°C also dramatically increased the solubility of all the fusion proteins. We purified hGH from MBP-, PDIb'a'-, or Trx-tagged hGH expressed at 18°C in E. coli using simple chromatographic techniques and compared the final purity, yield, and activity of hGH to assess the impact of each partner protein. Purified hGH was highly pure on silver-stained gel and contained very low levels of endotoxin. On average, ∼37 mg, ∼12 mg, and ∼7 mg of hGH were obtained from 500 mL-cell cultures of Trx-hGH, MBP-hGH, and PDIb'a'-hGH, respectively. Subsequently, hGH was analyzed using mass spectroscopy to confirm the presence of two intra-molecular disulfide bonds. The bioactivity of purified hGHs was demonstrated using Nb2-11 cell.

  14. BONDING ALUMINUM METALS

    Science.gov (United States)

    Noland, R.A.; Walker, D.E.

    1961-06-13

    A process is given for bonding aluminum to aluminum. Silicon powder is applied to at least one of the two surfaces of the two elements to be bonded, the two elements are assembled and rubbed against each other at room temperature whereby any oxide film is ruptured by the silicon crystals in the interface; thereafter heat and pressure are applied whereby an aluminum-silicon alloy is formed, squeezed out from the interface together with any oxide film, and the elements are bonded.

  15. Copper wire bonding

    CERN Document Server

    Chauhan, Preeti S; Zhong, ZhaoWei; Pecht, Michael G

    2014-01-01

    This critical volume provides an in-depth presentation of copper wire bonding technologies, processes and equipment, along with the economic benefits and risks.  Due to the increasing cost of materials used to make electronic components, the electronics industry has been rapidly moving from high cost gold to significantly lower cost copper as a wire bonding material.  However, copper wire bonding has several process and reliability concerns due to its material properties.  Copper Wire Bonding book lays out the challenges involved in replacing gold with copper as a wire bond material, and includes the bonding process changes—bond force, electric flame off, current and ultrasonic energy optimization, and bonding tools and equipment changes for first and second bond formation.  In addition, the bond–pad metallurgies and the use of bare and palladium-coated copper wires on aluminum are presented, and gold, nickel and palladium surface finishes are discussed.  The book also discusses best practices and re...

  16. Multi-terminal memtransistors from polycrystalline monolayer molybdenum disulfide

    Science.gov (United States)

    Sangwan, Vinod K.; Lee, Hong-Sub; Bergeron, Hadallia; Balla, Itamar; Beck, Megan E.; Chen, Kan-Sheng; Hersam, Mark C.

    2018-02-01

    Memristors are two-terminal passive circuit elements that have been developed for use in non-volatile resistive random-access memory and may also be useful in neuromorphic computing. Memristors have higher endurance and faster read/write times than flash memory and can provide multi-bit data storage. However, although two-terminal memristors have demonstrated capacity for basic neural functions, synapses in the human brain outnumber neurons by more than a thousandfold, which implies that multi-terminal memristors are needed to perform complex functions such as heterosynaptic plasticity. Previous attempts to move beyond two-terminal memristors, such as the three-terminal Widrow-Hoff memristor and field-effect transistors with nanoionic gates or floating gates, did not achieve memristive switching in the transistor. Here we report the experimental realization of a multi-terminal hybrid memristor and transistor (that is, a memtransistor) using polycrystalline monolayer molybdenum disulfide (MoS2) in a scalable fabrication process. The two-dimensional MoS2 memtransistors show gate tunability in individual resistance states by four orders of magnitude, as well as large switching ratios, high cycling endurance and long-term retention of states. In addition to conventional neural learning behaviour of long-term potentiation/depression, six-terminal MoS2 memtransistors have gate-tunable heterosynaptic functionality, which is not achievable using two-terminal memristors. For example, the conductance between a pair of floating electrodes (pre- and post-synaptic neurons) is varied by a factor of about ten by applying voltage pulses to modulatory terminals. In situ scanning probe microscopy, cryogenic charge transport measurements and device modelling reveal that the bias-induced motion of MoS2 defects drives resistive switching by dynamically varying Schottky barrier heights. Overall, the seamless integration of a memristor and transistor into one multi-terminal device could

  17. New analogs of the CART peptide with anorexigenic potency: the importance of individual disulfide bridges.

    Science.gov (United States)

    Blechová, Miroslava; Nagelová, Veronika; Záková, Lenka; Demianová, Zuzana; Zelezná, Blanka; Maletínská, Lenka

    2013-01-01

    The CART (cocaine- and amphetamine-regulated transcript) peptide is an anorexigenic neuropeptide that acts in the hypothalamus. The receptor and the mechanism of action of this peptide are still unknown. In our previous study, we showed that the CART peptide binds specifically to PC12 rat pheochromocytoma cells in both the native and differentiated into neuronal phenotype. Two biologically active forms, CART(55-102) and CART(61-102), with equal biological activity, contain three disulfide bridges. To clarify the importance of each of these disulfide bridges in maintaining the biological activity of CART(61-102), an Ala scan at particular S-S bridges forming cysteines was performed, and analogs with only one or two disulfide bridges were synthesized. In this study, a stabilized CART(61-102) analog with norleucine instead of methionine at position 67 was also prepared and was found to bind to PC12 cells with an anorexigenic potency similar to that of CART(61-102). The binding study revealed that out of all analogs tested, [Ala(68,86)]CART(61-102), which contains two disulfide bridges (positions 74-94 and 88-101), preserved a high affinity to both native PC12 cells and those that had been differentiated into neurons. In food intake and behavioral tests with mice after intracerebroventricular administration, this analog showed strong and long-lasting anorexigenic potency. Therefore, the disulfide bridge between cysteines 68 and 86 in CART(61-102) can be omitted without a loss of biological activity, but the preservation of two other disulfide bridges and the full-length peptide are essential for biological activity. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. The impact of disulfide bond dynamics in wheat gluten protein on the development of fermented pastry crumb.

    Science.gov (United States)

    Ooms, Nand; Jansens, Koen J A; Pareyt, Bram; Reyniers, Stijn; Brijs, Kristof; Delcour, Jan A

    2018-03-01

    Gluten proteins functionality during pastry production was examined by including redox agents in the ingredient bill. Addition of reducing and oxidizing agents respectively increased and decreased dough height during fermentation. The presence of large gas bubbles in the samples with oxidizing agents may have caused a 'stacking'-effect and a more effective dough lift. During baking, the level of extractable proteins decreased to comparable values for all samples, except when potassium iodate (KIO 3 ) was used in the recipe. As a result of its use, a lower level of gliadin was incorporated into the gluten polymer and dough layers tended to 'slide' apart during baking, thereby causing collapse. Most likely, KIO 3 caused glutenin oxidation within each individual dough layer to such extent during the dough stage that insufficient thiol groups were available for forming dough layer interconnections during baking, after margarine melting. Furthermore, addition of redox agents impacted the product's crumb structure. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Cyclic voltammetric study of the redox system of glutathione using the disulfide bond reductant tris(2-carboxyethyl)phosphine

    Czech Academy of Sciences Publication Activity Database

    Kizek, René; Vacek, Jan; Trnková, L.; Jelen, František

    2004-01-01

    Roč. 63, 1-2 (2004), s. 19-24 ISSN 1567-5394 R&D Projects: GA AV ČR IAA1163201; GA ČR GA203/02/0422 Institutional research plan: CEZ:AV0Z5004920 Keywords : voltammetry * hanging mercury drop electrode (HMDE) * glutathione (GSH, GSSG) Subject RIV: BO - Biophysics Impact factor: 2.261, year: 2004

  20. High-molecular-weight polymers containing biodegradable disulfide bonds: synthesis and in vitro verification of intracellular degradation

    Czech Academy of Sciences Publication Activity Database

    Etrych, Tomáš; Kovář, Lubomír; Šubr, Vladimír; Braunová, Alena; Pechar, Michal; Chytil, Petr; Říhová, Blanka; Ulbrich, Karel

    2010-01-01

    Roč. 25, č. 1 (2010), s. 5-26 ISSN 0883-9115 R&D Projects: GA AV ČR IAA400500806; GA AV ČR KAN200200651 Institutional research plan: CEZ:AV0Z40500505; CEZ:AV0Z50200510 Keywords : water-soluble polymers * reductive degradation * HPMA copolymers Subject RIV: CD - Macromolecular Chemistry Impact factor: 2.610, year: 2010

  1. Acid-Induced Cold Gelation of Globular Proteins: Effects of Protein Aggregate Characteristics and Disulfide Bonding on Rheological Properties

    NARCIS (Netherlands)

    Alting, A.C.; Weijers, M.; Hoog, E.H.A. de; Pijpekamp, A.M. van de; Cohen Stuart, M.A.; Hamer, R.J.; Kruif, C.G. de; Visschers, R.W.

    2004-01-01

    The process of cold gelation of ovalbumin and the properties of the resulting cold-set gels were compared to those of whey protein isolate. Under the chosen heating conditions, most protein was organized in aggregates. For both protein preparations, the aggregates consisted of covalently linked

  2. UV-C irradiation disrupts platelet surface disulfide bonds and activates the platelet integrin alphaIIbbeta3

    NARCIS (Netherlands)

    Verhaar, Robin; Dekkers, David W. C.; de Cuyper, Iris M.; Ginsberg, Mark H.; de Korte, Dirk; Verhoeven, Arthur J.

    2008-01-01

    UV-C irradiation has been shown to be effective for pathogen reduction in platelet concentrates, but preliminary work indicated that UV-C irradiation of platelets can induce platelet aggregation. In this study, the mechanism underlying this phenomenon was investigated. Irradiation of platelets with

  3. Synthesis of the proteinase inhibitor LEKTI domain 6 by the fragment condensation method and regioselective disulfide bond formation.

    Science.gov (United States)

    Vasileiou, Zoe; Barlos, Kostas K; Gatos, Dimitrios; Adermann, Knut; Deraison, Celine; Barlos, Kleomenis

    2010-01-01

    Proteinase inhibitors are of high pharmaceutical interest and are drug candidates for a variety of indications. Specific kallikrein inhibitors are important for their antitumor activity and their potential application to the treatment of skin diseases. In this study we describe the synthesis of domain 6 of the kallikrein inhibitor Lympho-Epithilial Kazal-Type Inhibitor (LEKTI) by the fragment condensation method and site-directed cystine bridge formation. To obtain the linear LEKTI precursor, the condensation was best performed in solution, coupling the protected fragment 1-22 to 23-68. This method yielded LEKTI domain 6 of high purity and equipotent to the recombinantly produced peptide. (c) 2010 Wiley Periodicals, Inc.

  4. On the healing mechanism of sol-gel derived hybrid materials containing dynamic di-sulfide bonds

    NARCIS (Netherlands)

    AbdolahZadeh, M.; Esteves, A.C.C.; Van der Zwaag, S.; Garcia Espallargas, S.J.

    2013-01-01

    Sol-gel technology is increasingly being used in coatings for corrosion protection and adhesion improvement. So far, the self-healing concept in sol-gel coatings has only been approached from extrinsic healing perspective (i.e. use of nano and micro carriers of corrosion inhibitors) [1]. Despite the

  5. Beta-Cyclodextrin Duplexes That Are Connected through Two Disulfide Bonds: Potent Hosts for the Complexation of Organic Molecules

    Czech Academy of Sciences Publication Activity Database

    Grishina, Anastasia; Stanchev, Stancho; Kumprecht, Lukáš; Buděšínský, Miloš; Pojarová, Michaela; Dušek, Michal; Rumlová, Michaela; Křížová, Ivana; Rulíšek, Lubomír; Kraus, Tomáš

    2012-01-01

    Roč. 18, č. 39 (2012), s. 12292-12304 ISSN 0947-6539 Grant - others:AV ČR(CZ) M200550904 Institutional support: RVO:61388963 ; RVO:68378271 Keywords : cucurbiturils * cyclodextrins * duplexes * host-guest systems * sulfur Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 5.831, year: 2012

  6. Transversely Compressed Bonded Joints

    DEFF Research Database (Denmark)

    Hansen, Christian Skodborg; Schmidt, Jacob Wittrup; Stang, Henrik

    2012-01-01

    The load capacity of bonded joints can be increased if transverse pressure is applied at the interface. The transverse pressure is assumed to introduce a Coulomb-friction contribution to the cohesive law for the interface. Response and load capacity for a bonded single-lap joint was derived using...

  7. Cytoplasmic glutathione redox status determines survival upon exposure to the thiol-oxidant 4,4'-dipyridyl disulfide

    DEFF Research Database (Denmark)

    López-Mirabal, H Reynaldo; Thorsen, Michael; Kielland-Brandt, Morten C

    2007-01-01

    Dipyridyl disulfide (DPS) is a highly reactive thiol oxidant that functions as electron acceptor in thiol-disulfide exchange reactions. DPS is very toxic to yeasts, impairing growth at low micromolar concentrations. The genes TRX2 (thioredoxin), SOD1 (superoxide dismutase), GSH1 (gamma-glutamyl-c......Dipyridyl disulfide (DPS) is a highly reactive thiol oxidant that functions as electron acceptor in thiol-disulfide exchange reactions. DPS is very toxic to yeasts, impairing growth at low micromolar concentrations. The genes TRX2 (thioredoxin), SOD1 (superoxide dismutase), GSH1 (gamma...... antioxidant pools of glutathione (GSH) and thioredoxin are required for resistance to DPS. We found that DPS-sensitive mutants display increases in the disulfide form of GSH (GSSG) during DPS exposure that roughly correlate with their more oxidizing GSH redox potential in the cytosol and their degree of DPS...

  8. Corporate Bonds in Denmark

    DEFF Research Database (Denmark)

    Tell, Michael

    2015-01-01

    Corporate financing is the choice between capital generated by the corporation and capital from external investors. However, since the financial crisis shook the markets in 2007–2008, financing opportunities through the classical means of financing have decreased. As a result, corporations have...... to think in alternative ways such as issuing corporate bonds. A market for corporate bonds exists in countries such as Norway, Germany, France, the United Kingdom and the United States, while Denmark is still behind in this trend. Some large Danish corporations have instead used foreign corporate bonds...... markets. However, NASDAQ OMX has introduced the First North Bond Market in December 2012 and new regulatory framework came into place in 2014, which may contribute to a Danish based corporate bond market. The purpose of this article is to present the regulatory changes in Denmark in relation to corporate...

  9. Dynamic thiol/disulfide homeostasis and effects of smoking on homeostasis parameters in patients with psoriasis.

    Science.gov (United States)

    Emre, Selma; Demirseren, Duriye Deniz; Alisik, Murat; Aktas, Akin; Neselioglu, Salim; Erel, Ozcan

    2017-12-01

    Recently, increased reactive oxygen species (ROS), reduced antioxidant capacity, and oxidative stress have been suggested in the pathogenesis of psoriasis. The aim of this study to evaluate the thiol/disulfide homeostasis in patients with psoriasis. Ninety patients with psoriasis who did not receive any systemic treatment in the last six  months were included in the study. Seventy-six age and gender-matched healthy volunteers served as control group. Thiol/disulfide homeostasis was measured in venous blood samples obtained from patient and control groups. Native thiol and total thiol levels were significantly higher in patients than in control group. When thiol/disulfide hemostasis parameters and clinical and demographic characteristics were compared, a negative correlation was detected between native thiol and total thiol with age. The levels of total thiols had also negative correlation with PASI and duration of the disease. When we divided the patients into smokers and non-smokers, native thiol and total thiol levels were significantly higher in smokers than in controls, whereas native thiol and total thiol levels were comparable in non-smoker patients and controls. Thiol/disulfide balance shifted towards thiol in psoriasis patients and this may be responsible for increased keratinocyte proliferation in the pathogenesis of psoriasis.

  10. Differential Labeling of Free and Disulfide-Bound Thiol Functions in Proteins

    NARCIS (Netherlands)

    Seiwert, B.; Hayen, H.; Karst, U.

    2008-01-01

    A method for the simultaneous determination of the number of free cysteine groups and disulfide-bound cysteine groups in proteins has been developed based on the sequential labeling of free and bound thiol functionalities with two ferrocene-based maleimide reagents. Liquid

  11. New thiol-responsive mono-cleavable block copolymer micelles labeled with single disulfides.

    Science.gov (United States)

    Sourkohi, Behnoush Khorsand; Schmidt, Rolf; Oh, Jung Kwon

    2011-10-18

    Thiol-responsive symmetric triblock copolymers having single disulfide linkages in the middle blocks (called mono-cleavable block copolymers, ss-ABP(2)) were synthesized by atom transfer radical polymerization in the presence of a disulfide-labeled difunctional Br-initiator. These brush-like triblock copolymers consist of a hydrophobic polyacrylate block having pendent oligo(propylene oxide) and a hydrophilic polymethacrylate block having pendent oligo(ethylene oxide). Gel permeation chromatography and (1)H NMR results confirmed the synthesis of well-defined mono-cleavable block copolymers and revealed that polymerizations were well controlled. Because of amphiphilic nature, these copolymers self-assembled to form colloidally stable micelles above critical micellar concentration of 0.032 mg · mL(-1). In response to reductive reactions, disulfides in thiol-responsive micelles were cleaved. Atomic force microscopy and dynamic light scattering analysis suggested that the cleavage of disulfides caused dissociation of micelles to smaller-sized assembled structures in water. Moreover, in a biomedical perspective, the mono-cleavable block copolymer micelles are not cytotoxic and thus biocompatible. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Differential expression of disulfide reductase enzymes in a free-living platyhelminth (Dugesia dorotocephala.

    Directory of Open Access Journals (Sweden)

    Alberto Guevara-Flores

    Full Text Available A search of the disulfide reductase activities expressed in the adult stage of the free-living platyhelminth Dugesia dorotocephala was carried out. Using GSSG or DTNB as substrates, it was possible to obtain a purified fraction containing both GSSG and DTNB reductase activities. Through the purification procedure, both disulfide reductase activities were obtained in the same chromatographic peak. By mass spectrometry analysis of peptide fragments obtained after tryptic digestion of the purified fraction, the presence of glutathione reductase (GR, thioredoxin-glutathione reductase (TGR, and a putative thioredoxin reductase (TrxR was detected. Using the gold compound auranofin to selectively inhibit the GSSG reductase activity of TGR, it was found that barely 5% of the total GR activity in the D. dorotocephala extract can be assigned to GR. Such strategy did allow us to determine the kinetic parameters for both GR and TGR. Although It was not possible to discriminate DTNB reductase activity due to TrxR from that of TGR, a chromatofocusing experiment with a D. dorotocephala extract resulted in the obtention of a minor protein fraction enriched in TrxR, strongly suggesting its presence as a functional protein. Thus, unlike its parasitic counterparts, in the free-living platyhelminth lineage the three disulfide reductases are present as functional proteins, albeit TGR is still the major disulfide reductase involved in the reduction of both Trx and GSSG. This fact suggests the development of TGR in parasitic flatworms was not linked to a parasitic mode of life.

  13. Selective removal of heavy metal ions by disulfide linked polymer networks

    Energy Technology Data Exchange (ETDEWEB)

    Ko, Dongah [Department of Environmental Engineering, Technical University of Denmark, Miljøvej 113, 2800 Kgs. Lyngby (Denmark); Lee, Joo Sung [Graduate School of EEWS, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141 (Korea, Republic of); Patel, Hasmukh A. [Department of Chemistry, Northwestern University, Evanston, IL 60208 (United States); Jakobsen, Mogens H. [Department of Micro and Nano technology, Technical University of Denmark, Ørsteds Plads, 345B, 2800 Kgs. Lyngby (Denmark); Hwang, Yuhoon [Department of Environmental Engineering, Seoul National University of Science and Technology, 232 Gongreung-ro, Nowon-gu, Seoul 01811 (Korea, Republic of); Yavuz, Cafer T. [Graduate School of EEWS, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141 (Korea, Republic of); Hansen, Hans Chr. Bruun [Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg, Thorvaldsensvej 40, 1871 Frederiksberg C (Denmark); Andersen, Henrik R., E-mail: henrik@ndersen.net [Department of Environmental Engineering, Technical University of Denmark, Miljøvej 113, 2800 Kgs. Lyngby (Denmark)

    2017-06-15

    Highlights: • Disulfide/thiol polymer networks are promising as sorbent for heavy metals. • Rapid sorption and high Langmuir affinity constant (a{sub L}) for stormwater treatment. • Selective sorption for copper, cadmium, and zinc in the presence of calcium. • Reusability likely due to structure stability of disulfide linked polymer networks. - Abstract: Heavy metal contaminated surface water is one of the oldest pollution problems, which is critical to ecosystems and human health. We devised disulfide linked polymer networks and employed as a sorbent for removing heavy metal ions from contaminated water. Although the polymer network material has a moderate surface area, it demonstrated cadmium removal efficiency equivalent to highly porous activated carbon while it showed 16 times faster sorption kinetics compared to activated carbon, owing to the high affinity of cadmium towards disulfide and thiol functionality in the polymer network. The metal sorption mechanism on polymer network was studied by sorption kinetics, effect of pH, and metal complexation. We observed that the metal ions–copper, cadmium, and zinc showed high binding affinity in polymer network, even in the presence of competing cations like calcium in water.

  14. Research of the technology of obtaining pure and disperse molybdenum disulfide from molybdenum concentrate

    International Nuclear Information System (INIS)

    Hovsepyan, A.H.; Israyelyan, S.M.

    2009-01-01

    The technology of obtaining pure and disperse molybdenum disulfide is worked out. The processes of refinement from the flotation reagents and deslimation by means of decantation, refinement of molybdenite concentrate from impurities by selective leaching methods are studied. The optimal regime of technological process is chosen

  15. Decontamination of Oils Contaminated with Polychlorinated Biphenyls and Dibenzyl Disulfide Using Polar Aprotic Solvents

    Czech Academy of Sciences Publication Activity Database

    Kaštánek, František; Matějková, Martina; Spáčilová, Lucie; Maléterová, Ywetta; Kaštánek, P.; Šolcová, Olga

    2015-01-01

    Roč. 4, č. 2 (2015), s. 41-48 ISSN 2319-5967 R&D Projects: GA TA ČR(CZ) TA04020151 Institutional support: RVO:67985858 Keywords : corrosive sulfur * dibenzyl disulfide * polar aprotic solvents Subject RIV: CI - Industrial Chemistry, Chemical Engineering http://www.ijesit.com/Volume%204/Issue%202/IJESIT201502_06.pdf

  16. Synthesis of tetraalkyl thiuram disulfides using different oxidants in recycling solvent mixture

    Directory of Open Access Journals (Sweden)

    Milosavljević Milutin M.

    2012-01-01

    Full Text Available A new optimized laboratory synthesis of tetraalkyl thiuram disulfides, starting from dialkyl amines and carbon disulfide in presence of three oxidants (hydrogen peroxide, potassium peroxodisulfate and sodium hypochlorite and appropriate reaction medium: two mixtures of isopropyl alcohol - water used in two consecutive syntheses, was presented in this work. First synthesis was performed in a recycled azeotropic mixture of isopropyl alcohol - water 87.7% - 12.3%, and second in a filtrate obtained after first synthesis, which was a mixture of isopropyl alcohol - water 70.4% - 29.6%. After the second synthesis and filtration, recycled azeotropic mixture isopropyl alcohol - water 87.7% - 12.3% was regenerated from the filtrate by rectification. Considering this, the technology for beneficial use of recycling isopropyl alcohol - water mixture as reaction medium for tetraalkyl thiuram disulfides synthesis was developed. Such concept contributes to extraordinary economical benefit of implemented optimal laboratory synthesis at semi-industrial level. High yields of tetraalkyl thiuram disulfides syntheses were obtained at both laboratory and semiindustrial level. Structure and purity of synthesized compounds were confirmed by elemental analysis, as well as FTIR, 1H and 13C NMR, and MS spectral data.

  17. Efficacy of HOCl scavenging by sulfur-containing compounds: antioxidant activity of glutathione disulfide?

    NARCIS (Netherlands)

    den Hartog, G.J.M.; Haenen, G.R.M.M.; Vegt, E.; van der Vijgh, W.J.F.; Bast, A.

    2002-01-01

    Efficacy of HOCl scavenging by sulfur-containing compounds: antioxidant activity of glutathione disulfide? den Hartog GJ, Haenen GR, Vegt E, van der Vijgh WJ, Bast A. Department of Pharmacology and Toxicology, Maastricht University, The Netherlands. Hypochlorous acid (HOCl) is a bactericidal

  18. Bond markets in Africa

    Directory of Open Access Journals (Sweden)

    Yibin Mu

    2013-07-01

    Full Text Available African bond markets have been steadily growing in recent years, but nonetheless remain undeveloped. African countries would benefit from greater access to financing and deeper financial markets. This paper compiles a unique set of data on government securities and corporate bond markets in Africa. It then applies an econometric model to analyze the key determinants of African government securities market and corporate bond market capitalization. Government securities market capitalization is directly related to better institutions and interest rate volatility, and inversely related to smaller fiscal deficits, higher interest rate spreads, exchange rate volatility, and current and capital account openness. Corporate bond market capitalization is directly linked to economic size, the level of development of the economy and financial markets, better institutions, and interest rate volatility, and inversely related to higher interest rate spreads and current account openness. Policy implications follow.

  19. Handbook of wafer bonding

    CERN Document Server

    Ramm, Peter; Taklo, Maaike M V

    2011-01-01

    Written by an author and editor team from microsystems companies and industry-near research organizations, this handbook and reference presents dependable, first-hand information on bonding technologies.In the first part, researchers from companies and institutions around the world discuss the most reliable and reproducible technologies for the production of bonded wafers. The second part is devoted to current and emerging applications, including microresonators, biosensors and precise measuring devices.

  20. Diffusion bonding techniques

    International Nuclear Information System (INIS)

    Peters, R.D.

    1978-01-01

    The applications of diffusion bonding at the General Electric Neutron Devices Department are briefly discussed, with particular emphasis on the gold/gold or gold/indium joints made between metallized alumina ceramic parts in the vacuum switch tube and the crystal resonator programs. Fixtures which use the differential expansion of dissimilar metals are described and compared to one that uses hydraulic pressure to apply the necessary bonding force

  1. Effects of polybrominated diphenyl ethers (PBDEs) and their derivatives on protein disulfide isomerase activity and growth hormone release of GH3 cells.

    Science.gov (United States)

    Hashimoto, Shoko; Yoshimura, Hiromi; Okada, Kazushi; Uramaru, Naoto; Sugihara, Kazumi; Kitamura, Shigeyuki; Imaoka, Susumu

    2012-03-19

    Polybrominated diphenyl ethers (PBDEs) have been used in a variety of consumer products such as flame retardants and recently have been known to be widespread environmental pollutants, which probably affect biological functions of mammalian cells. However, the risk posed by PBDE metabolites has not been clarified. Our previous study suggested that bisphenol A (BPA), an endocrine-disrupting chemical, binds to protein disulfide isomerase (PDI) and inhibits its activity. PDI is an isomerase enzyme in the endoplasmic reticulum and facilitates the formation or cleavage of disulfide bonds. PDI consists of a, b, b', and a' domains and the c region, with the a and a' domains having isomerase active sites. In the present study, we tested the effects of 10 kinds of PBDE compounds and their metabolites on PDI. OH-PBDEs specifically inhibited the isomerase activity of PDI, with 4'-OH-PBDE more effective than 2' (or 2)-OH-PBDEs. 4'-OH-PBDE inhibited the isomerase activity of the b'a'c fragment but not that of ab and a'c, suggesting that the b' domain of PDI is essential for the inhibition by 4'-OH-PBDE. We also investigated the effects of these chemicals on the production of growth hormone (GH) in GH3 cells. In GH3 cells, levels of mRNA and protein of GH stimulated by T(3) were reduced by 4'-OH-PBDE and 4'-MeO-PBDE. The reduction in GH expression caused by these compounds was not changed by the overexpression or knockdown of PDI in GH3 cells, while these manipulations of PDI levels significantly suppressed the expression of GH. These results suggest that the biological effects of PBDEs differed depending on their brominated and hydroxylated positions. © 2011 American Chemical Society

  2. Disulfide-induced self-assembled targets: A novel strategy for the label free colorimetric detection of DNAs/RNAs via unmodified gold nanoparticles

    Science.gov (United States)

    Shokri, Ehsan; Hosseini, Morteza; Davari, Mehdi D.; Ganjali, Mohammad R.; Peppelenbosch, Maikel P.; Rezaee, Farhad

    2017-04-01

    A modified non-cross-linking gold-nanoparticles (Au-NPs) aggregation strategy has been developed for the label free colorimetric detection of DNAs/RNAs based on self-assembling target species in the presence of thiolated probes. Two complementary thiol- modified probes, each of which specifically binds at one half of the target introduced SH groups at both ends of dsDNA. Continuous disulfide bond formation at 3‧ and 5‧ terminals of targets leads to the self-assembly of dsDNAs into the sulfur- rich and flexible products with different lengths. These products have a high affinity for the surface of Au-NPs and efficiently protect the surface from salt induced aggregation. To evaluate the assay efficacy, a small part of the citrus tristeza virus (CTV) genome was targeted, leading to a detection limit of about 5 × 10-9 mol.L-1 over a linear ranged from 20 × 10-9 to 10 × 10-7 mol.L-1. This approach also exhibits good reproducibility and recovery levels in the presence of plant total RNA or human plasma total circulating RNA extracts. Self-assembled targets can be then sensitively distinguished from non-assembled or mismatched targets after gel electrophoresis. The disulfide reaction method and integrating self-assembled DNAs/RNAs targets with bare AuNPs as a sensitive indicator provide us a powerful and simple visual detection tool for a wide range of applications.

  3. Inhibition of the functional interplay between endoplasmic reticulum (ER) oxidoreduclin-1α (Ero1α) and protein-disulfide isomerase (PDI) by the endocrine disruptor bisphenol A.

    Science.gov (United States)

    Okumura, Masaki; Kadokura, Hiroshi; Hashimoto, Shoko; Yutani, Katsuhide; Kanemura, Shingo; Hikima, Takaaki; Hidaka, Yuji; Ito, Len; Shiba, Kohei; Masui, Shoji; Imai, Daiki; Imaoka, Susumu; Yamaguchi, Hiroshi; Inaba, Kenji

    2014-09-26

    Bisphenol A (BPA) is an endocrine disruptor that may have adverse effects on human health. We recently isolated protein-disulfide isomerase (PDI) as a BPA-binding protein from rat brain homogenates and found that BPA markedly inhibited PDI activity. To elucidate mechanisms of this inhibition, detailed structural, biophysical, and functional analyses of PDI were performed in the presence of BPA. BPA binding to PDI induced significant rearrangement of the N-terminal thioredoxin domain of PDI, resulting in more compact overall structure. This conformational change led to closure of the substrate-binding pocket in b' domain, preventing PDI from binding to unfolded proteins. The b' domain also plays an essential role in the interplay between PDI and ER oxidoreduclin 1α (Ero1α), a flavoenzyme responsible for reoxidation of PDI. We show that BPA inhibited Ero1α-catalyzed PDI oxidation presumably by inhibiting the interaction between the b' domain of PDI and Ero1α; the phenol groups of BPA probably compete with a highly conserved tryptophan residue, located in the protruding β-hairpin of Ero1α, for binding to PDI. Consistently, BPA slowed down the reoxidation of PDI and caused the reduction of PDI in HeLa cells, indicating that BPA has a great impact on the redox homeostasis of PDI within cells. However, BPA had no effect on the interaction between PDI and peroxiredoxin-4 (Prx4), another PDI family oxidase, suggesting that the interaction between Prx4 and PDI is different from that of Ero1α and PDI. These results indicate that BPA, a widely distributed and potentially harmful chemical, inhibits Ero1-PDI-mediated disulfide bond formation. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Inhibition of the Functional Interplay between Endoplasmic Reticulum (ER) Oxidoreduclin-1α (Ero1α) and Protein-disulfide Isomerase (PDI) by the Endocrine Disruptor Bisphenol A*

    Science.gov (United States)

    Okumura, Masaki; Kadokura, Hiroshi; Hashimoto, Shoko; Yutani, Katsuhide; Kanemura, Shingo; Hikima, Takaaki; Hidaka, Yuji; Ito, Len; Shiba, Kohei; Masui, Shoji; Imai, Daiki; Imaoka, Susumu; Yamaguchi, Hiroshi; Inaba, Kenji

    2014-01-01

    Bisphenol A (BPA) is an endocrine disruptor that may have adverse effects on human health. We recently isolated protein-disulfide isomerase (PDI) as a BPA-binding protein from rat brain homogenates and found that BPA markedly inhibited PDI activity. To elucidate mechanisms of this inhibition, detailed structural, biophysical, and functional analyses of PDI were performed in the presence of BPA. BPA binding to PDI induced significant rearrangement of the N-terminal thioredoxin domain of PDI, resulting in more compact overall structure. This conformational change led to closure of the substrate-binding pocket in b′ domain, preventing PDI from binding to unfolded proteins. The b′ domain also plays an essential role in the interplay between PDI and ER oxidoreduclin 1α (Ero1α), a flavoenzyme responsible for reoxidation of PDI. We show that BPA inhibited Ero1α-catalyzed PDI oxidation presumably by inhibiting the interaction between the b′ domain of PDI and Ero1α; the phenol groups of BPA probably compete with a highly conserved tryptophan residue, located in the protruding β-hairpin of Ero1α, for binding to PDI. Consistently, BPA slowed down the reoxidation of PDI and caused the reduction of PDI in HeLa cells, indicating that BPA has a great impact on the redox homeostasis of PDI within cells. However, BPA had no effect on the interaction between PDI and peroxiredoxin-4 (Prx4), another PDI family oxidase, suggesting that the interaction between Prx4 and PDI is different from that of Ero1α and PDI. These results indicate that BPA, a widely distributed and potentially harmful chemical, inhibits Ero1-PDI-mediated disulfide bond formation. PMID:25122773

  5. Structural studies of polypeptides: Mechanism of immunoglobin catalysis and helix propagation in hybrid sequence, disulfide containing peptides

    Energy Technology Data Exchange (ETDEWEB)

    Storrs, Richard Wood [Univ. of California, Berkeley, CA (United States)

    1992-08-01

    Catalytic immunoglobin fragments were studied Nuclear Magnetic Resonance spectroscopy to identify amino acid residues responsible for the catalytic activity. Small, hybrid sequence peptides were analyzed for helix propagation following covalent initiation and for activity related to the protein from which the helical sequence was derived. Hydrolysis of p-nitrophenyl carbonates and esters by specific immunoglobins is thought to involve charge complementarity. The pK of the transition state analog P-nitrophenyl phosphate bound to the immunoglobin fragment was determined by 31P-NMR to verify the juxtaposition of a positively charged amino acid to the binding/catalytic site. Optical studies of immunoglobin mediated photoreversal of cis, syn cyclobutane thymine dimers implicated tryptophan as the photosensitizing chromophore. Research shows the chemical environment of a single tryptophan residue is altered upon binding of the thymine dimer. This tryptophan residue was localized to within 20 Å of the binding site through the use of a nitroxide paramagnetic species covalently attached to the thymine dimer. A hybrid sequence peptide was synthesized based on the bee venom peptide apamin in which the helical residues of apamin were replaced with those from the recognition helix of the bacteriophage 434 repressor protein. Oxidation of the disufide bonds occured uniformly in the proper 1-11, 3-15 orientation, stabilizing the 434 sequence in an α-helix. The glycine residue stopped helix propagation. Helix propagation in 2,2,2-trifluoroethanol mixtures was investigated in a second hybrid sequence peptide using the apamin-derived disulfide scaffold and the S-peptide sequence. The helix-stop signal previously observed was not observed in the NMR NOESY spectrum. Helical connectivities were seen throughout the S-peptide sequence. The apamin/S-peptide hybrid binded to the S-protein (residues 21-166 of ribonuclease A) and reconstituted enzymatic activity.

  6. Structural studies of polypeptides: Mechanism of immunoglobin catalysis and helix propagation in hybrid sequence, disulfide containing peptides

    Energy Technology Data Exchange (ETDEWEB)

    Storrs, R.W.

    1992-08-01

    Catalytic immunoglobin fragments were studied Nuclear Magnetic Resonance spectroscopy to identify amino acid residues responsible for the catalytic activity. Small, hybrid sequence peptides were analyzed for helix propagation following covalent initiation and for activity related to the protein from which the helical sequence was derived. Hydrolysis of p-nitrophenyl carbonates and esters by specific immunoglobins is thought to involve charge complementarity. The pK of the transition state analog P-nitrophenyl phosphate bound to the immunoglobin fragment was determined by [sup 31]P-NMR to verify the juxtaposition of a positively charged amino acid to the binding/catalytic site. Optical studies of immunoglobin mediated photoreversal of cis, syn cyclobutane thymine dimers implicated tryptophan as the photosensitizing chromophore. Research shows the chemical environment of a single tryptophan residue is altered upon binding of the thymine dimer. This tryptophan residue was localized to within 20 [Angstrom] of the binding site through the use of a nitroxide paramagnetic species covalently attached to the thymine dimer. A hybrid sequence peptide was synthesized based on the bee venom peptide apamin in which the helical residues of apamin were replaced with those from the recognition helix of the bacteriophage 434 repressor protein. Oxidation of the disufide bonds occured uniformly in the proper 1-11, 3-15 orientation, stabilizing the 434 sequence in an [alpha]-helix. The glycine residue stopped helix propagation. Helix propagation in 2,2,2-trifluoroethanol mixtures was investigated in a second hybrid sequence peptide using the apamin-derived disulfide scaffold and the S-peptide sequence. The helix-stop signal previously observed was not observed in the NMR NOESY spectrum. Helical connectivities were seen throughout the S-peptide sequence. The apamin/S-peptide hybrid binded to the S-protein (residues 21-166 of ribonuclease A) and reconstituted enzymatic activity.

  7. Evaluation of dynamic serum thiol/disulfide homeostasis in locally advanced and metastatic gastric cancer

    Directory of Open Access Journals (Sweden)

    Mutlu Hizal

    2018-04-01

    Full Text Available Background: Gastric cancer is one the most diagnosed cancer and the third leading cause of death from cancer worldwide. As an indicator of antioxidant capacity thiol/disulfide homeostasis regulates detoxification, cell signal mechanisms, apoptosis, transcription and antioxidant defense mechanisms. Disregulation of thiol/disulfide homeostasis identified in other cancer types by recent data. In this study, we aimed to evaluate the thiol/disulfide homeostasis in advanced gastric cancer patients. Methods: The patients who diagnosed with gastric cancer and healthy control subjects were included to study. Serum samples for the thiol-disulphide test were obtained at the time of diagnosis. Thiol-disulphide homeostasis tests were measured by the automated spectrophotometric method. Thiol-disulphide homeostasis was also measured according to clinical and laboratory features. Results: Thirty newly diagnosed advanced gastric adenocarcinoma patients and 28 healthy controls were enrolled in the study. The native thiol (NT and total thiol (TT levels of patients' group were significantly lower compared with controls (p = 0.001 and p < 0.001. In the CEA high (≥5.4 ng/ml group, DS/NT ratio were higher compared with CEA low (<5.4 ng/ml group (p = 0.024. In CA.19-9 high (≥28.3 kU/L group, both DS and DS/NT ratio were significantly higher compared with a CA19-9 low(<28.3 kU/L group (p < 0.05 both. The correlation between CEA and DS levels was also significant (p = 0.02. There was also a positive correlation between CEA levels and DS/NT ratio (p = 0.01. Conclusion: Derangements of thiol/disulfide homeostasis may have a role in gastric cancer pathogenesis and the higher level of oxidative stress may relate to extensive and aggressiveness of the advanced disease. The diagnostic and prognostic values of thiol/disulfide products need to identify with further studies. Keywords: Thiol, Disulfide, Oxidative stress, Gastric cancer, Metastatic

  8. Romanian government bond market

    Directory of Open Access Journals (Sweden)

    Cornelia POP

    2012-12-01

    Full Text Available The present paper aims to present the level of development reached by Romanian government bond market segment, as part of the country financial market. The analysis will be descriptive (the data series available for Romania are short, based on the secondary data offered by the official bodies involved in the process of issuing and trading the Romanian government bonds (Romanian Ministry of Public Finance, Romanian National Bank and Bucharest Stock Exchange, and also on secondary data provided by the Federation of European Stock Exchanges.To enhance the market credibility as a benchmark, a various combination of measures is necessary; among these measures are mentioned: the extension of the yield curve; the issuance calendars in order to improve transparency; increasing the disclosure of information on public debt issuance and statistics; holding regular meetings with dealers, institutional investors and rating agencies; introducing a system of primary dealers; establishing a repurchase (repo market in the government bond market. These measures will be discussed based on the evolution presented inside the paper.The paper conclude with the fact that, until now, the Romanian government bond market did not provide a benchmark for the domestic financial market and that further efforts are needed in order to increase the government bond market transparency and liquidity.

  9. Interstellar hydrogen bonding

    Science.gov (United States)

    Etim, Emmanuel E.; Gorai, Prasanta; Das, Ankan; Chakrabarti, Sandip K.; Arunan, Elangannan

    2018-06-01

    This paper reports the first extensive study of the existence and effects of interstellar hydrogen bonding. The reactions that occur on the surface of the interstellar dust grains are the dominant processes by which interstellar molecules are formed. Water molecules constitute about 70% of the interstellar ice. These water molecules serve as the platform for hydrogen bonding. High level quantum chemical simulations for the hydrogen bond interaction between 20 interstellar molecules (known and possible) and water are carried out using different ab-intio methods. It is evident that if the formation of these species is mainly governed by the ice phase reactions, there is a direct correlation between the binding energies of these complexes and the gas phase abundances of these interstellar molecules. Interstellar hydrogen bonding may cause lower gas abundance of the complex organic molecules (COMs) at the low temperature. From these results, ketenes whose less stable isomers that are more strongly bonded to the surface of the interstellar dust grains have been observed are proposed as suitable candidates for astronomical observations.

  10. Safe and Liquid Mortgage Bonds

    DEFF Research Database (Denmark)

    Dick-Nielsen, Jens; Gyntelberg, Jacob; Lund, Jesper

    This paper shows that strict match pass-through funding of covered bonds provides safe and liquid mortgage bonds. Despite a 30% drop in house prices during the 2008 global crisis Danish mortgage bonds remained as liquid as most European government bonds. The Danish pass-through system effectively...... eliminates credit risk from the investor's perspective. Similar to other safe bonds, funding liquidity becomes the main driver of mortgage bond liquidity and this creates commonality in liquidity across markets and countries. These findings have implications for how to design a robust mortgage bond system...

  11. Identification of Thioredoxin Disulfide Targets Using a Quantitative Proteomics Approach Based on Isotope-Coded Affinity Tags

    DEFF Research Database (Denmark)

    Hägglund, Per; Bunkenborg, Jakob; Maeda, Kenji

    2008-01-01

    Thioredoxin (Trx) is a ubiquitous protein disulfide reductase involved in a wide range of cellular redox processes. A large number of putative target proteins have been identified using proteomics approaches, but insight into target specificity at the molecular level is lacking since the reactivity...... of Trx toward individual disulfides has not been quantified. Here, a novel proteomics procedure is described for quantification of Trx-mediated target disulfide reduction based on thiol-specific differential labeling with the iodoacetamide-based isotope-coded affinity tag (ICAT) reagents. Briefly......, protein extract of embryos from germinated barley seeds was treated +/- Trx, and thiols released from target protein disulfides were irreversibly blocked with iodoacetamide. The remaining cysteine residues in the Trx-treated and the control (-Trx) samples were then chemically reduced and labeled...

  12. Hydrogen bonded supramolecular materials

    CERN Document Server

    Li, Zhan-Ting

    2015-01-01

    This book is an up-to-date text covering topics in utilizing hydrogen bonding for constructing functional architectures and supramolecular materials. The first chapter addresses the control of photo-induced electron and energy transfer. The second chapter summarizes the formation of nano-porous materials. The following two chapters introduce self-assembled gels, many of which exhibit unique functions. Other chapters cover the advances in supramolecular liquid crystals and the versatility of hydrogen bonding in tuning/improving the properties and performance of materials. This book is designed

  13. Continuing bonds and place.

    Science.gov (United States)

    Jonsson, Annika; Walter, Tony

    2017-08-01

    Where do people feel closest to those they have lost? This article explores how continuing bonds with a deceased person can be rooted in a particular place or places. Some conceptual resources are sketched, namely continuing bonds, place attachment, ancestral places, home, reminder theory, and loss of place. The authors use these concepts to analyze interview material with seven Swedes and five Britons who often thought warmly of the deceased as residing in a particular place and often performing characteristic actions. The destruction of such a place, by contrast, could create a troubling, haunting absence, complicating the deceased's absent-presence.

  14. Regional cerebral blood flow after long-term exposure to carbon disulfide

    International Nuclear Information System (INIS)

    Aaserud, O.; Russell, D.; Nyberg-Hansen, R.; Joergensen, E.B.; Gjerstad, L.; Rootwelt, K.; Nakstad, P.; Hommeren, O.J.; Tvedt, B.

    1992-01-01

    Sixteen former rayon viscose workers were investigated four years after the exposure to carbon disulfide was discontinued. Median age was 58 years (range 43-65 years), median exposure time was 17 years (range 10-35 years). Encephalopathy was diagnosed in altogether 14 workers. To further explore pathophysiological mechanisms, cerebrovascular investigations were employed. Doppler ultrasound examination of the precerebral vessels in 15 workers showed a slight stenosis of the left internal carotid artery in one. Regional cerebral blood flow investigation (rCBF) with single photon emission computerized tomography (SPECT) with Xenon-133 gas was performed in 14. There was no significant difference from a control group. Regional side-to-side asymmetries beyond reference limits were demonstrated in eight workers. The abnormalities were modest, but may indicate a tendency toward focal blood flow disturbances in workers with long-term exposure to carbon disulfide. (au)

  15. Reduction-Triggered Transformation of Crosslinking Modules of Disulfide-Containing Micelles with Chemically Tunable Rates.

    Science.gov (United States)

    Deng, Zhengyu; Yuan, Shuai; Xu, Ronald X; Liang, Haojun; Liu, Shiyong

    2018-05-16

    A dilemma exists between the circulation stability and cargo release/mass diffusion at desired sites for designing delivery nanocarriers and in vivo nanoreactors. We herein report disulfide-crosslinked (DCL) micelles exhibiting reduction-triggered switching of crosslinking modules and synchronized hydrophobic-to-hydrophilic transition. Tumor cell-targeted DCL micelles undergo cytoplasmic milieu-triggered disulfide cleavage and cascade self-immolative decaging reactions at chemically adjustable rates, generating primary amine moieties. Extensive amidation reactions with neighboring ester moieties then occur due to high local concentrations and suppression of apparent amine pKa within hydrophobic cores, leading to the transformation of crosslinking modules and formation of tracelessly crosslinked (TCL) micelles with hydrophilic cores inside live cells. We further integrate this design principle with theranostic nanocarriers for selective intracellular drug transport guided by enhanced magnetic resonance (MR) imaging performance. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Rethinking Cysteine Protective Groups: S-Alkylsulfonyl-l-Cysteines for Chemoselective Disulfide Formation.

    Science.gov (United States)

    Schäfer, Olga; Huesmann, David; Muhl, Christian; Barz, Matthias

    2016-12-12

    The ability to reversibly cross-link proteins and peptides grants the amino acid cysteine its unique role in nature as well as in peptide chemistry. We report a novel class of S-alkylsulfonyl-l-cysteines and N-carboxy anhydrides (NCA) thereof for peptide synthesis. The S-alkylsulfonyl group is stable against amines and thus enables its use under Fmoc chemistry conditions and the controlled polymerization of the corresponding NCAs yielding well-defined homo- as well as block co-polymers. Yet, thiols react immediately with the S-alkylsulfonyl group forming asymmetric disulfides. Therefore, we introduce the first reactive cysteine derivative for efficient and chemoselective disulfide formation in synthetic polypeptides, thus bypassing additional protective group cleavage steps. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. A Protein Disulfide Isomerase Gene Fusion Expression System That Increases the Extracellular Productivity of Bacillus brevis

    Science.gov (United States)

    Kajino, Tsutomu; Ohto, Chikara; Muramatsu, Masayoshi; Obata, Shusei; Udaka, Shigezo; Yamada, Yukio; Takahashi, Haruo

    2000-01-01

    We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system. PMID:10653729

  18. Selective removal of heavy metal ions by disulfide linked polymer networks

    DEFF Research Database (Denmark)

    Ko, Dongah; Sung Lee, Joo; Patel, Hasmukh A.

    2017-01-01

    Heavy metal contaminated surface water is one of the oldest pollution problems, which is critical to ecosystems and human health. We devised disulfide linked polymer networks and employed as a sorbent for removing heavy metal ions from contaminated water. Although the polymer network material has...... a moderate surface area, it demonstrated cadmium removal efficiency equivalent to highly porous activated carbon while it showed 16 times faster sorption kinetics compared to activated carbon, owing to the high affinity of cadmium towards disulfide and thiol functionality in the polymer network. The metal...... sorption mechanism on polymer network was studied by sorption kinetics, effect of pH, and metal complexation. We observed that the metal ions―copper, cadmium, and zinc showed high binding affinity in polymer network, even in the presence of competing cations like calcium in water....

  19. Autonomic healable waterborne organic-inorganic polyurethane hybrids based on aromatic disulfide moieties

    Directory of Open Access Journals (Sweden)

    R. H. Aguirresarobe

    2017-04-01

    Full Text Available Aromatic disulfide dynamic structures were incorporated as chain extenders in waterborne organic-inorganic polyurethane hybrids in order to provide autonomic healable characteristics. The synthesis was carried out following the acetone process methodology and the influence of the introduction of the healing agents in the polymer dispersion stability was analyzed. After the crosslinking process at room temperature, organic-inorganic hybrid films, which presented autonomic healing characteristics, were obtained. These features were evaluated by means of stress-strain tests and the films showed repetitive healing abilities. Thus, the optimum healing time at room temperature (25 °C as well as the influence of different parameters in the healing efficiency, such the aromatic disulfide concentration or the physical properties of the polymer matrix were analyzed.

  20. Enhancing the Thermal Resistance of a Novel Acidobacteria-Derived Phytase by Engineering of Disulfide Bridges.

    Science.gov (United States)

    Tan, Hao; Miao, Renyun; Liu, Tianhai; Cao, Xuelian; Wu, Xiang; Xie, Liyuan; Huang, Zhongqian; Peng, Weihong; Gan, Bingcheng

    2016-10-28

    A novel phytase of Acidobacteria was identified from a soil metagenome, cloned, overexpressed, and purified. It has low sequence similarity (phytases. At the optimum pH (2.5), the phytase shows an activity level of 1,792 μmol/min/mg at physiological temperature (37°C) and could retain 92% residual activity after 30 min, indicating the phytase is acidophilic and acidostable. However the phytase shows poor stability at high temperatures. To improve its thermal resistance, the enzyme was redesigned using Disulfide by Design 2.0, introducing four additional disulfide bridges. The half-life time of the engineered phytase at 60°C and 80°C, respectively, is 3.0× and 2.8× longer than the wild-type, and its activity and acidostability are not significantly affected.

  1. Investigation of the deposition and thermal behavior of striped phases of unsymmetric disulfide self-assembled monolayers on Au(111): The case of 11-hydroxyundecyl decyl disulfide

    Energy Technology Data Exchange (ETDEWEB)

    Albayrak, Erol [Department of Materials and Metallurgical Engineering, Ahi Evran University, Kırşehir 40000 (Turkey); Karabuga, Semistan [Department of Chemistry, Kahramanmaraş Sütçü İmam University, Kahramanmaraş 46030 (Turkey); Bracco, Gianangelo [CNR-IMEM and Department of Physics, University of Genoa, via Dodecaneso 33, Genoa 16146 (Italy); Danışman, M. Fatih, E-mail: danisman@metu.edu.tr [Department of Chemistry, Middle East Technical University, Ankara 06800 (Turkey)

    2015-01-07

    Self-assembled monolayers (SAMs) of unsymmetric disulfides on Au(111) are used to form mixed SAMs that can be utilized in many applications. Here, we have studied 11-hydroxyundecyl decyl disulfide (CH{sub 3}–(CH{sub 2}){sub 9}–S–S–(CH{sub 2}){sub 11}–OH, HDD) SAMs produced by supersonic molecular beam deposition and characterized by He diffraction. The film growth was monitored at different temperatures up to a coverage which corresponds to a full lying down phase and the diffraction analysis shows that below 250 K the phase is different from the phase measured above 300 K. During the annealing of the film, two phase transitions were observed, at 250 K and 350 K. The overall data suggest that the former is related to an irreversible phase separation of HDD above 250 K to decanethiolate (–S–(CH{sub 2}){sub 9}–CH{sub 3}, DTT) and hydroxyundecylthiolate (–S–(CH{sub 2}){sub 11}–OH, MUDT), while the latter to a reversible melting of the film. Above 450 K, the specular intensity shows an increase related to film desorption and different chemisorbed states were observed with energies in the same range as observed for decanethiol (H–S–(CH{sub 2}){sub 9}–CH{sub 3}, DT) and mercaptoundecanol (H–S–(CH{sub 2}){sub 11}–OH, MUD) SAMs.

  2. Structural Characterization and Disulfide Assignment of Spider Peptide Phα1β by Mass Spectrometry

    Science.gov (United States)

    Wormwood, Kelly L.; Ngounou Wetie, Armand Gatien; Gomez, Marcus Vinicius; Ju, Yue; Kowalski, Paul; Mihasan, Marius; Darie, Costel C.

    2018-05-01

    Native Phα1β is a peptide purified from the venom of the armed spider Phoneutria nigriventer that has been shown to have an extensive analgesic effect with fewer side effects than ω-conotoxin MVIIA. Recombinant Phα1β mimics the effects of the native Phα1β. Because of this, it has been suggested that Phα1β may have potential to be used as a therapeutic for controlling persistent pathological pain. The amino acid sequence of Phα1β is known; however, the exact structure and disulfide arrangement has yet to be determined. Determination of the disulfide linkages and exact structure could greatly assist in pharmacological analysis and determination of why this peptide is such an effective analgesic. Here, we used biochemical and mass spectrometry approaches to determine the disulfide linkages present in the recombinant Phα1β peptide. Using a combination of MALDI-MS, direct infusion ESI-MS, and nanoLC-MS/MS analysis of the undigested recombinant Phα1β peptide and digested with AspN, trypsin, or AspN/trypsin, we were able to identify and confirm all six disulfide linkages present in the peptide as Cys1-2, Cys3-4, Cys5-6, Cys7-8, Cys9-10, and Cys11-12. These results were also partially confirmed in the native Phα1β peptide. These experiments provide essential structural information about Phα1β and may assist in providing insight into the peptide's analgesic effect with very low side effects. [Figure not available: see fulltext.

  3. Structural Characterization and Disulfide Assignment of Spider Peptide Phα1β by Mass Spectrometry

    Science.gov (United States)

    Wormwood, Kelly L.; Ngounou Wetie, Armand Gatien; Gomez, Marcus Vinicius; Ju, Yue; Kowalski, Paul; Mihasan, Marius; Darie, Costel C.

    2018-04-01

    Native Phα1β is a peptide purified from the venom of the armed spider Phoneutria nigriventer that has been shown to have an extensive analgesic effect with fewer side effects than ω-conotoxin MVIIA. Recombinant Phα1β mimics the effects of the native Phα1β. Because of this, it has been suggested that Phα1β may have potential to be used as a therapeutic for controlling persistent pathological pain. The amino acid sequence of Phα1β is known; however, the exact structure and disulfide arrangement has yet to be determined. Determination of the disulfide linkages and exact structure could greatly assist in pharmacological analysis and determination of why this peptide is such an effective analgesic. Here, we used biochemical and mass spectrometry approaches to determine the disulfide linkages present in the recombinant Phα1β peptide. Using a combination of MALDI-MS, direct infusion ESI-MS, and nanoLC-MS/MS analysis of the undigested recombinant Phα1β peptide and digested with AspN, trypsin, or AspN/trypsin, we were able to identify and confirm all six disulfide linkages present in the peptide as Cys1-2, Cys3-4, Cys5-6, Cys7-8, Cys9-10, and Cys11-12. These results were also partially confirmed in the native Phα1β peptide. These experiments provide essential structural information about Phα1β and may assist in providing insight into the peptide's analgesic effect with very low side effects. [Figure not available: see fulltext.

  4. Photochemical synthesis of ultrafine organosilicon particles from trimethyl(2-propynyloxy)silane and carbon disulfide

    Czech Academy of Sciences Publication Activity Database

    Morita, H.; Nozawa, R.; Bastl, Zdeněk; Šubrt, Jan; Pola, Josef

    2006-01-01

    Roč. 179, 1-2 (2006), s. 142-148 ISSN 1010-6030 Grant - others:MEXT(JP) 767/15085203 Institutional research plan: CEZ:AV0Z40400503; CEZ:AV0Z40320502; CEZ:AV0Z40720504 Keywords : ultrafine particles * photo-polymerization * trimethyl(2-propynyloxy)silane * carbon disulfide Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.098, year: 2006

  5. Thiol/Disulfide system plays a crucial role in redox protection in the acidophilic iron-oxidizing bacterium Leptospirillum ferriphilum.

    Directory of Open Access Journals (Sweden)

    Javiera Norambuena

    Full Text Available Thiol/disulfide systems are involved in the maintenance of the redox status of proteins and other molecules that contain thiol/disulfide groups. Leptospirillum ferriphilum DSM14647, an acidophilic bacterium that uses Fe(2+ as electron donor, and withstands very high concentrations of iron and other redox active metals, is a good model to study how acidophiles preserve the thiol/disulfide balance. We studied the composition of thiol/disulfide systems and their role in the oxidative stress response in this extremophile bacterium. Bioinformatic analysis using genomic data and enzymatic assays using protein extracts from cells grown under oxidative stress revealed that the major thiol/disulfide system from L. ferriphilum are a cytoplasmic thioredoxin system (composed by thioredoxins Trx and thioredoxin reductase TR, periplasmic thiol oxidation system (DsbA/DsbB and a c-type cytochrome maturation system (DsbD/DsbE. Upon exposure of L. ferriphilum to reactive oxygen species (ROS-generating compounds, transcriptional activation of the genes encoding Trxs and the TR enzyme, which results in an increase of the corresponding activity, was observed. Altogether these data suggest that the thioredoxin-based thiol/disulfide system plays an important role in redox protection of L. ferriphilum favoring the survival of this microorganism under extreme environmental oxidative conditions.

  6. An analytic study of molybdenum disulfide nanofluids using the modern approach of Atangana-Baleanu fractional derivatives

    Science.gov (United States)

    Ali Abro, Kashif; Hussain, Mukkarum; Mahmood Baig, Mirza

    2017-10-01

    The significance of the different shapes of molybdenum disulfide nanoparticles contained in ethylene glycol has recently attracted researchers, because of the numerical or experimental analyses on the shapes of molybdenum disulfide and the lack of fractionalized analytic approaches. This work is dedicated to examining the shape impacts of molybdenum disulfide nanofluids in the mixed convection flow with magnetic field and a porous medium. Ethylene glycol is chosen as the base fluid in which molybdenum disulfide nanoparticles are suspended. Non-spherically shaped molybdenum disulfide nanoparticles, namely, platelet, blade, cylinder and brick, are utilized in this analysis. The modeling of the problem is characterized by employing the modern approach of Atangana-Baleanu fractional derivatives and the governing partial differential equations are solved via Laplace transforms with inversion. Solutions are obtained for temperature distribution and velocity field and expressed in terms of compact form of M-function, Mba(T) . In the end, a figures are drawn to compare the different non-spherically shaped molybdenum disulfide nanoparticles. Furthermore, the Atangana-Baleanu fractional derivatives model has been compared with ordinary derivatives models and discussed graphically by setting various rheological parameters.

  7. Abiotic synthesis of organic compounds from carbon disulfide under hydrothermal conditions.

    Science.gov (United States)

    Rushdi, Ahmed I; Simoneit, Bernd R T

    2005-12-01

    Abiotic formation of organic compounds under hydrothermal conditions is of interest to bio, geo-, and cosmochemists. Oceanic sulfur-rich hydrothermal systems have been proposed as settings for the abiotic synthesis of organic compounds. Carbon disulfide is a common component of magmatic and hot spring gases, and is present in marine and terrestrial hydrothermal systems. Thus, its reactivity should be considered as another carbon source in addition to carbon dioxide in reductive aqueous thermosynthesis. We have examined the formation of organic compounds in aqueous solutions of carbon disulfide and oxalic acid at 175 degrees C for 5 and 72 h. The synthesis products from carbon disulfide in acidic aqueous solutions yielded a series of organic sulfur compounds. The major compounds after 5 h of reaction included dimethyl polysulfides (54.5%), methyl perthioacetate (27.6%), dimethyl trithiocarbonate (6.8%), trithianes (2.7%), hexathiepane (1.4%), trithiolanes (0.8%), and trithiacycloheptanes (0.3%). The main compounds after 72 h of reaction consisted of trithiacycloheptanes (39.4%), pentathiepane (11.6%), tetrathiocyclooctanes (11.5%), trithiolanes (10.6%), tetrathianes (4.4%), trithianes (1.2%), dimethyl trisulfide (1.1%), and numerous minor compounds. It is concluded that the abiotic formation of aliphatic straight-chain and cyclic polysulfides is possible under hydrothermal conditions and warrants further studies.

  8. Resolution of Disulfide Heterogeneity in Nogo Receptor 1 Fusion Proteins by Molecular Engineering

    Energy Technology Data Exchange (ETDEWEB)

    P Weinreb; D Wen; F Qian; C Wildes; E Garber; L Walus; M Jung; J Wang; J Relton; et al.

    2011-12-31

    NgRI (Nogo-66 receptor) is part of a signalling complex that inhibits axon regeneration in the central nervous system. Truncated soluble versions of NgRI have been used successfully to promote axon regeneration in animal models of spinal-cord injury, raising interest in this protein as a potential therapeutic target. The LRR (leucine-rich repeat) regions in NgRI are flanked by N- and C-terminal disulfide-containing 'cap' domains (LRRNT and LRRCT respectively). In the present work we show that, although functionally active, the NgRI(310)-Fc fusion protein contains mislinked and heterogeneous disulfide patterns in the LRRCT domain, and we report the generation of a series of variant molecules specifically designed to prevent this heterogeneity. Using these variants we explored the effects of modifying the NgRI truncation site or the spacing between the NgRI and Fc domains, or replacing cysteines within the NgRI or IgG hinge regions. One variant, which incorporates replacements of Cys{sup 266} and Cys{sup 309} with alanine residues, completely eliminated disulfide scrambling while maintaining functional in vitro and in vivo efficacy. This modified NgRI-Fc molecule represents a significantly improved candidate for further pharmaceutical development, and may serve as a useful model for the optimization of other IgG fusion proteins made from LRR proteins.

  9. Metallic and highly conducting two-dimensional atomic arrays of sulfur enabled by molybdenum disulfide nanotemplate

    Science.gov (United States)

    Zhu, Shuze; Geng, Xiumei; Han, Yang; Benamara, Mourad; Chen, Liao; Li, Jingxiao; Bilgin, Ismail; Zhu, Hongli

    2017-10-01

    Element sulfur in nature is an insulating solid. While it has been tested that one-dimensional sulfur chain is metallic and conducting, the investigation on two-dimensional sulfur remains elusive. We report that molybdenum disulfide layers are able to serve as the nanotemplate to facilitate the formation of two-dimensional sulfur. Density functional theory calculations suggest that confined in-between layers of molybdenum disulfide, sulfur atoms are able to form two-dimensional triangular arrays that are highly metallic. As a result, these arrays contribute to the high conductivity and metallic phase of the hybrid structures of molybdenum disulfide layers and two-dimensional sulfur arrays. The experimentally measured conductivity of such hybrid structures reaches up to 223 S/m. Multiple experimental results, including X-ray photoelectron spectroscopy (XPS), transition electron microscope (TEM), selected area electron diffraction (SAED), agree with the computational insights. Due to the excellent conductivity, the current density is linearly proportional to the scan rate until 30,000 mV s-1 without the attendance of conductive additives. Using such hybrid structures as electrode, the two-electrode supercapacitor cells yield a power density of 106 Wh kg-1 and energy density 47.5 Wh kg-1 in ionic liquid electrolytes. Our findings offer new insights into using two-dimensional materials and their Van der Waals heterostructures as nanotemplates to pattern foreign atoms for unprecedented material properties.

  10. Modeling on-column reduction of trisulfide bonds in monoclonal antibodies during protein A chromatography.

    Science.gov (United States)

    Ghose, Sanchayita; Rajshekaran, Rupshika; Labanca, Marisa; Conley, Lynn

    2017-01-06

    Trisulfides can be a common post-translational modification in many recombinant monoclonal antibodies. These are a source of product heterogeneity that add to the complexity of product characterization and hence, need to be reduced for consistent product quality. Trisulfide bonds can be converted to the regular disulfide bonds by incorporating a novel cysteine wash step during Protein A affinity chromatography. An empirical model is developed for this on-column reduction reaction to compare the reaction rates as a function of typical operating parameters such as temperature, cysteine concentration, reaction time and starting level of trisulfides. The model presented here is anticipated to assist in the development of optimal wash conditions for the Protein A step to effectively reduce trisulfides to desired levels. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Integration of European Bond Markets

    DEFF Research Database (Denmark)

    Christiansen, Charlotte

    2014-01-01

    I investigate the time variation in the integration of EU government bond markets. The integration is measured by the explanatory power of European factor portfolios for the individual bond markets for each year. The integration of the government bond markets is stronger for EMU than non-EMU memb......I investigate the time variation in the integration of EU government bond markets. The integration is measured by the explanatory power of European factor portfolios for the individual bond markets for each year. The integration of the government bond markets is stronger for EMU than non...

  12. Study of a photo-induced lysozyme-riboflavin bond

    International Nuclear Information System (INIS)

    Ferrer, I.; Silva, E.

    1985-01-01

    Irradiation of lysozyme in the presence of riboflavin results in the formation of a lysozyme-riboflavin adduct. Reduction and carboxymethylation of the four disulfide bonds as well as the chemical modification of the Tyr residues and the photochemical alteration of the His residue in lysozyme, do not affect the formation of the photo-induced lysozyme-riboflavin bond. When the lysozyme-riboflavin adduct was subjected to mild acid hydrolysis and ion exchange chromatography, the retention of a compound containing 14 C-riboflavin was observed. Free 14 C-ribboflavin, on the contrary is not retained by the column. The photo-oxidation of free Trp in the presence of 14 C-riboflavin, gave a compound which bound to the ion exchange resin like the above-mentioned derivative. The photo-oxidation of the Trp residues in lysozyme and in peptides obtained from lysozyme showed very high quantum yields, and these values were directly related to the incorporation of 14 C-riboflavin in these samples. (orig.)

  13. Study of a photo-induced lysozyme-riboflavin bond

    Energy Technology Data Exchange (ETDEWEB)

    Ferrer, I; Silva, E

    1985-01-01

    Irradiation of lysozyme in the presence of riboflavin results in the formation of a lysozyme-riboflavin adduct. Reduction and carboxymethylation of the four disulfide bonds as well as the chemical modification of the Tyr residues and the photochemical alteration of the His residue in lysozyme, do not affect the formation of the photo-induced lysozyme-riboflavin bond. When the lysozyme-riboflavin adduct was subjected to mild acid hydrolysis and ion exchange chromatography, the retention of a compound containing /sup 14/C-riboflavin was observed. Free /sup 14/C-riboflavin, on the contrary is not retained by the column. The photo-oxidation of free Trp in the presence of /sup 14/C-riboflavin, gave a compound which bound to the ion exchange resin like the above-mentioned derivative. The photo-oxidation of the Trp residues in lysozyme and in peptides obtained from lysozyme showed very high quantum yields, and these values were directly related to the incorporation of /sup 14/C-riboflavin in these samples.

  14. Amalgam shear bond strength to dentin using different bonding agents.

    Science.gov (United States)

    Vargas, M A; Denehy, G E; Ratananakin, T

    1994-01-01

    This study evaluated the shear bond strength of amalgam to dentin using five different bonding agents: Amalgambond Plus, Optibond, Imperva Dual, All-Bond 2, and Clearfil Liner Bond. Flat dentin surfaces obtained by grinding the occlusal portion of 50 human third molars were used for this study. To contain the amalgam on the tooth surface, cylindrical plastic molds were placed on the dentin and secured with sticky wax. The bonding agents were then applied according to the manufacturers' instructions or light activated and Tytin amalgam was condensed into the plastic molds. The samples were thermocycled and shear bond strengths were determined using an Instron Universal Testing Machine. Analysis by one-way ANOVA indicated significant difference between the five groups (P < 0.05). The bond strength of amalgam to dentin was significantly higher with Amalgambond Plus using the High-Performance Additive than with the other four bonding agents.

  15. Convertible bond valuation focusing on Chinese convertible bond market

    OpenAIRE

    Yang, Ke

    2010-01-01

    This paper mainly discusses the methods of valuation of convertible bonds in Chinese market. Different from common convertible bonds in European market, considering the complicate features of Chinese convertible bond, this paper represents specific pricing approaches for pricing convertible bonds with different provisions along with the increment of complexity of these provisions. More specifically, this paper represents the decomposing method and binomial tree method for pricing both of Non-...

  16. Comparison of shear bond strength of the stainless steel metallic brackets bonded by three bonding systems

    Directory of Open Access Journals (Sweden)

    Mehdi Ravadgar

    2013-09-01

    Full Text Available Introduction: In orthodontic treatment, it is essential to establish a satisfactory bond between enamel and bracket. After the self-etch primers (SEPs were introduced for the facilitation of bracket bonding in comparison to the conventional etch-and-bond system, multiple studies have been carried out on their shear bond strengths which have yielded different results. This study was aimed at comparing shear bond strengths of the stainless steel metallic brackets bonded by three bonding systems. Methods: In this experimental in vitro study, 60 extracted human maxillary premolar teeth were randomly divided into three equal groups: in the first group, Transbond XT (TBXT light cured composite was bonded with Transbond plus self-etching primer (TPSEP in the second group, TBXT composite was bonded with the conventional method of acid etching and in the third group, the self cured composite Unite TM bonding adhesive was bonded with the conventional method of acid etching. In all the groups, Standard edgewise-022 metallic brackets (American Orthodontics, Sheboygan, USA were used. Twenty-four hours after the completion of thermocycling, shear bond strength of brackets was measured by Universal Testing Machine (Zwick. In order to compare the shear bond strengths of the groups, the variance analysis test (ANOVA was adopted and p≤0.05 was considered as a significant level. Results: Based on megapascal, the average shear bond strength for the first, second, and third groups was 8.27±1.9, 9.78±2, and 8.92±2.5, respectively. There was no significant difference in the shear bond strength of the groups. Conclusions: Since TPSEP shear bond strength is approximately at the level of the conventional method of acid etching and within the desirable range for orthodontic brackets shear bond strength, applying TPSEP can serve as a substitute for the conventional method of etch and bond, particularly in orthodontic operations.

  17. Comparison of shear bond strength of the stainless steel metallic brackets bonded by three bonding systems

    Directory of Open Access Journals (Sweden)

    Mehdi Ravadgar

    2013-09-01

    Full Text Available Introduction: In orthodontic treatment, it is essential to establish a satisfactory bond between enamel and bracket. After the self-etch primers (SEPs were introduced for the facilitation of bracket bonding in comparison to the conventional etch-and-bond system, multiple studies have been carried out on their shear bond strengths which have yielded different results. This study was aimed at comparing shear bond strengths of the stainless steel metallic brackets bonded by three bonding systems. Methods: In this experimental in vitro study, 60 extracted human maxillary premolar teeth were randomly divided into three equal groups: in the first group, Transbond XT (TBXT light cured composite was bonded with Transbond plus self-etching primer (TPSEP; in the second group, TBXT composite was bonded with the conventional method of acid etching; and in the third group, the self cured composite Unite TM bonding adhesive was bonded with the conventional method of acid etching. In all the groups, Standard edgewise-022 metallic brackets (American Orthodontics, Sheboygan, USA were used. Twenty-four hours after the completion of thermocycling, shear bond strength of brackets was measured by Universal Testing Machine (Zwick. In order to compare the shear bond strengths of the groups, the variance analysis test (ANOVA was adopted and p≤0.05 was considered as a significant level. Results: Based on megapascal, the average shear bond strength for the first, second, and third groups was 8.27±1.9, 9.78±2, and 8.92±2.5, respectively. There was no significant difference in the shear bond strength of the groups. Conclusions: Since TPSEP shear bond strength is approximately at the level of the conventional method of acid etching and within the desirable range for orthodontic brackets shear bond strength, applying TPSEP can serve as a substitute for the conventional method of etch and bond, particularly in orthodontic operations.

  18. Why are Hydrogen Bonds Directional?

    Indian Academy of Sciences (India)

    century and most chemists appear to think of 'chemi- cal bond' as ..... These complexes, in their global min- ima, have ... taneously act as hydrogen bond donor and acceptor displaying ... also has a local minimum, which is linear and similar to.

  19. Bond yield curve construction

    Directory of Open Access Journals (Sweden)

    Kožul Nataša

    2014-01-01

    Full Text Available In the broadest sense, yield curve indicates the market's view of the evolution of interest rates over time. However, given that cost of borrowing it closely linked to creditworthiness (ability to repay, different yield curves will apply to different currencies, market sectors, or even individual issuers. As government borrowing is indicative of interest rate levels available to other market players in a particular country, and considering that bond issuance still remains the dominant form of sovereign debt, this paper describes yield curve construction using bonds. The relationship between zero-coupon yield, par yield and yield to maturity is given and their usage in determining curve discount factors is described. Their usage in deriving forward rates and pricing related derivative instruments is also discussed.

  20. Corporate Hybrid Bonds

    OpenAIRE

    Ahlberg, Johan; Jansson, Anton

    2016-01-01

    Hybrid securities do not constitute a new phenomenon in the Swedish capital markets. Most commonly, hybrids issued by Swedish real estate companies in recent years are preference shares. Corporate hybrid bonds on the other hand may be considered as somewhat of a new-born child in the family of hybrid instruments. These do, as all other hybrid securities, share some equity-like and some debt-like characteristics. Nevertheless, since 2013 the interest for the instrument has grown rapidly and ha...

  1. Hybrid Cat Bonds

    OpenAIRE

    Barrieu, Pauline; Louberge, Henri

    2009-01-01

    Natural catastrophes attract regularly the attention of media and have become a source of public concern. From a financial viewpoint, natural catastrophes represent idiosyncratic risks, diversifiable at the world level. But for reasons analyzed in this paper reinsurance markets are unable to cope with this risk completely. Insurance-linked securities, such as cat bonds, have been issued to complete the international risk transfer process, but their development is disappointing so far. This pa...

  2. Optimal Investment in Structured Bonds

    DEFF Research Database (Denmark)

    Jessen, Pernille; Jørgensen, Peter Løchte

    The paper examines the role of structured bonds in the optimal portfolio of a small retail investor. We consider the typical structured bond essentially repacking an exotic option and a zero coupon bond, i.e. an investment with portfolio insurance. The optimal portfolio is found when the investment...

  3. 46 CFR Sec. 10 - Bonds.

    Science.gov (United States)

    2010-10-01

    ... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 10 Bonds. (a... awarded work and the furnishing of the performance and payment bonds required by Article 14 of the NSA... of the NSA-LUMPSUMREP Contract, the standard form of individual performance bond (Standard Form 25...

  4. The chemical bond in inorganic chemistry the bond valence model

    CERN Document Server

    Brown, I David

    2016-01-01

    The bond valence model is a version of the ionic model in which the chemical constraints are expressed in terms of localized chemical bonds formed by the valence charge of the atoms. Theorems derived from the properties of the electrostatic flux predict the rules obeyed by both ionic and covalent bonds. They make quantitative predictions of coordination number, crystal structure, bond lengths and bond angles. Bond stability depends on the matching of the bonding strengths of the atoms, while the conflicting requirements of chemistry and space lead to the structural instabilities responsible for the unusual physical properties displayed by some materials. The model has applications in many fields ranging from mineralogy to molecular biology.

  5. Hydrogen bonding in ionic liquids.

    Science.gov (United States)

    Hunt, Patricia A; Ashworth, Claire R; Matthews, Richard P

    2015-03-07

    Ionic liquids (IL) and hydrogen bonding (H-bonding) are two diverse fields for which there is a developing recognition of significant overlap. Doubly ionic H-bonds occur when a H-bond forms between a cation and anion, and are a key feature of ILs. Doubly ionic H-bonds represent a wide area of H-bonding which has yet to be fully recognised, characterised or explored. H-bonds in ILs (both protic and aprotic) are bifurcated and chelating, and unlike many molecular liquids a significant variety of distinct H-bonds are formed between different types and numbers of donor and acceptor sites within a given IL. Traditional more neutral H-bonds can also be formed in functionalised ILs, adding a further level of complexity. Ab initio computed parameters; association energies, partial charges, density descriptors as encompassed by the QTAIM methodology (ρBCP), qualitative molecular orbital theory and NBO analysis provide established and robust mechanisms for understanding and interpreting traditional neutral and ionic H-bonds. In this review the applicability and extension of these parameters to describe and quantify the doubly ionic H-bond has been explored. Estimating the H-bonding energy is difficult because at a fundamental level the H-bond and ionic interaction are coupled. The NBO and QTAIM methodologies, unlike the total energy, are local descriptors and therefore can be used to directly compare neutral, ionic and doubly ionic H-bonds. The charged nature of the ions influences the ionic characteristics of the H-bond and vice versa, in addition the close association of the ions leads to enhanced orbital overlap and covalent contributions. The charge on the ions raises the energy of the Ylp and lowers the energy of the X-H σ* NBOs resulting in greater charge transfer, strengthening the H-bond. Using this range of parameters and comparing doubly ionic H-bonds to more traditional neutral and ionic H-bonds it is clear that doubly ionic H-bonds cover the full range of weak

  6. Study of the helium cross-section of unsymmetric disulfide self-assembled monolayers on Au(111)

    Energy Technology Data Exchange (ETDEWEB)

    Albayrak, Erol [Department of Materials and Metallurgical Engineering, Ahi Evran University, Kırşehir 40000 (Turkey); Karabuga, Semistan [Department of Chemistry, Kahramanmaraş Sütçü İmam University, Kahramanmaraş 46030 (Turkey); Bracco, Gianangelo [CNR-IMEM and Department of Physics, University of Genoa, Via Dodecaneso 33, Genoa 16146 (Italy); Danışman, M. Fatih, E-mail: danisman@metu.edu.tr [Department of Chemistry, Middle East Technical University, Ankara 06800 (Turkey)

    2016-12-30

    Highlights: • Unsymmetrtic disulfide (HDD and HOD) self assembled monolayers were grown on Au(111) by supersonic molecular beam deposition. • Helium scattering cross sections for these two different unsymmetric disulfides were determined. • A common low temperature film phase was observed for the studied disulfides. - Abstract: We have investigated the formation of self-assembled monolayers (SAMs) of 11-hydroxyundecyl decyl disulfide (CH{sub 3}-(CH{sub 2}){sub 9}-S-S-(CH{sub 2}){sub 11}-OH, HDD) and 11-hydroxyundecyl octadecyl disulfide (CH{sub 3}-(CH{sub 2}){sub 17}-S-S-(CH{sub 2}){sub 11}-OH, HOD) produced by supersonic molecular beam deposition (SMBD). The study has been carried out by means of helium diffraction at very low film coverage. In this regime helium single molecule cross sections have been estimated in a temperature range between 100 K and 450 K. The results show a different behavior above 300 K that has been interpreted as the starting of mobility with the formation of two thiolate moieties either linked by a gold adatom or distant enough to prevent cross section overlapping. Finally, helium diffraction patterns measured at 80 K for the SAMs grown at 200 K are discussed and the results support the proposed hypothesis of molecular dissociation based on the cross section data.

  7. Kinetic study of the interaction of glutathione with four antitumor disulfides: possible mechanism for cellular glutathione depletion.

    Science.gov (United States)

    Kirkpatrick, D L

    1989-01-01

    The reactions between the cellular tripeptide, glutathione (GSH) and four disulfide derivatives of 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) (compounds 1-4) were studied kinetically. The decyl and phenyl derivatives of 6-MP and 6-TG were reacted with GSH in phosphate buffer (pH 7.4 or 6.0) at 25.0 degrees C and were monitored spectrophotometrically by observing the release of 6-MP and 6-TG. Second order kinetics were observed, with rate constants of 142, 564, 4174 and 429 M-1 s-1 being measured for compounds 1-4, respectively. When the reactions were carried out in the presence of GSH-S-transferase the rates were enhanced 1.3-5.4 times those observed in the absence of enzyme. Products of the reactions were isolated by chromatography and tentatively identified by TLC or fast atom bombardment mass spectrometry. It was observed that GSH reacted with each disulfide in a 1:1 manner, forming a mixed disulfide between GSH and decanethiol or thiophenol while releasing 6-MP or 6-TG. It was concluded that the reported depletion of GSH from EMT6 cells after exposure to these disulfides could be due to their reaction with GSH, and the formation of the mixed disulfides.

  8. Kinetics and mechanisms of thiol-disulfide exchange covering direct substitution and thiol oxidation-mediated pathways.

    Science.gov (United States)

    Nagy, Péter

    2013-05-01

    Disulfides are important building blocks in the secondary and tertiary structures of proteins, serving as inter- and intra-subunit cross links. Disulfides are also the major products of thiol oxidation, a process that has primary roles in defense mechanisms against oxidative stress and in redox regulation of cell signaling. Although disulfides are relatively stable, their reduction, isomerisation, and interconversion as well as their production reactions are catalyzed by delicate enzyme machineries, providing a dynamic system in biology. Redox homeostasis, a thermodynamic parameter that determines which reactions can occur in cellular compartments, is also balanced by the thiol-disulfide pool. However, it is the kinetic properties of the reactions that best represent cell dynamics, because the partitioning of the possible reactions depends on kinetic parameters. This review is focused on the kinetics and mechanisms of thiol-disulfide substitution and redox reactions. It summarizes the challenges and advances that are associated with kinetic investigations in small molecular and enzymatic systems from a rigorous chemical perspective using biological examples. The most important parameters that influence reaction rates are discussed in detail. Kinetic studies of proteins are more challenging than small molecules, and quite often investigators are forced to sacrifice the rigor of the experimental approach to obtain the important kinetic and mechanistic information. However, recent technological advances allow a more comprehensive analysis of enzymatic systems via using the systematic kinetics apparatus that was developed for small molecule reactions, which is expected to provide further insight into the cell's machinery.

  9. Differential regulation of tissue thiol-disulfide redox status in a murine model of peritonitis

    Directory of Open Access Journals (Sweden)

    Benton Shana M

    2012-10-01

    Full Text Available Abstract Background Glutathione (GSH/glutathione disulfide (GSSG and cysteine (Cys/cystine (CySS are major redox pools with important roles in cytoprotection. We determined the impact of septic peritonitis on thiol-disulfide redox status in mice. Methods FVB/N mice (6–12 week old; 8/group underwent laparotomy with cecal ligation and puncture (CLP or laparotomy alone (control. Sections of ileum, colon, lung and liver were obtained and GSH, GSSG, Cys and CySS concentrations determined by HPLC 24 h after laparotomy. Redox potential [Eh in millivolts (mV] of the GSH/GSSG and Cys/CySS pools was calculated using the Nernst equation. Data were analyzed by ANOVA (mean ± SE. Results GSH/GSSG Eh in ileum, colon, and liver was significantly oxidized in septic mice versus control mice (ileum: septic −202±4 versus control −228±2 mV; colon: -195±8 versus −214±1 mV; and liver: -194±3 vs. -210±1 mV, all Ph was unchanged with CLP, while liver and lung Cys/CySS Eh became significantly more reducing (liver: septic = −103±3 versus control −90±2 mV; lung: -101±5 versus −81±1 mV, each P Conclusions Septic peritonitis induced by CLP oxidizes ileal and colonic GSH/GSSG redox but Cys/CySS Eh remains unchanged in these intestinal tissues. In liver, CLP oxidizes the GSH/GSSG redox pool and CyS/CySS Eh becomes more reducing; in lung, CLP does not alter GSH/GSSG Eh, and Cys/CySS Eh is less oxidized. CLP-induced infection/inflammation differentially regulates major thiol-disulfide redox pools in this murine model.

  10. Thiol/disulfide homeostasis in pregnant women with obstructive sleep apnea syndrome.

    Science.gov (United States)

    Üstündağ, Yasemin; Demirci, Hakan; Balık, Rifat; Erel, Ozcan; Özaydın, Fahri; Kücük, Bilgen; Ertaş, Dilber; Ustunyurt, Emin

    2017-11-27

    Repetitive episodes of hypoxia and reoxygenation during sleep in patients with obstructive sleep apnea syndrome (OSAS) resemble an ischemia-reperfusion injury. We aimed to test the hypothesis that oxidative stress occurs in pregnant women with OSAS. We also aimed to compare thiol/disulfide homeostasis with ischemia-modified albumin (IMA) and total antioxidant capacity (TAC) as markers of ischemia-reperfusion injury in pregnant women with and without OSAS and healthy control. This study included 29 pregnant women with OSAS, 30 women without OSAS in the third trimester applying for periodic examinations, and 30 healthy women. Serum IMA and TAC (using the ferric reducing power of plasma method) were measured. Serum thiol/disulfide homeostasis was determined by a novel automated method. The mean age of the pregnant women with OSAS was 31.0 ± 4.7 years with a mean gestational age of 36.5 ± 3.0 weeks. The mean age of pregnant women without OSAS was 29.8 ± 4.9 years with a mean gestational age of 36.9 ± 2.7 weeks. The mean age of the nonpregnant control group was 29.7 ± 6.4 years. Both native thiol (291 ± 29 μmol/L versus 314 ± 30 μmol/L; p = .018) and total thiol (325 ± 32 versus 350 ± 32, p = .025) levels were lower in pregnant women with OSAS compared to pregnant women without OSAS, respectively (p total thiol levels were lower in pregnant women with OSAS compared to those without OSAS. However, dynamic thiol/disulfide homeostasis parameters cannot provide valuable information to discriminate OSAS in pregnant women.

  11. Influence of Disulfide Connectivity on Structure and Bioactivity of α-Conotoxin TxIA

    Directory of Open Access Journals (Sweden)

    Yong Wu

    2014-01-01

    Full Text Available Cone snails express a sophisticated arsenal of small bioactive peptides known as conopeptides or conotoxins (CTxs. Through evolutionary selection, these peptides have gained the ability to interact with a range of ion channels and receptors, such as nicotinic acetylcholine receptors (nAChRs. Here, we used reversed-phase high performance liquid chromatography (RP-HPLC and electrospray ionization-mass spectrometry (ESI-MS to explore the venom peptide diversity of Conus textile, a species of cone snail native to Hainan, China. One fraction of C. textile crude venom potently blocked α3β2 nAChRs. Subsequent purification, synthesis, and tandem mass spectrometric analysis demonstrated that the most active compound in this fraction was identical to α-CTx TxIA, an antagonist of α3β2 nAChRs. Then three disulfide isoforms of α-CTx TxIA were synthesized and their activities were investigated systematically for the first time. As we observed, disulfide isomerisation was particularly important for α-CTx TxIA potency. Although both globular and ribbon isomers showed similar retention times in RP-HPLC, globular TxIA potently inhibited α3β2 nAChRs with an IC50 of 5.4 nM, while ribbon TxIA had an IC50 of 430 nM. In contrast, beads isomer had little activity towards α3β2 nAChRs. Two-step oxidation synthesis produced the highest yield of α-CTx TxIA native globular isomer, while a one-step production process based on random oxidation folding was not suitable. In summary, this study demonstrated the relationship between conotoxin activity and disulfide connectivity on α-CTx TxIA.

  12. Influence of the degree of crosslinking on the depolymerization of disulfide polymer

    International Nuclear Information System (INIS)

    Rekalicj, J.V.; Radosavljevicj, D.S.; Popovicj, E.M.; Stashicj, L.

    1976-01-01

    The action of nucleophilic reagents (hydrogen sulfide ion, dithionite ion and hydrazine) on disulfide polymers prepd. from bis-2-chloroethyl formal and 1,2,3-trichloropropane, taken in various mol rations is studied. The depolymerization efficiency is higher with hydrazine and dithionite than with a mixt. of sodium hydrogen sulfide and sodium sulfite. An interpretation of the results is given, attempting to correlate the content of SH-groups in the obtained product with the same quantity in some defined compds. which can be present after the depolymerization

  13. Defect-Mediated Lithium Adsorption and Diffusion on Monolayer Molybdenum Disulfide

    OpenAIRE

    Sun, Xiaoli; Wang, Zhiguo; Fu, Yong Qing

    2015-01-01

    Monolayer Molybdenum Disulfide (MoS2) is a promising anode material for lithium ion batteries because of its high capacities. In this work, first principle calculations based on spin density functional theory were performed to investigate adsorption and diffusion of lithium on monolayer MoS2 with defects, such as single- and few-atom vacancies, antisite, and grain boundary. The values of adsorption energies on the monolayer MoS2 with the defects were increased compared to those on the pristin...

  14. Antiradiation compounds XV: condensations of carbon disulfide with amino, chloro, cyanomethyl, and sulfonamido heterocycles

    International Nuclear Information System (INIS)

    Foye, W.O.; Kauffman, J.M.; Lanzillo, J.J.; LaSala, E.F.

    1975-01-01

    Condensations of carbon disulfide were carried out with amino, chloro, and diamino heterocycles to give condensed ring thiazoline-2-thiones and imidazoline-2-thiones, with cyanomethyl heterocycles to give dithio acid derivatives, and with heterocyclic sulfonamides to give sulfonyldithiocarbamates. Of several examples tested, pyrido[3,2-d]thiazoline-2-thione, disodium 2-(5-chloro-2-thienyl)-3,3-dimercaptoacrylonitrile, triethylammonium 4-sulfamoylphenyldithiocarbamate, ammonium β-phenethyldithiocarbamate, and methyl N-(thiophene-2-sulfonyl)dithiocarbamate, only the last-named compound showed any radiation protection for mice. Several compounds gave negative tests for antimalarial activity

  15. Disulfide high mobility group box-1 causes bladder pain through bladder Toll-like receptor 4.

    Science.gov (United States)

    Ma, Fei; Kouzoukas, Dimitrios E; Meyer-Siegler, Katherine L; Westlund, Karin N; Hunt, David E; Vera, Pedro L

    2017-05-25

    Bladder pain is a prominent symptom in several urological conditions (e.g. infection, painful bladder syndrome/interstitial cystitis, cancer). Understanding the mechanism of bladder pain is important, particularly when the pain is not accompanied by bladder pathology. Stimulation of protease activated receptor 4 (PAR4) in the urothelium results in bladder pain through release of urothelial high mobility group box-1 (HMGB1). HGMB1 has two functionally active redox states (disulfide and all-thiol) and it is not known which form elicits bladder pain. Therefore, we investigated whether intravesical administration of specific HMGB1 redox forms caused abdominal mechanical hypersensitivity, micturition changes, and bladder inflammation in female C57BL/6 mice 24 hours post-administration. Moreover, we determined which of the specific HMGB1 receptors, Toll-like receptor 4 (TLR4) or receptor for advanced glycation end products (RAGE), mediate HMGB1-induced changes. Disulfide HMGB1 elicited abdominal mechanical hypersensitivity 24 hours after intravesical (5, 10, 20 μg/150 μl) instillation. In contrast, all-thiol HMGB1 did not produce abdominal mechanical hypersensitivity in any of the doses tested (1, 2, 5, 10, 20 μg/150 μl). Both HMGB1 redox forms caused micturition changes only at the highest dose tested (20 μg/150 μl) while eliciting mild bladder edema and reactive changes at all doses. We subsequently tested whether the effects of intravesical disulfide HMGB1 (10 μg/150 μl; a dose that did not produce inflammation) were prevented by systemic (i.p.) or local (intravesical) administration of either a TLR4 antagonist (TAK-242) or a RAGE antagonist (FPS-ZM1). Systemic administration of either TAK-242 (3 mg/kg) or FPS-ZM1 (10 mg/kg) prevented HMGB1 induced abdominal mechanical hypersensitivity while only intravesical TLR4 antagonist pretreatment (1.5 mg/ml; not RAGE) had this effect. The disulfide form of HMGB1 mediates bladder pain directly (not

  16. Synthesis of tetramethylthiuram disulfide ({sup 35}S); Synthese du disulfure de tetramethylthiurame ({sup 35}S)

    Energy Technology Data Exchange (ETDEWEB)

    Bentov, M [Commissariat a l' Energie Atomique, Saclay (France). Centre d' Etudes Nucleaires

    1958-07-01

    Tetramethylthiuram disulfide ({sup 35}S) has been prepared by exchange between elementary sulfur ({sup 35}S) and sodium N-dithiocarbamate followed by an oxidation with potassium ferricyanide. The method allows to obtain rapidly a pure product with relatively high activity. The radioactive exchange is 82 per cent. (author) [French] Le disurfure de tetramethylthiurame ({sup 35}S) a ete prepare par echange entre le soufre elementaire ({sup 35}S) et le N-dimethyldithiocarbamate de soude suivi d'une oxydation par le ferricyanure de potasse. La methode permet d'obtenir rapidement un produit pur avec une activite relativement haute. L'echange radioactif effectue est de 82 pour cent. (auteur)

  17. The disulfide-rich Metridia luciferase refolded from E. coli inclusion bodies reveals the properties of a native folded enzyme produced in insect cells.

    Science.gov (United States)

    Markova, Svetlana V; Larionova, Marina D; Gorbunova, Darya A; Vysotski, Eugene S

    2017-10-01

    The bioluminescence of a marine copepod Metridia longa is determined by a small secreted coelenterazine-dependent luciferase that uses coelenterazine as a substrate of enzymatic reaction to generate light (λ max =480nm). To date, four different isoforms of the luciferase differing in size, sequences, and properties have been cloned by functional screening. All of them contain ten conserved Cys residues that suggests up to five SS intramolecular bonds per luciferase molecule. Whereas the use of copepod luciferases as bioluminescent reporters in biomedical research in vivo is growing from year to year, their application for in vitro assays is still limited by the difficulty in obtaining significant amounts of luciferase. The most cost-effective host for producing recombinant proteins is Escherichia coli. However, prokaryotic and eukaryotic cells maintain the reductive environment in cytoplasm that hinders the disulfide bond formation and consequently the proper folding of luciferase. Here we report the expression of the MLuc7 isoform of M. longa luciferase in E. coli cells and the efficient procedure for refolding from inclusion bodies yielding a high-active monomeric protein. Furthermore, in a set of identical experiments we demonstrate that bioluminescent and structural features of MLuc7 produced in bacterial cells are identical to those of MLuc7 isoform produced from culture medium of insect cells. Although the yield of high-purity protein is only 6mg/L, the application of E. coli cells to produce the luciferase is simpler and more cost-effective than the use of insect cells. We expect that the suggested technology of Metridia luciferase production allows obtaining of sufficient amounts of protein both for the development of novel in vitro analytical assays with the use of MLuc7 as a label and for structural studies. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. MTH1745, a protein disulfide isomerase-like protein from thermophilic archaea, Methanothermobacter thermoautotrophicum involving in stress response.

    Science.gov (United States)

    Ding, Xia; Lv, Zhen-Mei; Zhao, Yang; Min, Hang; Yang, Wei-Jun

    2008-01-01

    MTH1745 is a putative protein disulfide isomerase characterized with 151 amino acid residues and a CPAC active-site from the anaerobic archaea Methanothermobacter thermoautotrophicum. The potential functions of MTH1745 are not clear. In the present study, we show a crucial role of MTH1745 in protecting cells against stress which may be related to its functions as a disulfide isomerase and its chaperone properties. Using real-time polymerase chain reaction analyses, the level of MTH1745 messenger RNA (mRNA) in the thermophilic archaea M. thermoautotrophicum was found to be stress-induced in that it was significantly higher under low (50 degrees C) and high (70 degrees C) growth temperatures than under the optimal growth temperature for the organism (65 degrees C). Additionally, the expression of MTH1745 mRNA was up-regulated by cold shock (4 degrees C). Furthermore, the survival of MTH1745 expressing Escherichia coli cells was markedly higher than that of control cells in response to heat shock (51.0 degrees C). These results indicated that MTH1745 plays an important role in the resistance of stress. By assay of enzyme activities in vitro, MTH1745 also exhibited a chaperone function by promoting the functional folding of citrate synthase after thermodenaturation. On the other hand, MTH1745 was also shown to function as a disulfide isomerase on the refolding of denatured and reduced ribonuclease A. On the basis of its single thioredoxin domain, function as a disulfide isomerase, and its chaperone activity, we suggest that MTH1745 may be an ancient protein disulfide isomerase. These studies may provide clues to the understanding of the function of protein disulfide isomerase in archaea.

  19. Intracellular drug delivery nanocarriers of glutathione-responsive degradable block copolymers having pendant disulfide linkages.

    Science.gov (United States)

    Khorsand, Behnoush; Lapointe, Gabriel; Brett, Christopher; Oh, Jung Kwon

    2013-06-10

    Self-assembled micelles of amphiphilic block copolymers (ABPs) with stimuli-responsive degradation (SRD) properties have a great promise as nanotherapeutics exhibiting enhanced release of encapsulated therapeutics into targeted cells. Here, thiol-responsive degradable micelles based on a new ABP consisting of a pendant disulfide-labeled methacrylate polymer block (PHMssEt) and a hydrophilic poly(ethylene oxide) (PEO) block were investigated as effective intracellular nanocarriers of anticancer drugs. In response to glutathione (GSH) as a cellular trigger, the cleavage of pendant disulfide linkages in hydrophobic PHMssEt blocks of micellar cores caused the destabilization of self-assembled micelles due to change in hydrophobic/hydrophilic balance. Such GSH-triggered micellar destabilization changed their size distribution with an appearance of large aggregates and led to enhanced release of encapsulated anticancer drugs. Cell culture results from flow cytometry and confocal laser scanning microscopy for cellular uptake as well as cell viability measurements for high anticancer efficacy suggest that new GSH-responsive degradable PEO-b-PHMssEt micelles offer versatility in multifunctional drug delivery applications.

  20. Structure of α-conotoxin BuIA: influences of disulfide connectivity on structural dynamics

    Directory of Open Access Journals (Sweden)

    Craik David J

    2007-04-01

    Full Text Available Abstract Background α-Conotoxins have exciting therapeutic potential based on their high selectivity and affinity for nicotinic acetylcholine receptors. The spacing between the cysteine residues in α-conotoxins is variable, leading to the classification of sub-families. BuIA is the only α-conotoxin containing a 4/4 cysteine spacing and thus it is of significant interest to examine the structure of this conotoxin. Results In the current study we show the native globular disulfide connectivity of BuIA displays multiple conformations in solution whereas the non-native ribbon isomer has a single well-defined conformation. Despite having multiple conformations in solution the globular form of BuIA displays activity at the nicotinic acetylcholine receptor, contrasting with the lack of activity of the structurally well-defined ribbon isomer. Conclusion These findings are opposite to the general trends observed for α-conotoxins where the native isomers have well-defined structures and the ribbon isomers are generally disordered. This study thus highlights the influence of the disulfide connectivity of BuIA on the dynamics of the three-dimensional structure.

  1. Polymeric redox-responsive delivery systems bearing ammonium salts cross-linked via disulfides

    Directory of Open Access Journals (Sweden)

    Christian Dollendorf

    2013-08-01

    Full Text Available A redox-responsive polycationic system was synthesized via copolymerization of N,N-diethylacrylamide (DEAAm and 2-(dimethylaminoethyl methacrylate (DMAEMA. N,N’-bis(4-chlorobutanoylcystamine was used as disulfide-containing cross-linker to form networks by the quaternization of tertiary amine groups. The insoluble cationic hydrogels become soluble by reduction of disulfide to mercaptanes by use of dithiothreitol (DTT, tris(2-carboxyethylphosphine (TCEP or cysteamine, respectively. The soluble polymeric system can be cross-linked again by using oxygen or hydrogen peroxide under basic conditions. The redox-responsive polymer networks can be used for molecular inclusion and controlled release. As an example, phenolphthalein, methylene blue and reactive orange 16 were included into the network. After treatment with DTT a release of the dye could be recognized. Physical properties of the cross-linked materials, e.g., glass transition temperature (Tg, swelling behavior and cloud points (Tc were investigated. Redox-responsive behavior was further analyzed by rheological measurements.

  2. An antiviral disulfide compound blocks interaction between arenavirus Z protein and cellular promyelocytic leukemia protein

    International Nuclear Information System (INIS)

    Garcia, C.C.; Topisirovic, I.; Djavani, M.; Borden, K.L.B.; Damonte, E.B.; Salvato, M.S.

    2010-01-01

    The promyelocytic leukemia protein (PML) forms nuclear bodies (NB) that can be redistributed by virus infection. In particular, lymphocytic choriomeningitis virus (LCMV) influences disruption of PML NB through the interaction of PML with the arenaviral Z protein. In a previous report, we have shown that the disulfide compound NSC20625 has antiviral and virucidal properties against arenaviruses, inducing unfolding and oligomerization of Z without affecting cellular RING-containing proteins such as the PML. Here, we further studied the effect of the zinc-finger-reactive disulfide NSC20625 on PML-Z interaction. In HepG2 cells infected with LCMV or transiently transfected with Z protein constructs, treatment with NSC20625 restored PML distribution from a diffuse-cytoplasmic pattern to punctate, discrete NB which appeared identical to NB found in control, uninfected cells. Similar results were obtained in cells transfected with a construct expressing a Z mutant in zinc-binding site 2 of the RING domain, confirming that this Z-PML interaction requires the integrity of only one zinc-binding site. Altogether, these results show that the compound NSC20625 suppressed Z-mediated PML NB disruption and may be used as a tool for designing novel antiviral strategies against arenavirus infection.

  3. Ophthalmological and angiographic findings in workers exposed to carbon disulfide (author's transl)

    Energy Technology Data Exchange (ETDEWEB)

    Savic, S.

    1982-01-01

    Microaneurysms are important in the diagnosis of vascular changes caused by carbon disulfide. They can be diagnosed by ophtholmoscopy, angiography or angioscopy. In our opinion even a careful ophthalmoscopic investigation is sufficient for diagnosis, so that angiography is not absolutely necessary for any mass survey. The incidence of microaneurysms correlates with the duration (both daily and total) as well as with the intensity of exposure to carbon disulfide. The quantity correlates closely with the intensity of exposure. The incidence of microaneurysms is not correlated to age; however it was found to be highest in 40-50-year-old men working with staple fibers, whereas in the spinning department it occurred in 50-55-old men. Microaneurysms are found equally frequently in active workers and invalids. There was no difference between the two groups with regard to degenerative changes of the macula. However, the changes found in the eyes of men from the staple fiber department were more pronounced than in those from the spinning department.

  4. Degradation of ethyl mercaptan and its major intermediate diethyl disulfide by Pseudomonas sp. strain WL2.

    Science.gov (United States)

    Wang, Xiangqian; Wu, Chao; Liu, Nan; Li, Sujing; Li, Wei; Chen, Jianmeng; Chen, Dongzhi

    2015-04-01

    A Pseudomonas sp. strain WL2 that is able to efficiently metabolize ethyl mercaptan (EM) into diethyl disulfide (DEDS) through enzymatic oxidation was isolated from the activated sludge of a pharmaceutical wastewater plant. One hundred percent removal of 113.5 mg L(-1) EM and 110.3 mg L(-1) DEDS were obtained within 14 and 32 h, respectively. A putative EM degradation pathway that involved the catabolism via DEDS was proposed, which indicated DEDS were further mineralized into carbon dioxide (CO2), bacterial cells, and sulfate (SO4 (2-)) through the transformation of element sulfur and ethyl aldehyde. Degradation kinetics for EM and DEDS with different initial concentrations by strain WL2 were evaluated using Haldane-Andrews model with maximum specific degradation rates of 3.13 and 1.33 g g(-1) h(-1), respectively, and maximum degradation rate constants of 0.522 and 0.175 h(-1) using pseudo-first-order kinetic model were obtained. Results obtained that aerobic degradation of EM by strain WL2 was more efficient than those from previous studies. Substrate range studies of strain WL2 demonstrated its ability to degrade several mercaptans, disulfides, aldehydes, and methanol. All the results obtained highlight the potential of strain WL2 for the use in the biodegradation of volatile organic sulfur compounds (VOSCs).

  5. Rhodium-Catalyzed Insertion Reaction of PhP Group of Pentaphenylcyclopentaphosphine with Acyclic and Cyclic Disulfides.

    Science.gov (United States)

    Arisawa, Mieko; Sawahata, Kyosuke; Yamada, Tomoki; Sarkar, Debayan; Yamaguchi, Masahiko

    2018-02-16

    Organophosphorus compounds with a phosphorus atom attached to a phenyl group and two organothio/organoseleno groups were synthesized using the rhodium-catalyzed insertion reaction of the PhP group of pentaphenylcyclopentaphosphine (PhP) 5 with acyclic disulfides and diselenides. The method was applied to the synthesis of heterocyclic compounds containing the S-P-S group by the reaction of (PhP) 5 and cyclic disulfides such as 1,2-dithietes, 1,2-dithiocane, 1,4,5-dithiopane, and 1,2-dithiolanes.

  6. Scaling of the critical free length for progressive unfolding of self-bonded graphene

    Energy Technology Data Exchange (ETDEWEB)

    Kwan, Kenny; Cranford, Steven W., E-mail: s.cranford@neu.edu [Laboratory of Nanotechnology in Civil Engineering (NICE), Department of Civil and Environmental Engineering, Northeastern University, 400 Snell Engineering, 360 Huntington Avenue, Boston, Massachusetts 02115 (United States)

    2014-05-19

    Like filled pasta, rolled or folded graphene can form a large nanocapsule surrounding a hollow interior. Use as a molecular carrier, however, requires understanding of the opening of such vessels. Here, we investigate a monolayer sheet of graphene as a theoretical trial platform for such a nanocapsule. The graphene is bonded to itself via aligned disulfide (S-S) bonds. Through theoretical analysis and atomistic modeling, we probe the critical nonbonded length (free length, L{sub crit}) that induces fracture-like progressive unfolding as a function of folding radius (R{sub i}). We show a clear linear scaling relationship between the length and radius, which can be used to determine the necessary bond density to predict mechanical opening/closing. However, stochastic dissipated energy limits any exact elastic formulation, and the required energy far exceeds the dissociation energy of the S-S bond. We account for the necessary dissipated kinetic energy through a simple scaling factor (Ω), which agrees well with computational results.

  7. Fatigue aging of adhesive bonds

    International Nuclear Information System (INIS)

    DeLollis, N.J.

    1979-01-01

    A year long study has been made of the effect of fatigue on the bond between two epoxy encapsulant formulations and a fused alumina disc. The variables studied included isothermal aging at temperatures up to and including the cure temperature and cyclic thermal aging from +74 to -54 0 C. The encapsulants were glass microballoon filled epoxies differing only in curing agents. One was cured with an aromatic amine eutectic (Shell Curing Agent Z). The other was cured with diethanolamine. The Z cured encapsulant bond failed completely at the bond interface with little or no aging; infrared evidence indicated a soluble interlayer as a possible cause of failure. The diethanolamine cured encapsulant survived a year of isothermal aging with little or no evidence of bond degradation. Cyclic thermal aging resulted in gradual bond failure with time. An extrapolation of the cyclic aging data indicates that the stresses induced by thermal cycling would result in complete bond failure in about 1200 days

  8. What is a hydrogen bond?

    Indian Academy of Sciences (India)

    First page Back Continue Last page Overview Graphics. What is a hydrogen bond? Precise definition of a hydrogen bond is still elusive!1. Several criteria are listed usually for X-H•••Y, X and Y initially thought to be F, O and N only1. Structural: The X-Y bond length is less than the sum of their van der Waals radii. X-H•••Y is ...

  9. Composite interlayer for diffusion bonding

    International Nuclear Information System (INIS)

    1976-01-01

    A ductile interlayer is described, which is useful for transient liquid phase diffusion bonding of metallic articles; the interlayer consisting of a melting point depressant and a plurality of ductile lamellae which are free from carbides, aluminides and borides. The composition and fabrication of the lamellae, and the process for bonding the metallic articles, depend on the composition of the metals to be bonded, and are exemplified in the specification. (U.K.)

  10. Wafer bonding applications and technology

    CERN Document Server

    Gösele, Ulrich

    2004-01-01

    During the past decade direct wafer bonding has developed into a mature materials integration technology. This book presents state-of-the-art reviews of the most important applications of wafer bonding written by experts from industry and academia. The topics include bonding-based fabrication methods of silicon-on-insulator, photonic crystals, VCSELs, SiGe-based FETs, MEMS together with hybrid integration and laser lift-off. The non-specialist will learn about the basics of wafer bonding and its various application areas, while the researcher in the field will find up-to-date information about this fast-moving area, including relevant patent information.

  11. A simplified indirect bonding technique

    Directory of Open Access Journals (Sweden)

    Radha Katiyar

    2014-01-01

    Full Text Available With the advent of lingual orthodontics, indirect bonding technique has become an integral part of practice. It involves placement of brackets initially on the models and then their transfer to teeth with the help of transfer trays. Problems encountered with current indirect bonding techniques used are (1 the possibility of adhesive flash remaining around the base of the brackets which requires removal (2 longer time required for the adhesive to gain enough bond strength for secure tray removal. The new simplified indirect bonding technique presented here overcomes both these problems.

  12. Human Bond Communication

    DEFF Research Database (Denmark)

    Prasad, Ramjee

    2016-01-01

    Modern dexterous communication technology is progressively enabling humans to communicate their information through them with speech (aural) and media (optical) as underpinning essence. Humans realize this kind of aural and optical information by their optical and auditory senses. However, due...... to certain constraints, the ability to incorporate the other three sensory features namely, olfactory, gustatory, and tactile are still far from reality. Human bond communication is a novel concept that incorporates olfactory, gustatory, and tactile that will allow more expressive and holistic sensory...... information exchange through communication techniques for more human sentiment centric communication. This concept endorses the need of inclusion of other three senses and proposes an innovative approach of holistic communication for future communication network....

  13. 30 CFR 281.33 - Bonds and bonding requirements.

    Science.gov (United States)

    2010-07-01

    ... 30 Mineral Resources 2 2010-07-01 2010-07-01 false Bonds and bonding requirements. 281.33 Section 281.33 Mineral Resources MINERALS MANAGEMENT SERVICE, DEPARTMENT OF THE INTERIOR OFFSHORE LEASING OF MINERALS OTHER THAN OIL, GAS, AND SULPHUR IN THE OUTER CONTINENTAL SHELF Financial Considerations § 281.33...

  14. 29 CFR 2580.412-19 - Term of the bond, discovery period, other bond clauses.

    Science.gov (United States)

    2010-07-01

    ... SECURITY ACT OF 1974 TEMPORARY BONDING RULES General Bond Rules § 2580.412-19 Term of the bond, discovery... 29 Labor 9 2010-07-01 2010-07-01 false Term of the bond, discovery period, other bond clauses... new bond must be obtained each year. There is nothing in the Act that prohibits a bond for a term...

  15. O hydrogen bonds in alkaloids

    Indian Academy of Sciences (India)

    An overview of general classification scheme, medicinal importance and crystal structure analysis with emphasis on the role of hydrogen bonding in some alkaloids is presented in this paper. The article is based on a general kind of survey while crystallographic analysis and role of hydrogen bonding are limited to only ...

  16. Distance criterion for hydrogen bond

    Indian Academy of Sciences (India)

    First page Back Continue Last page Overview Graphics. Distance criterion for hydrogen bond. In a D-H ...A contact, the D...A distance must be less than the sum of van der Waals Radii of the D and A atoms, for it to be a hydrogen bond.

  17. Optimal Investment in Structured Bonds

    DEFF Research Database (Denmark)

    Jessen, Pernille; Jørgensen, Peter Løchte

    2012-01-01

    of the article is to provide possible explanations for the puzzle of why small retail investors hold structured bonds. The investment universe consists of a stock index, a risk-free bank account, and a structured bond containing an option written on another index. We apply expected utility maximization...

  18. Fusion-bonded fluidic interconnects

    NARCIS (Netherlands)

    Fazal, I.; Elwenspoek, Michael Curt

    2008-01-01

    A new approach to realize fluidic interconnects based on the fusion bonding of glass tubes with silicon is presented. Fusion bond strength analyses have been carried out. Experiments with plain silicon wafers and coated with silicon oxide and silicon nitride are performed. The obtained results are

  19. Interchange reaction of disulfides and denaturation of oxytocin by copper(II)/ascorbic acid/O2 system.

    Science.gov (United States)

    Inoue, H; Hirobe, M

    1987-05-29

    The interchange reaction of disulfides was caused by the copper(II)/ascorbic acid/O2 system. The incubation of two symmetric disulfides, L-cystinyl-bis-L-phenylalanine (PP) and L-cystinyl-bis-L-tyrosine (TT), with L-ascorbic acid and CuSO4 in potassium phosphate buffer (pH 7.2, 50 mM) resulted in the formation of an asymmetric disulfide, L-cystinyl-L-phenylalanine-L-tyrosine (PT), and the final ratio of PP:PT:TT was 1:2:1. As the reaction was inhibited by catalase and DMSO only at the initial time, hydroxyl radical generated by the copper(II)/ascorbic acid/O2 system seemed to be responsible for the initiation of the reaction. Oxytocin and insulin were denatured by this system, and catalase and DMSO similarly inhibited these denaturations. As the composition of amino acids was unchanged after the reaction, hydroxyl radical was thought to cause the cleavage and/or interchange reaction of disulfides to denature the peptides.

  20. Simultaneous electrochemical determination of L-cysteine and L-cysteine disulfide at carbon ionic liquid electrode.

    Science.gov (United States)

    Safavi, Afsaneh; Ahmadi, Raheleh; Mahyari, Farzaneh Aghakhani

    2014-04-01

    A linear sweep voltammetric method is used for direct simultaneous determination of L-cysteine and L-cysteine disulfide (cystine) based on carbon ionic liquid electrode. With carbon ionic liquid electrode as a high performance electrode, two oxidation peaks for L-cysteine (0.62 V) and L-cysteine disulfide (1.3 V) were observed with a significant separation of about 680 mV (vs. Ag/AgCl) in phosphate buffer solution (pH 6.0). The linear ranges were obtained as 1.0-450 and 5.0-700 μM and detection limits were estimated to be 0.298 and 4.258 μM for L-cysteine and L-cysteine disulfide, respectively. This composite electrode was applied for simultaneous determination of L-cysteine and L-cysteine disulfide in two real samples, artificial urine and nutrient broth. Satisfactory results were obtained which clearly indicate the applicability of the proposed electrode for simultaneous determination of these compounds in complex matrices.

  1. Fast and efficient green synthesis of thiosulfonate S-esters by microwave-supported permanganate oxidation of symmetrical disulfides

    DEFF Research Database (Denmark)

    Thi, Luu Thi Xuan; Thi Nguyen, Thao-Tran; Le, Thach Ngoc

    2015-01-01

    Potassium permanganate absorbed on copper(II) sulfate pentahydrate has been found to be an efficient, inexpensive, and green oxidation agent for the synthesis of “symmetrical” thiosulfonate S-esters by oxidation of the corresponding symmetrical disulfides. The oxidation reactions were carried out...

  2. Single Layer Molybdenum Disulfide under Direct Out-of-Plane Compression: Low-Stress Band-Gap Engineering

    Czech Academy of Sciences Publication Activity Database

    Álvarez, M. P.; del Corro, Elena; Morales-García, A.; Kavan, Ladislav; Kalbáč, Martin; Frank, Otakar

    2015-01-01

    Roč. 15, č. 5 (2015), s. 3139-3146 ISSN 1530-6984 R&D Projects: GA ČR GA14-15357S; GA MŠk LL1301 Institutional support: RVO:61388955 Keywords : Molybdenum disulfide * band gap engineering * out-of-plane compression Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 13.779, year: 2015

  3. The effects of morphology re-arrangements on the pseudocapacitive properties of mesoporous molybdenum disulfide (MoS2) nanoflakes

    CSIR Research Space (South Africa)

    Khawula, TNY

    2016-07-01

    Full Text Available Mesoporous molybdenum disulfide (MoS(sub2)) with different morphologies have been prepared via hydrothermal method using different solvents, water or water/acetone mixture. The MoS(sub2) obtained with water alone gave a graphene-like nanoflakes (g...

  4. Step-wise addition of disulfide bridge in firefly luciferase controls color shift through a flexible loop: a thermodynamic perspective.

    Science.gov (United States)

    Nazari, Mahboobeh; Hosseinkhani, Saman; Hassani, Leila

    2013-02-01

    Multi-color bioluminescence is developed using the introduction of single/double disulfide bridges in firefly luciferase. The bioluminescence reaction, which uses luciferin, Mg(2+)-ATP and molecular oxygen to yield an electronically excited oxyluciferin, is carried out by the luciferase and emits visible light. The bioluminescence color of firefly luciferases is determined by the luciferase sequence and assay conditions. It has been proposed that the stability of a protein may increase through the introduction of a disulfide bridge that decreases the configurational entropy of unfolding. Single and double disulfide bridges are introduced into Photinus pyralis firefly luciferase to make separate mutant enzymes with a single/double bridge (C(81)-A(105)C, L(306)C-L(309)C, P(451)C-V(469)C; C(81)-A(105)C/P(451)C-V(469)C, and A(296)C-A(326)C/P(451)C-V(469)C). By introduction of disulfide bridges using site-directed mutagenesis in Photinus pyralis luciferase the color of emitted light was changed to red or kept in different extents. The bioluminescence color shift occurred with displacement of a critical loop in the luciferase structure without any change in green emitter mutants. Thermodynamic analysis revealed that among mutants, L(306)C-L(309)C shows a remarkable stability against urea denaturation and also a considerable increase in kinetic stability and a clear shift in bioluminescence spectra towards red.

  5. 40 CFR 63.500 - Back-end process provisions-carbon disulfide limitations for styrene butadiene rubber by emulsion...

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 9 2010-07-01 2010-07-01 false Back-end process provisions-carbon disulfide limitations for styrene butadiene rubber by emulsion processes. 63.500 Section 63.500 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) NATIONAL EMISSION STANDARDS FOR HAZARDOUS AIR POLLUTANTS FOR...

  6. Roles of the redox-active disulfide and histidine residues forming a catalytic dyad in reactions catalyzed by 2-ketopropyl coenzyme M oxidoreductase/carboxylase.

    Science.gov (United States)

    Kofoed, Melissa A; Wampler, David A; Pandey, Arti S; Peters, John W; Ensign, Scott A

    2011-09-01

    NADPH:2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC), an atypical member of the disulfide oxidoreductase (DSOR) family of enzymes, catalyzes the reductive cleavage and carboxylation of 2-ketopropyl-coenzyme M [2-(2-ketopropylthio)ethanesulfonate; 2-KPC] to form acetoacetate and coenzyme M (CoM) in the bacterial pathway of propylene metabolism. Structural studies of 2-KPCC from Xanthobacter autotrophicus strain Py2 have revealed a distinctive active-site architecture that includes a putative catalytic triad consisting of two histidine residues that are hydrogen bonded to an ordered water molecule proposed to stabilize enolacetone formed from dithiol-mediated 2-KPC thioether bond cleavage. Site-directed mutants of 2-KPCC were constructed to test the tenets of the mechanism proposed from studies of the native enzyme. Mutagenesis of the interchange thiol of 2-KPCC (C82A) abolished all redox-dependent reactions of 2-KPCC (2-KPC carboxylation or protonation). The air-oxidized C82A mutant, as well as wild-type 2-KPCC, exhibited the characteristic charge transfer absorbance seen in site-directed variants of other DSOR enzymes but with a pK(a) value for C87 (8.8) four units higher (i.e., four orders of magnitude less acidic) than that for the flavin thiol of canonical DSOR enzymes. The same higher pK(a) value was observed in native 2-KPCC when the interchange thiol was alkylated by the CoM analog 2-bromoethanesulfonate. Mutagenesis of the flavin thiol (C87A) also resulted in an inactive enzyme for steady-state redox-dependent reactions, but this variant catalyzed a single-turnover reaction producing a 0.8:1 ratio of product to enzyme. Mutagenesis of the histidine proximal to the ordered water (H137A) led to nearly complete loss of redox-dependent 2-KPCC reactions, while mutagenesis of the distal histidine (H84A) reduced these activities by 58 to 76%. A redox-independent reaction of 2-KPCC (acetoacetate decarboxylation) was not decreased for any of the

  7. SOCIAL BONDING: REGULATION BY NEUROPEPTIDES

    Directory of Open Access Journals (Sweden)

    Claudia eLieberwirth

    2014-06-01

    Full Text Available Affiliative social relationships (e.g., among spouses, family members, and friends play an essential role in human society. These relationships affect psychological, physiological, and behavioral functions. As positive and enduring bonds are critical for the overall well-being of humans, it is not surprising that considerable effort has been made to study the neurobiological mechanisms that underlie social bonding behaviors. The present review details the involvement of the nonapeptides, oxytocin (OT and arginine vasopressin (AVP, in the regulation of social bonding in mammals including humans. In particular, we will discuss the role of OT and AVP in the formation of social bonds between partners of a mating pair as well as between parents and their offspring. Furthermore, the role of OT and AVP in the formation of interpersonal bonding involving trust is also discussed.

  8. Fusion-bonded fluidic interconnects

    International Nuclear Information System (INIS)

    Fazal, I; Elwenspoek, M C

    2008-01-01

    A new approach to realize fluidic interconnects based on the fusion bonding of glass tubes with silicon is presented. Fusion bond strength analyses have been carried out. Experiments with plain silicon wafers and coated with silicon oxide and silicon nitride are performed. The obtained results are discussed in terms of the homogeneity and strength of fusion bond. High pressure testing shows that the bond strength is large enough for most applications of fluidic interconnects. The bond strength for 525 µm thick silicon, with glass tubes having an outer diameter of 6 mm and with a wall thickness of 2 mm, is more than 60 bars after annealing at a temperature of 800 °C

  9. Sibling bereavement and continuing bonds.

    Science.gov (United States)

    Packman, Wendy; Horsley, Heidi; Davies, Betty; Kramer, Robin

    2006-11-01

    Historically, from a Freudian and medical model perspective, emotional disengagement from the deceased was seen as essential to the successful adaptation of bereavement. A major shift in the bereavement literature has occurred and it is now generally accepted that despite the permanence of physical separation, the bereaved remains involved and connected to the deceased and can be emotionally sustained through continuing bonds. The majority of literature has focused on adults and on the nature of continuing bonds following the death of a spouse. In this article, the authors demonstrate how the continuing bonds concept applies to the sibling relationship. We describe the unique continued relationship formed by bereaved children and adolescents following a sibling loss, highlight the factors that influence the siblings continuing bonds expressions, and offer clinical interventions. In our view, mental health professionals can play an important role in helping parents encourage activities that may facilitate the creation and maintenance of continuing bonds in their children.

  10. Structure phenomena in the bond zone of explosively bonded plates

    International Nuclear Information System (INIS)

    Livne, Z.

    1979-12-01

    In the bond areas of couples of explosively bonded plates, there are often zones, generally designated as ''molten pockets'', which have undergone melting and solidification. The object of the present study was to investigate molten pockets, which have a decisive effect on bond quality. The experimental samples for the study were chosen in consideration of the mutual behaviour of the plates constituting the couples, according to their equilibrium phase diagrams. To facilitate the investigation, large plates were bonded under conditions that enabled to to obtain wavy bond zones that included relatively large molten pockets. To clarify the complex nature of molten pockets and their surroundings, a wide variety of methods were employed. It was found that the shape and composition of molten pockets largely depend upon the mechanism of formation of both the bond wave and the molten pockets. It was also found that the composition of molten pockets is not homogeneous, which is manifest in the modification of the composition of the pockets, the solidification morphology, the phases, which have been identified by X-ray diffraction, and the bond strenght and hardness. Moreover, the different solidification morphologies revealed by metallography were found to depend upon the types of plates bonded, the bonding conditions and the location of pockets in the wavy interface. For molten pockets, cooling rates of 10 4 to 10 5 (degC/sec) have been deduced from interdendritic spacing, and found to be in good agreement with calculations after a mathematical model. It seems that the fast cooling rates and the steep temperature gradients are at the origin of the particular solidification phenomena observed in molten pockets

  11. Dentin-bonding agents

    Directory of Open Access Journals (Sweden)

    João Carlos Gomes

    2008-01-01

    Full Text Available New dental restorative materials have been developed to meet not only the functional demands, but esthetics as well, and in the last few years an enormous range of new materials has appeared for use in dentistry. Among them, several adhesive systems, and different operative techniques for each group materials. Therefore, is indispensable for the professional to know about the properties, characteristics, and association of these materials with the dental structures, in order to select and use them correctly. Should conventional self-etching adhesive systems be used? This question encouraged this literature review to be conducted, with the aim of comparing the conventional adhesive systems with the self-etching systems and to look for scientific data that would help professionals to choose which adhesive system to use. When compared to conventional systems, it was noted that the self-etching systems show less sensitivity to technique, especially as regards errors the operator could commit. The self-etching systems, particularly the 2-step type, have shown equivalent values of bond strength, marginal microleakage and performance, therefore, will be an option for direct composite resin restorations in posterior teeth.

  12. Atomic force microscopy studies on molybdenum disulfide flakes as sodium-ion anodes.

    Science.gov (United States)

    Lacey, Steven D; Wan, Jiayu; von Wald Cresce, Arthur; Russell, Selena M; Dai, Jiaqi; Bao, Wenzhong; Xu, Kang; Hu, Liangbing

    2015-02-11

    A microscale battery comprised of mechanically exfoliated molybdenum disulfide (MoS2) flakes with copper connections and a sodium metal reference was created and investigated as an intercalation model using in situ atomic force microscopy in a dry room environment. While an ethylene carbonate-based electrolyte with a low vapor pressure allowed topographical observations in an open cell configuration, the planar microbattery was used to conduct in situ measurements to understand the structural changes and the concomitant solid electrolyte interphase (SEI) formation at the nanoscale. Topographical observations demonstrated permanent wrinkling behavior of MoS2 electrodes upon sodiation at 0.4 V. SEI formation occurred quickly on both flake edges and planes at voltages before sodium intercalation. Force spectroscopy measurements provided quantitative data on the SEI thickness for MoS2 electrodes in sodium-ion batteries for the first time.

  13. Highly Stretchable Supercapacitors Based on Aligned Carbon Nanotube/Molybdenum Disulfide Composites.

    Science.gov (United States)

    Lv, Tian; Yao, Yao; Li, Ning; Chen, Tao

    2016-08-01

    Stretchable supercapacitors that can sustain their performance under unpredictable tensile force are important elements for practical applications of various portable and wearable electronics. However, the stretchability of most reported supercapacitors was often lower than 100 % because of the limitation of the electrodes used. Herein we developed all-solid-state supercapacitors with a stretchability as high as 240 % by using aligned carbon nanotube composites with compact structure as electrodes. By combined with pseudocapacitive molybdenum disulfide nanosheets, the newly developed supercapacitor showed a specific capacitance of 13.16 F cm(-3) , and also showed excellent cycling retention (98 %) after 10 000 charge-discharge cycles. This work also presents a general and effective approach in developing high-performance electrodes for flexible and stretchable electronics. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Enhanced photoresponse of monolayer molybdenum disulfide (MoS2) based on microcavity structure

    Science.gov (United States)

    Lu, Yanan; Yang, Guofeng; Wang, Fuxue; Lu, Naiyan

    2018-05-01

    There is an increasing interest in using monolayer molybdenum disulfide (MoS2) for optoelectronic devices because of its inherent direct band gap characteristics. However, the weak absorption of monolayer MoS2 restricts its applications, novel concepts need to be developed to address the weakness. In this work, monolayer MoS2 monolithically integrates with plane microcavity structure, which is formed by the top and bottom chirped distributed Bragg reflector (DBR), is demonstrated to improve the absorption of MoS2. The optical absorption is 17-fold enhanced, reaching values over 70% at work wavelength. Moreover, the monolayer MoS2-based photodetector device with microcavity presents a significantly increased photoresponse, demonstrating its promising prospects in MoS2-based optoelectronic devices.

  15. Simultaneous Disulfide and Boronic Acid Ester Exchange in Dynamic Combinatorial Libraries

    Directory of Open Access Journals (Sweden)

    Sanna L. Diemer

    2015-09-01

    Full Text Available Dynamic combinatorial chemistry has emerged as a promising tool for the discovery of complex receptors in supramolecular chemistry. At the heart of dynamic combinatorial chemistry are the reversible reactions that enable the exchange of building blocks between library members in dynamic combinatorial libraries (DCLs ensuring thermodynamic control over the system. If more than one reversible reaction operates in a single dynamic combinatorial library, the complexity of the system increases dramatically, and so does its possible applications. One can imagine two reversible reactions that operate simultaneously or two reversible reactions that operate independently. Both these scenarios have advantages and disadvantages. In this contribution, we show how disulfide exchange and boronic ester transesterification can function simultaneous in dynamic combinatorial libraries under appropriate conditions. We describe the detailed studies necessary to establish suitable reaction conditions and highlight the analytical techniques appropriate to study this type of system.

  16. Simple one-pot synthesis of thioureas from amine, carbon disulfide and oxidants in water

    Directory of Open Access Journals (Sweden)

    Milosavljević Milutin M.

    2016-01-01

    Full Text Available The present study reports the new facile methodology for synthesis of symmetrical and asymmetrical thioureas by an one-pot reaction of amine, carbon disulfide and oxidants: hydrogen peroxide, ethylenediamine tetraacetic acid (EDTA/sodium percarbonate system or air. The structures of the synthesized compounds were confirmed by IR, 1H and 13C NMR and MS methods. Reaction mechanism has been proposed on the basis of reaction intermediate isolation and their structure determination. The synthetic benefits of the presented methods is reflected in the operational simplicity, mild reaction conditions, short reaction times, recycling of solvent, high purity and yield of products, absence of dangerous by-products and technological applicability at industrial scale. Considering commercial importance of the thioureas, it can be emphasized that implementation of the optimal synthesis of thiourea, based on presented methods, at industrial level of production would provide concurrent alternative to existing technologies in use. [Projekat Ministarstva nauke Republike Srbije, br. 172013

  17. In situ aquifer bioremediation of organics including cyanide and carbon disulfide

    International Nuclear Information System (INIS)

    Abou-Rizk, J.A.M.; Leavitt, M.E.; Graves, D.A.

    1995-01-01

    Low levels (< 1 mg/L) of acetone, cyanide, phenol, naphthalene, 2-methylnaphthalene, and carbon disulfide from an inactive industrial landfill were found above background levels in a shallow aquifer at an eastern coastal site. In situ biodegradation was evaluated for treatment of these contaminants. Two soil samples and three groundwater samples were taken from the site for a laboratory bioassessment and a biotreatability test. The positive results of the bioassessment suggested moving forward with biotreatability testing. Biotreatability test results indicated suitable site conditions for bioremediation and that all the contaminants of concern at the site could be biodegraded to nondetect or very low levels (< 50 microg/L) with oxygen only; i.e., addition of nutrients was not required. Pilot-scale testing was undertaken on site to provide information for full-scale design, including oxygen requirements and air injection well spacing. This report describes the approach, the results, and their impact on the full-scale remediation system

  18. Identification of intra- and intermolecular disulfide bridges in the multidrug resistance transporter ABCG2

    DEFF Research Database (Denmark)

    Henriksen, Ulla Birk; Fog, Jacob U; Litman, Thomas

    2005-01-01

    cysteines predicted to be on the extracellular face of ABCG2. Upon mutation of Cys-592 or Cys-608 to alanine (C592A and C608A), ABCG2 migrated as a dimer in SDS-PAGE under non-reducing conditions; however, mutation of Cys-603 to Ala (C603A) caused the transporter to migrate as a single monomeric band....... Despite this change, C603A displayed efficient membrane targeting and preserved transport function. Because the transporter migrated as a dimer in SDS-PAGE, when only Cys-603 was present (C592A-C608A), the data suggest that Cys-603 forms a symmetrical intermolecular disulfide bridge in the ABCG2 homodimer...

  19. Spin-Polarized Tunneling through Chemical Vapor Deposited Multilayer Molybdenum Disulfide.

    Science.gov (United States)

    Dankert, André; Pashaei, Parham; Kamalakar, M Venkata; Gaur, Anand P S; Sahoo, Satyaprakash; Rungger, Ivan; Narayan, Awadhesh; Dolui, Kapildeb; Hoque, Md Anamul; Patel, Ram Shanker; de Jong, Michel P; Katiyar, Ram S; Sanvito, Stefano; Dash, Saroj P

    2017-06-27

    The two-dimensional (2D) semiconductor molybdenum disulfide (MoS 2 ) has attracted widespread attention for its extraordinary electrical-, optical-, spin-, and valley-related properties. Here, we report on spin-polarized tunneling through chemical vapor deposited multilayer MoS 2 (∼7 nm) at room temperature in a vertically fabricated spin-valve device. A tunnel magnetoresistance (TMR) of 0.5-2% has been observed, corresponding to spin polarization of 5-10% in the measured temperature range of 300-75 K. First-principles calculations for ideal junctions result in a TMR up to 8% and a spin polarization of 26%. The detailed measurements at different temperature, bias voltages, and density functional theory calculations provide information about spin transport mechanisms in vertical multilayer MoS 2 spin-valve devices. These findings form a platform for exploring spin functionalities in 2D semiconductors and understanding the basic phenomena that control their performance.

  20. Room temperature humidity sensor based on polyaniline-tungsten disulfide composite

    Science.gov (United States)

    Manjunatha, S.; Chethan, B.; Ravikiran, Y. T.; Machappa, T.

    2018-05-01

    Polyaniline-tungsten disulfide (PANI-WS2) composite was synthesized using in situ polymerization technique by adding finely grinded powder of WS2 during the polymerization of aniline. Field emission scanning electron microscopy (FESEM) images showed the granular morphology with porous nature. Energy dispersive X-ray spectroscopy (EDX) confirmed the presence of carbon, nitrogen, chlorine of PANI, tungsten and sulfur elements of WS2. Humidity sensing property of the composite was investigated by plotting change in its resistance with different relative humidity environments ranging from 10 to 97% RH. Decrease in resistance of the composite was observed with increase in relative humidity. Maximum sensing response of the composite was found to be 88.46%. Response and recovery times of the composite at 97%RH were fair enough to fabricate a sensor based on it. Stability of the composite with respect to the humidity sensing behavior was observed to be unchanged even after two months.

  1. Few-layer molybdenum disulfide transistors and circuits for high-speed flexible electronics

    Science.gov (United States)

    Cheng, Rui; Jiang, Shan; Chen, Yu; Liu, Yuan; Weiss, Nathan; Cheng, Hung-Chieh; Wu, Hao; Huang, Yu; Duan, Xiangfeng

    2014-01-01

    Two-dimensional layered materials, such as molybdenum disulfide, are emerging as an exciting material system for future electronics due to their unique electronic properties and atomically thin geometry. Here we report a systematic investigation of MoS2 transistors with optimized contact and device geometry, to achieve self-aligned devices with performance including an intrinsic gain over 30, an intrinsic cut-off frequency fT up to 42 GHz and a maximum oscillation frequency fMAX up to 50 GHz, exceeding the reported values for MoS2 transistors to date (fT ~ 0.9 GHz, fMAX ~ 1 GHz). Our results show that logic inverters or radio frequency amplifiers can be formed by integrating multiple MoS2 transistors on quartz or flexible substrates with voltage gain in the gigahertz regime. This study demonstrates the potential of two-dimensional layered semiconductors for high-speed flexible electronics. PMID:25295573

  2. Quantum transport model for zigzag molybdenum disulfide nanoribbon structures : A full quantum framework

    International Nuclear Information System (INIS)

    Chen, Chun-Nan; Shyu, Feng-Lin; Chung, Hsien-Ching; Lin, Chiun-Yan; Wu, Jhao-Ying

    2016-01-01

    Mainly based on non-equilibrium Green’s function technique in combination with the three-band model, a full atomistic-scale and full quantum method for solving quantum transport problems of a zigzag-edge molybdenum disulfide nanoribbon (zMoSNR) structure is proposed here. For transport calculations, the relational expressions of a zMoSNR crystalline solid and its whole device structure are derived in detail and in its integrity. By adopting the complex-band structure method, the boundary treatment of this open boundary system within the non-equilibrium Green’s function framework is so straightforward and quite sophisticated. The transmission function, conductance, and density of states of zMoSNR devices are calculated using the proposed method. The important findings in zMoSNR devices such as conductance quantization, van Hove singularities in the density of states, and contact interaction on channel are presented and explored in detail.

  3. Quantum transport model for zigzag molybdenum disulfide nanoribbon structures : A full quantum framework

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Chun-Nan, E-mail: quantum@mail.tku.edu.tw, E-mail: ccn1114@kimo.com [Quantum Engineering Laboratory, Department of Physics, Tamkang University, Tamsui, New Taipei 25137, Taiwan (China); Shyu, Feng-Lin [Department of Physics, R.O.C. Military Academy, Kaohsiung 830, Taiwan (China); Chung, Hsien-Ching; Lin, Chiun-Yan [Department of Physics, National Cheng Kung University, Tainan 70101, Taiwan (China); Wu, Jhao-Ying [Center of General Studies, National Kaohsiung Marine University, Kaohsiung 811, Taiwan (China)

    2016-08-15

    Mainly based on non-equilibrium Green’s function technique in combination with the three-band model, a full atomistic-scale and full quantum method for solving quantum transport problems of a zigzag-edge molybdenum disulfide nanoribbon (zMoSNR) structure is proposed here. For transport calculations, the relational expressions of a zMoSNR crystalline solid and its whole device structure are derived in detail and in its integrity. By adopting the complex-band structure method, the boundary treatment of this open boundary system within the non-equilibrium Green’s function framework is so straightforward and quite sophisticated. The transmission function, conductance, and density of states of zMoSNR devices are calculated using the proposed method. The important findings in zMoSNR devices such as conductance quantization, van Hove singularities in the density of states, and contact interaction on channel are presented and explored in detail.

  4. Determination of glutathione and glutathione disulfide in biological samples: an in-depth review.

    Science.gov (United States)

    Monostori, Péter; Wittmann, Gyula; Karg, Eszter; Túri, Sándor

    2009-10-15

    Glutathione (GSH) is a thiol-containing tripeptide, which plays central roles in the defence against oxidative damage and in signaling pathways. Upon oxidation, GSH is transformed to glutathione disulfide (GSSG). The concentrations of GSH and GSSG and their molar ratio are indicators of cell functionality and oxidative stress. Assessment of redox homeostasis in various clinical states and medical applications for restoration of the glutathione status are of growing importance. This review is intended to provide a state-of-the-art overview of issues relating to sample pretreatment and choices for the separation and detection of GSH and GSSG. High-performance liquid chromatography, capillary electrophoresis and gas chromatography (as techniques with a separation step) with photometric, fluorimetric, electrochemical and mass spectrometric detection are discussed, stress being laid on novel approaches.

  5. Alkali metal and alkali metal hydroxide intercalates of the layered transition metal disulfides

    International Nuclear Information System (INIS)

    Kanzaki, Y.; Konuma, M.; Matsumoto, O.

    1981-01-01

    The intercalation reaction of some layered transition metal disulfides with alkali metals, alkali metal hydroxides, and tetraalkylammonium hydroxides were investigated. The alkali metal intercalates were prepared in the respective metal-hexamethylphosphoric triamide solutions in vaccuo, and the hydroxide intercalates in aqueous hydroxide solutions. According to the intercalation reaction, the c-lattice parameter was increased, and the increase indicated the expansion of the interlayer distance. In the case of alkali metal intercalates, the expansion of the interlayer distance increased continuously, corresponding to the atomic radius of the alkali metal. On the other hand, the hydroxide intercalates showed discrete expansion corresponding to the effective ionic radius of the intercalated cation. All intercalates of TaS 2 amd NbS 2 were superconductors. The expansion of the interlayer distance tended to increase the superconducting transition temperature in the intercalates of TaS 2 and vice versa in those of NbS 2 . (orig.)

  6. Basal-plane thermal conductivity of few-layer molybdenum disulfide

    International Nuclear Information System (INIS)

    Jo, Insun; Ou, Eric; Shi, Li; Pettes, Michael Thompson; Wu, Wei

    2014-01-01

    We report the in-plane thermal conductivity of suspended exfoliated few-layer molybdenum disulfide (MoS 2 ) samples that were measured by suspended micro-devices with integrated resistance thermometers. The obtained room-temperature thermal conductivity values are (44–50) and (48–52) W m −1 K −1 for two samples that are 4 and 7 layers thick, respectively. For both samples, the peak thermal conductivity occurs at a temperature close to 120 K, above which the thermal conductivity is dominated by intrinsic phonon-phonon scattering although phonon scattering by surface disorders can still play an important role in these samples especially at low temperatures

  7. Kinetics and Mechanisms of Thiol–Disulfide Exchange Covering Direct Substitution and Thiol Oxidation-Mediated Pathways

    Science.gov (United States)

    2013-01-01

    Abstract Significance: Disulfides are important building blocks in the secondary and tertiary structures of proteins, serving as inter- and intra-subunit cross links. Disulfides are also the major products of thiol oxidation, a process that has primary roles in defense mechanisms against oxidative stress and in redox regulation of cell signaling. Although disulfides are relatively stable, their reduction, isomerisation, and interconversion as well as their production reactions are catalyzed by delicate enzyme machineries, providing a dynamic system in biology. Redox homeostasis, a thermodynamic parameter that determines which reactions can occur in cellular compartments, is also balanced by the thiol–disulfide pool. However, it is the kinetic properties of the reactions that best represent cell dynamics, because the partitioning of the possible reactions depends on kinetic parameters. Critical Issues: This review is focused on the kinetics and mechanisms of thiol–disulfide substitution and redox reactions. It summarizes the challenges and advances that are associated with kinetic investigations in small molecular and enzymatic systems from a rigorous chemical perspective using biological examples. The most important parameters that influence reaction rates are discussed in detail. Recent Advances and Future Directions: Kinetic studies of proteins are more challenging than small molecules, and quite often investigators are forced to sacrifice the rigor of the experimental approach to obtain the important kinetic and mechanistic information. However, recent technological advances allow a more comprehensive analysis of enzymatic systems via using the systematic kinetics apparatus that was developed for small molecule reactions, which is expected to provide further insight into the cell's machinery. Antioxid. Redox Signal. 18, 1623–1641. PMID:23075118

  8. Disulfide scrambling in superoxide dismutase 1 reduces its cytotoxic effect in cultured cells and promotes protein aggregation.

    Directory of Open Access Journals (Sweden)

    Lina Leinartaitė

    Full Text Available Mutations in the gene coding for superoxide dismutase 1 (SOD1 are associated with familiar forms of the neurodegenerative disease amyotrophic lateral sclerosis (ALS. These mutations are believed to result in a "gain of toxic function", leading to neuronal degeneration. The exact mechanism is still unknown, but misfolding/aggregation events are generally acknowledged as important pathological events in this process. Recently, we observed that demetallated apoSOD1, with cysteine 6 and 111 substituted for alanine, is toxic to cultured neuroblastoma cells. This toxicity depended on an intact, high affinity Zn(2+ site. It was therefor contradictory to discover that wild-type apoSOD1 was not toxic, despite of its high affinity for Zn(2+. This inconsistency was hypothesized to originate from erroneous disulfide formation involving C6 and C111. Using high resolution non-reducing SDS-PAGE, we have in this study demonstrated that the inability of wild-type apoSOD1 to cause cell death stems from formation of non-native intra-molecular disulfides. Moreover, monomeric apoSOD1 variants capable of such disulfide scrambling aggregated into ThT positive oligomers under physiological conditions without agitation. The oligomers were stabilized by inter-molecular disulfides and morphologically resembled what has in other neurodegenerative diseases been termed protofibrils. Disulfide scrambling thus appears to be an important event for misfolding and aggregation of SOD1, but may also be significant for protein function involving cysteines, e.g. mitochondrial import and copper loading.

  9. Insights into the coal extractive solvent N-methyl-2-pyrrolidone + carbon disulfide

    Energy Technology Data Exchange (ETDEWEB)

    Santiago Aparicio; Mara J. Davila; Rafael Alcalde [University of Burgos, Burgos (Spain). Department of Chemistry

    2009-03-15

    A wide set of experimental and computational tools were used to characterize the N-methyl-2-pyrrolidone (NMP) + carbon disulfide mixed solvent in the full composition range. The interest in this solvent rose from its very efficient use for coal extraction through a mechanism still not fully understood. Thermophysical properties at ambient pressure together with pressure-volume-temperature (PVT) behavior were measured with the objective of providing the required data for the industrial use of the mixed fluid and to get insight into the fluid structure at the molecular level. NMR, FTIR, and solvatochromic studies were performed together with microwave dielectric relaxation spectroscopy (DRS) measurements, thus providing more information on the fluid's structure and allowing one to relate the molecular level behavior with the measured macroscopic properties. Moreover, density functional theory (DFT) and classical molecular dynamics simulations (MD) were used to obtain a detailed picture of the intermolecular interactions within the fluid, at short and long ranges, and of other relevant features leading to the structure of the studied system. The whole study leads to a fluid's picture in which carbon disulfide hinders the development of NMP/NMP intermolecular dipolar interactions, thus increasing the monomer population. We should remark that some properties reported in this work are in remarkable disagreement with previously reported studies, the most important one being the positive excess molar volume in the whole pressure-temperature range studied, which contrasts with the negative values reported in the literature. Previously reported properties are hardly justified with a coherent molecular level picture, whereas the whole collection of properties reported in this work leads to a more reasonable fluid's structure. 56 refs., 17 figs., 2 tabs.

  10. Reversible end-to-end assembly of gold nanorods using a disulfide-modified polypeptide

    International Nuclear Information System (INIS)

    Walker, David A; Gupta, Vinay K

    2008-01-01

    Directing the self-assembly of colloidal particles into nanostructures is of great interest in nanotechnology. Here, reversible end-to-end assembly of gold nanorods (GNR) is induced by pH-dependent changes in the secondary conformation of a disulfide-modified poly(L-glutamic acid) (SSPLGA). The disulfide anchoring group drives chemisorption of the polyacid onto the end of the gold nanorods in an ethanolic solution. A layer of poly(vinyl pyrrolidone) is adsorbed on the positively charged, surfactant-stabilized GNR to screen the surfactant bilayer charge and provide stability for dispersion of the GNR in ethanol. For comparison, irreversible end-to-end assembly using a bidentate ligand, namely 1,6-hexanedithiol, is also performed. Characterization of the modified GNR and its end-to-end linking behavior using SSPLGA and hexanedithiol is performed using dynamic light scattering (DLS), UV-vis absorption spectroscopy and transmission electron microscopy (TEM). Experimental results show that, in a colloidal solution of GNR-SSPLGA at a pH∼3.5, where the PLGA is in an α-helical conformation, the modified GNR self-assemble into one-dimensional nanostructures. The linking behavior can be reversed by increasing the pH (>8.5) to drive the conformation of the polypeptide to a random coil and this reversal with pH occurs rapidly within minutes. Cycling the pH multiple times between low and high pH values can be used to drive the formation of the nanostructures of the GNR and disperse them in solution.

  11. Roll bonding of strained aluminium

    DEFF Research Database (Denmark)

    Staun, Jakob M.

    2003-01-01

    This report investigates roll bonding of pre-strained (å ~ 4) aluminium sheets to produce high strain material from high purity aluminium (99.996%) and commercial pure aluminium (99.6%). The degree of bonding is investigated by optical microscopy and ultrasonic scanning. Under the right...... of the cross rolled volume fraction is found. To further asses this effect, and the anisotropy, it is necessary to acquire knowledge about both texture and microstructure, e.g. by TEM. Roll bonding of pre-strained aluminium is found to be a possible alternative to ARB in the quest for ultra-fine grained...

  12. Direct Bonded Pontic (Laporan Kasus

    Directory of Open Access Journals (Sweden)

    Suhandi Sidjaja

    2015-10-01

    Full Text Available Advanced science and technology in dentistry enable dental practitioners to modified she bonding techniques in tooth replacement. A pontic made of composite resin bonded to etched enamel of the adjacent teeth can be used in the replacement of one missing anterior tooth with a virgin or sowed adpicent tooth. The advantages of this technique include a one visit treatment, cow cost, good esthetics, less side effects and easy repair or rebounding. Clinical evaluation showed a high success rate therefore with a proper diagnosis and a perfect skill of the direct bonded technique this treatment can be used as an alternative restoration.

  13. Self-healing polymer gels based on dynamic covalent bonds%基于动态共价键的可自愈合聚合物凝胶

    Institute of Scientific and Technical Information of China (English)

    张云飞; 邓国华

    2012-01-01

    简要介绍了动态共价键既具有普通共价键的高强度和稳定性,又能像分子间作用力(如氢键)那样可逆地断裂和重组的特点,以及基于动态共价键构筑智能凝胶材料的优势。综述了多种动态共价键,如芳香基苯并呋喃酮二聚体(diarylbibenzo furanone,DABBF)、三硫酯(trithiocarbonate,TTC)、芳基硼酸酯、酰腙键(acylhydrazone bond)、双硫键(disulfide bond)等的结构及其动态化学,以及应用它们合成聚合物凝胶的方法、凝胶的自愈合机理和性能。提出了发现和采用多种动态共价键构筑可自愈合聚合物凝胶的趋势,为此须解决多种动态共价键的相容性、凝胶自愈合机理与性能的光谱表征等问题,并加强应用研究。%Dynamic covalent bonds have high mechanical strength and stability like ordinary covalent bonds and can reversibly break and rebuild like intermolecular forces(such as hydrogen bonding).The properties of dynamic covalent bonds are introduced.The advantages of building smart gels based on dynamic covalent bonds are described.Specifically,the structure and dynamic chemistry of diarylbibenzo furanone(DABBF),trithiocarbonate(TTC),phenylboronic aciddiol ester bond,acylhydrazone bond and disulfide bond are reviewed.The methods of utilizing those dynamic covalent bonds to construct dynamic gels with self-healing properties,including the healing mechanisms,are presented.Combining two or more covalent bonds to construct dynamic gels with more complex responsiveness are proposed.Problems,such as compatibility of the dynamic covalent bonds,spectroscopic methods for characterizing self-healing mechanisms and capabilities,and application-oriented systems need to be further investigated.

  14. Internal motion time scales of a small, highly stable and disulfide-rich protein: A 15N, 13C NMR and molecular dynamics study

    International Nuclear Information System (INIS)

    Guenneugues, Marc; Gilquin, Bernard; Wolff, Nicolas; Menez, Andre; Zinn-Justin, Sophie

    1999-01-01

    Motions of the backbone CαHα and threonine CβHβ bonds of toxin α were investigated using natural abundance 13C NMR and molecular dynamics. Measurement of the 13C longitudinal and transverse relaxation rates employed ACCORDION techniques together with coherence selection by pulsed field gradients and sensitivity enhancement through the use of preservation of equivalent pathway, thus allowing a considerable reduction of the required spectrometer time. 13C R1, R2, 1H → 13C NOE were obtained, as well as the variations of R1ρ(90 deg.) as a function of the rf field strength. These data were compared to those recorded by 1H and 15N NMR on a labelled sample of the toxin [Guenneugues et al. (1997) Biochemistry, 36, 16097-16108]. Both sets of data showed that picosecond to nanosecond time scale motions are well correlated to the secondary structure of the protein. This was further reinforced by the analysis of a 1 ns molecular dynamics simulation in water. Several CαHα and threonine CβHβ experimentally exhibit fast motions with a correlation time longer than 500 ps, that cannot be sampled along the simulation. In addition, the backbone exhibits motions on the microsecond to millisecond time scale on more than half of its length. Thus, toxin α, a highly stable protein (Tm=75 deg. C at acidic pH) containing 61 amino acids and 4 disulfides, shows important internal motions on time scales ranging from 0.1-0.5 ps, to 10-100 ps, 1 ns, and about 30 μs to 10 ms

  15. dimensional architectures via hydrogen bonds

    Indian Academy of Sciences (India)

    Administrator

    organization and has potential applications in the field of magnetism ... The concepts of crystal engineering ... 4. However, the utilization of hydrogen bond supramolecular syn- ... sembling the coordination networks by designing the ligands ...

  16. Hydrogen bonding in tight environments

    DEFF Research Database (Denmark)

    Pirrotta, Alessandro; Solomon, Gemma C.; Franco, Ignacio

    2016-01-01

    The single-molecule force spectroscopy of a prototypical class of hydrogen-bonded complexes is computationally investigated. The complexes consist of derivatives of a barbituric acid and a Hamilton receptor that can form up to six simultaneous hydrogen bonds. The force-extension (F-L) isotherms...... of the host-guest complexes are simulated using classical molecular dynamics and the MM3 force field, for which a refined set of hydrogen bond parameters was developed from MP2 ab initio computations. The F-L curves exhibit peaks that signal conformational changes during elongation, the most prominent...... of which is in the 60-180 pN range and corresponds to the force required to break the hydrogen bonds. These peaks in the F-L curves are shown to be sensitive to relatively small changes in the chemical structure of the host molecule. Thermodynamic insights into the supramolecular assembly were obtained...

  17. Adhesives for orthodontic bracket bonding

    Directory of Open Access Journals (Sweden)

    Déborah Daniella Diniz Fonseca

    2010-04-01

    Full Text Available The advent of acid etching, introduced by Buonocore in 1955, brought the possibility of bonding between the bracket base and enamel, contributing to more esthetic and conservative orthodontics. This direct bracket bonding technique has brought benefits such as reduced cost and time in performing the treatment, as well as making it easier to perform oral hygiene. The aim of this study was to conduct a survey of published studies on orthodontic bracket bonding to dental enamel. It was verified that resin composites and glass ionomer are the most studied and researched materials for this purpose. Resin-modified glass ionomer, with its biocompatibility, capacity of releasing fluoride and no need for acid etching on the tooth structure, has become increasingly popular among dentists. However, due to the esthetic and mechanical properties of light polymerizable resin composite, it continues to be one of the adhesives of choice in the bracket bonding technique and its use is widely disseminated.

  18. Method to improve commercial bonded SOI material

    Science.gov (United States)

    Maris, Humphrey John; Sadana, Devendra Kumar

    2000-07-11

    A method of improving the bonding characteristics of a previously bonded silicon on insulator (SOI) structure is provided. The improvement in the bonding characteristics is achieved in the present invention by, optionally, forming an oxide cap layer on the silicon surface of the bonded SOI structure and then annealing either the uncapped or oxide capped structure in a slightly oxidizing ambient at temperatures greater than 1200.degree. C. Also provided herein is a method for detecting the bonding characteristics of previously bonded SOI structures. According to this aspect of the present invention, a pico-second laser pulse technique is employed to determine the bonding imperfections of previously bonded SOI structures.

  19. Comparative evaluation of shear bond strength of metallic brackets bonded with two different bonding agents under dry conditions and with saliva contamination

    Directory of Open Access Journals (Sweden)

    Mashallah Khanehmasjedi

    2017-02-01

    Conclusion: Application of Single Bond and Assure bonding agents resulted in adequate bond strength of brackets to tooth structures. Contamination with saliva significantly decreased the bond strength of Assure bonding agent compared with dry conditions.

  20. Proposal of new bonding technique 'Instantaneous Liquid Phase (ILP) Bonding'

    International Nuclear Information System (INIS)

    Zhang, Yue-Chang; Nakagawa, Hiroji; Matsuda, Fukuhisa.

    1987-01-01

    A new bonding technique named ''Instantaneous Liquid Phase (ILP) bonding'' suitable mainly for welding dissimilar materials was proposed by which instantaneous melting of one or two of the faying surfaces is utilized. The processes of ILP bonding are mainly consisted of three stages, namely the first stage forming thin liquid layer by rapid heating, the second stage joining both specimens by thin liquid layer, and the third stage cooling the specimens rapidly to avoid the formation of brittle layer. The welding temperatures of the specimens to be welded in ILP bonding are generally differentiated from each other. ILP bonding was applied for a variety of combinations of dissimilar materials of aluminum, aluminum alloys, titanium, titanium alloy, carbon steel, austenitic stainless steel, copper and tungsten, and for similar materials of stainless steel and nickel-base alloy. There were no microvoids in these welding joints, and the formation of brittle layer at the bonding interface was suppressed. The welded joints of Al + Ti, Cu + carbon steel and Cu + austenitic stainless steel showed the fracture in base metal having lower tensile strength. Further, the welded joints of Al + carbon steel, Al alloy + Ti, Al alloy + carbon steel or + austenitic stainless steel, Ti + carbon steel or + austenitic stainless steel showed better tensile properties in the comparison with diffusion welding. Furthermore, ILP bonding was available for welding same materials susceptible to hot cracking. Because of the existence of liquid layer, the welding pressure required was extremely low, and preparation of faying surface by simple tooling or polishing by no.80 emery paper was enough. The change in specimen length before and after welding was relatively little, only depending on the thickness of liquid layer. The welding time was very short, and thus high welding efficiency was obtained. (author)

  1. Shear Bond Strength of Orthodontic Brackets Bonded to Zirconium Crowns.

    Science.gov (United States)

    Mehmeti, Blerim; Azizi, Bleron; Kelmendi, Jeta; Iljazi-Shahiqi, Donika; Alar, Željko; Anić-Milošević, Sandra

    2017-06-01

    An increasing demand for esthetic restorations has resulted in an increased use of all-ceramic restorations, such as zirconium. However, one of the challenges the orthodontist must be willing to face is how to increase bond strength between the brackets and various ceramic restorations.Bond strength can beaffected bybracket type, by the material that bracketsaremade of, and their base surface design or retention mode. ​: A im: of this study was to perform a comparative analysis of the shear bond strength (SBS) of metallic and ceramic orthodontic brackets bonded to all-zirconium ceramic surfaces used for prosthetic restorations, and also to evaluate the fracture mode of these two types of orthodontic brackets. Twenty samples/semi-crowns of all-zirconium ceramic, on which orthodontic brackets were bonded, 10 metallic and 10 ceramic polycrystalline brackets, were prepared for this research. SBS has been testedby Universal Testing Machine, with a load applied using a knife edged rod moving at a fixed rate of 1 mm/min, until failure occurred. The force required to debond the brackets was recorded in Newton, then SBS was calculated to MPa. In addition, the samples were analyzed using a digital camera magnifier to determine Adhesive Remnant Index (ARI). Statistical data were processed using t-test, and the level of significance was set at α = 0.05. Higher shear bond strength values were observed in metallic brackets bonded to zirconium crowns compared tothoseof ceramic brackets, with a significant difference. During the test, two of the ceramic brackets were partially or totally damaged. Metallic brackets, compared to ceramic polycrystalline brackets, seemed tocreate stronger adhesion with all-zirconium surfaces due to their better retention mode. Also, ceramic brackets showed higher fragility during debonding.

  2. Voltage-assisted polymer wafer bonding

    International Nuclear Information System (INIS)

    Varsanik, J S; Bernstein, J J

    2012-01-01

    Polymer wafer bonding is a widely used process for fabrication of microfluidic devices. However, best practices for polymer bonds do not achieve sufficient bond strength for many applications. By applying a voltage to a polymer bond in a process called voltage-assisted bonding, bond strength is shown to improve dramatically for two polymers (Cytop™ and poly(methyl methacrylate)). Several experiments were performed to provide a starting point for further exploration of this technique. An optimal voltage range is experimentally observed with a reduction in bonding strength at higher voltages. Additionally, voltage-assisted bonding is shown to reduce void diameter due to bond defects. An electrostatic force model is proposed to explain the improved bond characteristics. This process can be used to improve bond strength for most polymers. (paper)

  3. Optimising hydrogen bonding in solid wood

    DEFF Research Database (Denmark)

    Engelund, Emil Tang

    2009-01-01

    The chemical bonds of wood are both covalent bonds within the wood polymers and hydrogen bonds within and between the polymers. Both types of bonds are responsible for the coherence, strength and stiffness of the material. The hydrogen bonds are more easily modified by changes in load, moisture...... and temperature distorting the internal bonding state. A problem arises when studying hydrogen bonding in wood since matched wood specimens of the same species will have very different internal bonding states. Thus, possible changes in the bonding state due to some applied treatment such as conditioning...... maintaining 100 % moisture content of the wood. The hypothesis was that this would enable a fast stress relaxation as a result of reorganization of bonds, since moisture plasticizes the material and temperature promotes faster kinetics. Hereby, all past bond distortions caused by various moisture, temperature...

  4. Quantitation of a PEGylated protein in monkey serum by UHPLC-HRMS using a surrogate disulfide-containing peptide: A new approach to bioanalysis and in vivo stability evaluation of disulfide-rich protein therapeutics.

    Science.gov (United States)

    Zheng, Naiyu; Zeng, Jianing; Manney, Amy; Williams, Lakenya; Aubry, Anne-Françoise; Voronin, Kimberly; Buzescu, Adela; Zhang, Yan J; Allentoff, Alban; Xu, Carrie; Shen, Hongwu; Warner, William; Arnold, Mark E

    2016-04-15

    To quantify a therapeutic PEGylated protein in monkey serum as well as to monitor its potential in vivo instability and methionine oxidation, a novel ultra high performance liquid chromatography-high resolution mass spectrometric (UHPLC-HRMS) assay was developed using a surrogate disulfide-containing peptide, DCP(SS), and a confirmatory peptide, CP, a disulfide-free peptide. DCP(SS) was obtained by eliminating the step of reduction/alkylation before trypsin digestion. It contains an intact disulfide linkage between two peptide sequences that are essential for drug function but susceptible to potential in vivo cleavages. HRMS-based single ion monitoring (SIM) on a Q Exactive™ mass spectrometer was employed to improve assay specificity and sensitivity for DCP(SS) due to its poor fragmentation and low sensitivity with SRM detection. The assay has been validated for the protein drug in monkey serum using both surrogate peptides with excellent accuracy (within ±4.4%Dev) and precision (within 7.5%CV) with a lower limit of quantitation (LLOQ) at 10 ng mL(-1). The protein concentrations in monkey serum obtained from the DCP(SS)-based assay not only provided important pharmacokinetic parameters, but also confirmed in vivo stability of the peptide regions of interest by comparing drug concentrations with those obtained from the CP-based assay or from a ligand-binding assay (LBA). Furthermore, UHPLC-HRMS allowed simultaneous monitoring of the oxidized forms of both surrogate peptides to evaluate potential ex vivo/in vivo oxidation of one methionine present in each of both surrogate peptides. To the best of our knowledge, this is the first report of using a surrogate disulfide-containing peptide for LC-MS bioanalysis of a therapeutic protein. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Symmetric pseudocapacitors based on molybdenum disulfide (MoS2)-modified carbon nanospheres: correlating physicochemistry and synergistic interaction on energy storage

    CSIR Research Space (South Africa)

    Khawula, TNY

    2016-03-01

    Full Text Available Molybdenum disulfide-modified carbon nanospheres (MoS(sub2)/CNS) with two different morphologies (spherical and flower-like) have been synthesized using hydrothermal techniques and investigated as symmetric pseudocapacitors in an aqueous electrolyte...

  6. Continuous exposure to low-frequency noise and carbon disulfide: Combined effects on hearing.

    Science.gov (United States)

    Venet, Thomas; Carreres-Pons, Maria; Chalansonnet, Monique; Thomas, Aurélie; Merlen, Lise; Nunge, Hervé; Bonfanti, Elodie; Cosnier, Frédéric; Llorens, Jordi; Campo, Pierre

    2017-09-01

    Carbon disulfide (CS 2 ) is used in industry; it has been shown to have neurotoxic effects, causing central and distal axonopathies.However, it is not considered cochleotoxic as it does not affect hair cells in the organ of Corti, and the only auditory effects reported in the literature were confined to the low-frequency region. No reports on the effects of combined exposure to low-frequency noise and CS 2 have been published to date. This article focuses on the effects on rat hearing of combined exposure to noise with increasing concentrations of CS 2 (0, 63,250, and 500ppm, 6h per day, 5 days per week, for 4 weeks). The noise used was a low-frequency noise ranging from 0.5 to 2kHz at an intensity of 106dB SPL. Auditory function was tested using distortion product oto-acoustic emissions, which mainly reflects the cochlear performances. Exposure to noise alone caused an auditory deficit in a frequency area ranging from 3.6 to 6 kHz. The damaged area was approximately one octave (6kHz) above the highest frequency of the exposure noise (2.8kHz); it was a little wider than expected based on the noise spectrum.Consequently, since maximum hearing sensitivity is located around 8kHz in rats, low-frequency noise exposure can affect the cochlear regions detecting mid-range frequencies. Co-exposure to CS 2 (250-ppm and over) and noise increased the extent of the damaged frequency window since a significant auditory deficit was measured at 9.6kHz in these conditions.Moreover, the significance at 9.6kHz increased with the solvent concentrations. Histological data showed that neither hair cells nor ganglion cells were damaged by CS 2 . This discrepancy between functional and histological data is discussed. Like most aromatic solvents, carbon disulfide should be considered as a key parameter in hearing conservation régulations. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Species-Specific Thiol-Disulfide Equilibrium Constant: A Tool To Characterize Redox Transitions of Biological Importance.

    Science.gov (United States)

    Mirzahosseini, Arash; Somlyay, Máté; Noszál, Béla

    2015-08-13

    Microscopic redox equilibrium constants, a new species-specific type of physicochemical parameters, were introduced and determined to quantify thiol-disulfide equilibria of biological significance. The thiol-disulfide redox equilibria of glutathione with cysteamine, cysteine, and homocysteine were approached from both sides, and the equilibrium mixtures were analyzed by quantitative NMR methods to characterize the highly composite, co-dependent acid-base and redox equilibria. The directly obtained, pH-dependent, conditional constants were then decomposed by a new evaluation method, resulting in pH-independent, microscopic redox equilibrium constants for the first time. The 80 different, microscopic redox equilibrium constant values show close correlation with the respective thiolate basicities and provide sound means for the development of potent agents against oxidative stress.

  8. Self-homodimerization of an actinoporin by disulfide bridging reveals implications for their structure and pore formation.

    Science.gov (United States)

    Valle, Aisel; Pérez-Socas, Luis Benito; Canet, Liem; Hervis, Yadira de la Patria; de Armas-Guitart, German; Martins-de-Sa, Diogo; Lima, Jônatas Cunha Barbosa; Souza, Adolfo Carlos Barros; Barbosa, João Alexandre Ribeiro Gonçalves; de Freitas, Sonia Maria; Pazos, Isabel Fabiola

    2018-04-26

    The Trp111 to Cys mutant of sticholysin I, an actinoporin from Stichodactyla helianthus sea anemone, forms a homodimer via a disulfide bridge. The purified dimer is 193 times less hemolytic than the monomer. Ultracentrifugation, dynamic light scattering and size-exclusion chromatography demonstrate that monomers and dimers are the only independent oligomeric states encountered. Indeed, circular dichroism and fluorescence spectroscopies showed that Trp/Tyr residues participate in homodimerization and that the dimer is less thermostable than the monomer. A homodimer three-dimensional model was constructed and indicates that Trp147/Tyr137 are at the homodimer interface. Spectroscopy results validated the 3D-model and assigned 85° to the disulfide bridge dihedral angle responsible for dimerization. The homodimer model suggests that alterations in the membrane/carbohydrate-binding sites in one of the monomers, as result of dimerization, could explain the decrease in the homodimer ability to form pores.

  9. Dissulfeto de molibdênio, um material multifuncional e surpreendente Molybdenum disulfide, a multifunctional and remarkable material

    Directory of Open Access Journals (Sweden)

    Fernando Wypych

    2002-02-01

    Full Text Available The aim of this work is to review the chemical and physical properties of layered molybdenum disulfide. The three polymorphic/polytypic modifications of the compound were found, the polytypes 2H (molybdenite and 3R are semiconductors while the polymorph 1T is an electronic conductor. 2H-MoS2 has several important industrial applications as hydrotreatment catalysts, energy storage devices, solar cells, solid lubricants, among others. When intercalated, the 2H phase changes to a distorted 1T phase, producing unstable intercalation compounds that can be exfoliated in solution, producing single layers and consequently nanocomposites. The direct synthesis of the 1T phase produces stable intercalation compounds. Recently molybdenum disulfide was prepared as nanotubes and fulerene-like structures that bring new insights in the investigation of this important material.

  10. Protein and non-protein sulfhydryls and disulfides in gastric mucosa and liver after gastrotoxic chemicals and sucralfate: possible new targets of pharmacologic agents.

    Science.gov (United States)

    Nagy, Lajos; Nagata, Miki; Szabo, Sandor

    2007-04-14

    To investigate the role of major non-protein and protein sulfhydryls and disulfides in chemically induced gastric hemorrhagic mucosal lesions (HML) and the mechanism of gastroprotective effect of sucralfate. Rats were given 1 mL of 75% ethanol, 25% NaCl, 0.6 mol/L HCl, 0.2 mol/L NaOH or 1% ammonia solutions intragastrically (i.g.) and sacrificed 1, 3, 6 or 12 min later. Total (reduced and oxidized) glutathione (GSH + GSSG), glutathione disulfide (GSSG), protein free sulfhydryls (PSH), protein-glutathione mixed disulfides (PSSG) and protein cystine disulfides (PSSP) were measured in gastric mucosa and liver. Reduced glutathione (GSH) was depleted in the gastric mucosa after ethanol, HCl or NaCl exposure, while oxidized glutathione (GSSG) concentrations increased, except by HCl and NaOH exposure. Decreased levels of PSH after exposure to ethanol were observed, NaCl or NaOH while the total protein disulfides were increased. Ratios of reduced to oxidized glutathione or sulfhydrils to disulfides were decreased by all chemicals. No changes in thiol homeostasis were detected in the liver after i.g. abbreviation should be spelled out the first time here administration of ethanol. Sucralfate increased the concentrations of GSH and PSH and prevented the ethanol-induced changes in gastric mucosal thiol concentrations. Our modified methods are now suitable for direct measurements of major protein and non-protein thiols/disulfides in the gastric mucosa or liver. A common element in the pathogenesis of chemically induced HML and in the mechanism of gastroprotective drugs seems to be the decreased ratios of reduced and oxidized glutathione as well as protein sulfhydryls and disulfides.

  11. Protein and non-protein sulfhydryls and disulfides in gastric mucosa and liver after gastrotoxic chemicals and sucralfate: Possible new targets of pharmacologic agents

    Institute of Scientific and Technical Information of China (English)

    Lajos Nagy; Miki Nagata; Sandor Szabo

    2007-01-01

    AIM: To investigate the role of major non-protein and protein sulfhydryls and disulfides in chemically induced gastric hemorrhagic mucosal lesions (HML) and the mechanism of gastroprotective effect of sucralfate.METHODS: Rats were given 1 mL of 75% ethanol, 25%NaCl, 0.6 mol/L HCI, 0.2 mol/L NaOH or 1% ammonia solutions intragastrically (i.g.) and sacrificed 1, 3, 6 or 12 min later. Total (reduced and oxidized) glutathione (GSH + GSSG), glutathione disulfide (GSSG), protein free sulfhydryls (PSH), protein-glutathione mixed disulfides (PSSG) and protein cystine disulfides (PSSP) were measured in gastric mucosa and liver.RESULTS: Reduced glutathione (GSH) was depleted in the gastric mucosa after ethanol, HCI or NaCl exposure,while oxidized glutathione (GSSG) concentrations increased, except by HCI and NaOH exposure. Decreased levels of PSH after exposure to ethanol were observed,NaCl or NaOH while the total protein disulfides were increased. Ratios of reduced to oxidized glutathione or sulfhydrils to disulfides were decreased by all chemicals.No changes in thiol homeostasis were detected in the liver after i.g. abbreviation should be spelled out the first time here administration of ethanol. Sucralfate increased the concentrations of GSH and PSH and prevented the ethanol-induced changes in gastric mucosal thiol concentrations.CONCLUSION: Our modified methods are now suitable for direct measurements of major protein and nonprotein thiols/disulfides in the gastric mucosa or liver.A common element in the pathogenesis of chemically induced HML and in the mechanism of gastroprotective drugs seems to be the decreased ratios of reduced and oxidized glutathione as well as protein sulfhydryls and disulfides.

  12. "Vibrational bonding": a new type of chemical bond is discovered.

    Science.gov (United States)

    Rhodes, Christopher J; Macrae, Roderick M

    2015-01-01

    A long-sought but elusive new type of chemical bond, occurring on a minimum-free, purely repulsive potential energy surface, has recently been convincingly shown to be possible on the basis of high-level quantum-chemical calculations. This type of bond, termed a vibrational bond, forms because the total energy, including the dynamical energy of the nuclei, is lower than the total energy of the dissociated products, including their vibrational zero-point energy. For this to be the case, the ZPE of the product molecule must be very high, which is ensured by replacing a conventional hydrogen atom with its light isotope muonium (Mu, mass = 1/9 u) in the system Br-H-Br, a natural transition state in the reaction between Br and HBr. A paramagnetic species observed in the reaction Mu +Br2 has been proposed as a first experimental sighting of this species, but definitive identification remains challenging.

  13. Oxidative protein folding: from thiol-disulfide exchange reactions to the redox poise of the endoplasmic reticulum.

    Science.gov (United States)

    Hudson, Devin A; Gannon, Shawn A; Thorpe, Colin

    2015-03-01

    This review examines oxidative protein folding within the mammalian endoplasmic reticulum (ER) from an enzymological perspective. In protein disulfide isomerase-first (PDI-first) pathways of oxidative protein folding, PDI is the immediate oxidant of reduced client proteins and then addresses disulfide mispairings in a second isomerization phase. In PDI-second pathways the initial oxidation is PDI-independent. Evidence for the rapid reduction of PDI by reduced glutathione is presented in the context of PDI-first pathways. Strategies and challenges are discussed for determination of the concentrations of reduced and oxidized glutathione and of the ratios of PDI(red):PDI(ox). The preponderance of evidence suggests that the mammalian ER is more reducing than first envisaged. The average redox state of major PDI-family members is largely to almost totally reduced. These observations are consistent with model studies showing that oxidative protein folding proceeds most efficiently at a reducing redox poise consistent with a stoichiometric insertion of disulfides into client proteins. After a discussion of the use of natively encoded fluorescent probes to report the glutathione redox poise of the ER, this review concludes with an elaboration of a complementary strategy to discontinuously survey the redox state of as many redox-active disulfides as can be identified by ratiometric LC-MS-MS methods. Consortia of oxidoreductases that are in redox equilibrium can then be identified and compared to the glutathione redox poise of the ER to gain a more detailed understanding of the factors that influence oxidative protein folding within the secretory compartment. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Mutagenesis of the redox-active disulfide in mercuric ion reductase: Catalysis by mutant enzymes restricted to flavin redox chemistry

    International Nuclear Information System (INIS)

    Distefano, M.D.; Au, K.G.; Walsh, C.T.

    1989-01-01

    Mercuric reductase, a flavoenzyme that possesses a redox-active cystine, Cys 135 Cys 140 , catalyzes the reduction of Hg(II) to Hg(0) by NADPH. As a probe of mechanism, the authors have constructed mutants lacking a redox-active disulfide by eliminating Cys 135 (Ala 135 Cys 140 ), Cys 14 (Cys 135 Ala 140 ), or both (Ala 135 Ala 140 ). Additionally, they have made double mutants that lack Cys 135 (Ala 135 Cys 139 Cys 140 ) or Cys 140 (Cys 135 Cys 139 Ala 140 ) but introduce a new Cys in place of Gly 139 with the aim of constructing dithiol pairs in the active site that do not form a redox-active disulfide. The resulting mutant enzymes all lack redox-active disulfides and are hence restricted to FAD/FADH 2 redox chemistry. Each mutant enzyme possesses unique physical and spectroscopic properties that reflect subtle differences in the FAD microenvironment. Preliminary evidence for the Ala 135 Cys 139 Cys 14 mutant enzyme suggests that this protein forms a disulfide between the two adjacent Cys residues. Hg(II) titration experiments that correlate the extent of charge-transfer quenching with Hg(II) binding indicate that the Ala 135 Cys 140 protein binds Hg(II) with substantially less avidity than does the wild-type enzyme. All mutant mercuric reductases catalyze transhydrogenation and oxygen reduction reactions through obligatory reduced flavin intermediates at rates comparable to or greater than that of the wild-type enzyme. In multiple-turnover assays which monitored the production of Hg(0), two of the mutant enzymes were observed to proceed through at least 30 turnovers at rates ca. 1000-fold slower than that of wild-type mercuric reductase. They conclude that the Cys 135 and Cys 140 thiols serve as Hg(II) ligands that orient the Hg(II) for subsequent reduction by a reduced flavin intermediate

  15. Scan-rate dependence in protein calorimetry: the reversible transitions of Bacillus circulans xylanase and a disulfide-bridge mutant.

    OpenAIRE

    Davoodi, J.; Wakarchuk, W. W.; Surewicz, W. K.; Carey, P. R.

    1998-01-01

    The stabilities of Bacillus circulans xylanase and a disulfide-bridge-containing mutant (S100C/N148C) were investigated by differential scanning calorimetry (DSC) and thermal inactivation kinetics. The thermal denaturation of both proteins was found to be irreversible, and the apparent transition temperatures showed a considerable dependence upon scanning rate. In the presence of low (nondenaturing) concentrations of urea, calorimetric transitions were observed for both proteins in the second...

  16. Enamel Bond Strength of New Universal Adhesive Bonding Agents.

    Science.gov (United States)

    McLean, D E; Meyers, E J; Guillory, V L; Vandewalle, K S

    2015-01-01

    Universal bonding agents have been introduced for use as self-etch or etch-and-rinse adhesives depending on the dental substrate and clinician's preference. The purpose of this study was to evaluate the shear bond strength (SBS) of composite to enamel using universal adhesives compared to a self-etch adhesive when applied in self-etch and etch-and-rinse modes over time. Extracted human third molars were used to create 120 enamel specimens. The specimens were ground flat and randomly divided into three groups: two universal adhesives and one self-etch adhesive. Each group was then subdivided, with half the specimens bonded in self-etch mode and half in etch-and-rinse mode. The adhesives were applied as per manufacturers' instructions, and composite was bonded using a standardized mold and cured incrementally. The groups were further divided into two subgroups with 10 specimens each. One subgroup was stored for 24 hours and the second for six months in 37°C distilled water and tested in shear. Failure mode was also determined for each specimen. A three-way analysis of variance (ANOVA) found a significant difference between groups based on bonding agent (p0.05). Clearfil SE in etch-and-rinse and self-etch modes had more mixed fractures than either universal adhesive in either mode. Etching enamel significantly increased the SBS of composite to enamel. Clearfil SE had significantly greater bond strength to enamel than either universal adhesive, which were not significantly different from each other.

  17. Common Factors in International Bond Returns

    NARCIS (Netherlands)

    Driessen, J.J.A.G.; Melenberg, B.; Nijman, T.E.

    2000-01-01

    In this paper we estimate and interpret the factors that jointly determine bond returns of different maturities in the US, Germany and Japan.We analyze both currency-hedged and unhedged bond returns.For currency-hedged bond returns, we find that five factors explain 96.5% of the variation of bond

  18. 7 CFR 1726.27 - Contractor's bonds.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 11 2010-01-01 2010-01-01 false Contractor's bonds. 1726.27 Section 1726.27... AGRICULTURE ELECTRIC SYSTEM CONSTRUCTION POLICIES AND PROCEDURES General § 1726.27 Contractor's bonds. (a) RUS Form 168b, Contractor's Bond, shall be used when a contractor's bond is required by RUS Forms 200, 257...

  19. Oregon School Bond Manual. Fourth Edition.

    Science.gov (United States)

    Oregon State Dept. of Education, Salem.

    The manual is intended to guide attorneys and officials of school districts in the issuance and sale of school district bonds. Purchasers of school district bonds rely on the recommendations of accredited bond attorneys who render opinions concerning the validity and legality of bond issues offered for sale. This manual is designed to assist in…

  20. Oregon School Bond Manual. Fifth Edition.

    Science.gov (United States)

    Oregon State Dept. of Education, Salem.

    To help school districts comply with Oregon's school bond laws, this manual provides guidelines for school district attorneys and personnel in the issuance and sale of school bonds. The document describes the proper time sequence of the bonding procedure, including elections, school board authorizations, necessary certificates, bond registration…

  1. Design, Synthesis and Biological Evaluation of Brain-Targeted Thiamine Disulfide Prodrugs of Ampakine Compound LCX001

    Directory of Open Access Journals (Sweden)

    Dian Xiao

    2016-04-01

    Full Text Available Ampakine compounds have been shown to reverse opiate-induced respiratory depression by activation of amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA glutamate receptors. However, their pharmacological exploitations are hindered by low blood-brain barrier (BBB permeability and limited brain distribution. Here, we explored whether thiamine disulfide prodrugs with the ability of “lock-in” can be used to solve these problems. A series of thiamine disulfide prodrugs 7a–7f of ampakine compound LCX001 was synthesized and evaluated. The trials in vitro showed that prodrugs 7e, 7d, 7f possessed a certain stability in plasma and quickly decomposed in brain homogenate by the disulfide reductase. In vivo, prodrug 7e decreased the peripheral distribution of LCX001 and significantly increased brain distribution of LCX001 after i.v. administration. This compound showed 2.23- and 3.29-fold greater increases in the AUC0-t and MRT0-t of LCX001 in brain, respectively, than did LCX001 itself. A preliminary pharmacodynamic study indicated that the required molar dose of prodrug 7e was only one eighth that of LCX001 required to achieve the same effect in mice. These findings provide an important reference to evaluate the clinical outlook of ampakine compounds.

  2. Engineering nutritious proteins: improvement of stability in the designer protein MB-1 via introduction of disulfide bridges.

    Science.gov (United States)

    Doucet, Alain; Williams, Martin; Gagnon, Mylene C; Sasseville, Maxime; Beauregard, Marc

    2002-01-02

    Protein design is currently used for the creation of new proteins with desirable traits. In this laboratory the focus has been on the synthesis of proteins with high essential amino acid content having potential applications in animal nutrition. One of the limitations faced in this endeavor is achieving stable proteins despite a highly biased amino acid content. Reported here are the synthesis and characterization of two disulfide-bridged mutants derived from the MB-1 designer protein. Both mutants outperformed their parent protein MB-1 with their bridge formed, as shown by circular dichroism, size exclusion chromatography, thermal denaturation, and proteolytic degradation experiments. When the disulfide bridges were cleaved, the mutants' behavior changed: the mutants significantly unfolded, suggesting that the introduction of Cys residues was deleterious to MB-1-folding. In an attempt to compensate for the mutations used, a Tyr62-Trp mutation was performed, leading to an increase in bulk and hydrophobicity in the core. The Trp-containing disulfide-bridged mutants did not behave as well as the original MB-1Trp, suggesting that position 62 might not be adequate for a compensatory mutation.

  3. Design and introduction of a disulfide bridge in firefly luciferase: increase of thermostability and decrease of pH sensitivity.

    Science.gov (United States)

    Imani, Mehdi; Hosseinkhani, Saman; Ahmadian, Shahin; Nazari, Mahboobeh

    2010-08-01

    The thermal sensitivity and pH-sensitive spectral properties of firefly luciferase have hampered its application in a variety of fields. It is proposed that the stability of a protein can be increased by introduction of disulfide bridge that decreases the configurational entropy of unfolding. A disulfide bridge is introduced into Photinus pyralis firefly luciferase to make two separate mutant enzymes with a single bridge. Even though the A103C/S121C mutant showed remarkable thermal stability, its specific activity decreased, whereas the A296C/A326C mutant showed tremendous thermal stability, relative pH insensitivity and 7.3-fold increase of specific activity. Moreover, the bioluminescence emission spectrum of A296C/A326C was resistant against higher temperatures (37 degrees C). Far-UV CD analysis showed slight secondary structure changes for both mutants. Thermal denaturation analysis showed that conformational stabilities of A103C/S121C and A296C/A326C are more than native firefly luciferase. It is proposed that since A296 and A326 are situated in the vicinity of the enzyme active site microenvironment in comparison with A103 and S121, the formation of a disulfide bridge in this region has more impact on enzyme kinetic characteristics.

  4. Deriving the bond pricing equation

    Directory of Open Access Journals (Sweden)

    Kožul Nataša

    2014-01-01

    Full Text Available Given the recent focus on Eurozone debt crisis and the credit rating downgrade not only of US debt, but that of other countries and many UK major banking institutions, this paper aims to explain the concept of bond yield, its different measures and bond pricing equation. Yields on capital market instruments are rarely quoted on the same basis, which makes direct comparison between different as investment choices impossible. Some debt instruments are quoted on discount basis, whilst coupon-bearing ones accrue interest differently, offer different compounding opportunities, have different coupon payment frequencies, and manage non-business day maturity dates differently. Moreover, rules governing debt vary across countries, markets and currencies, making yield calculation and comparison a rather complex issue. Thus, some fundamental concepts applicable to debt instrument yield measurement, with focus on bond equation, are presented here. In addition, bond equation expressed in annuity form and used to apply Newton-Raphson algorithm to derive true bond yield is also shown.

  5. Welding, Bonding and Fastening, 1984

    Science.gov (United States)

    Buckley, J. D. (Editor); Stein, B. A. (Editor)

    1985-01-01

    A compilation of papers presented in a joint NASA, American Society for Metals, The George Washington University, American Welding Soceity, and Society of Manufacturing Engineers conference on Welding, Bonding, and Fastening at Langley Research Center, Hampton, VA, on October 23 to 25, 1984 is given. Papers were presented on technology developed in current research programs relevant to welding, bonding, and fastening of structural materials required in fabricating structures and mechanical systems used in the aerospace, hydrospace, and automotive industries. Topics covered in the conference included equipment, hardware and materials used when welding, brazing, and soldering, mechanical fastening, explosive welding, use of unique selected joining techniques, adhesives bonding, and nondestructive evaluation. A concept of the factory of the future was presented, followed by advanced welding techniques, automated equipment for welding, welding in a cryogenic atmosphere, blind fastening, stress corrosion resistant fasteners, fastening equipment, explosive welding of different configurations and materials, solid-state bonding, electron beam welding, new adhesives, effects of cryogenics on adhesives, and new techniques and equipment for adhesive bonding.

  6. Three methods to measure RH bond energies

    International Nuclear Information System (INIS)

    Berkowitz, J.; Ellison, G.B.; Gutman, D.

    1993-01-01

    In this paper the authors compare and contrast three powerful methods for experimentally measuring bond energies in polyatomic molecules. The methods are: radical kinetics; gas phase acidity cycles; and photoionization mass spectroscopy. The knowledge of the values of bond energies are a basic piece of information to a chemist. Chemical reactions involve the making and breaking of chemical bonds. It has been shown that comparable bonds in polyatomic molecules, compared to the same bonds in radicals, can be significantly different. These bond energies can be measured in terms of bond dissociation energies

  7. Recombinant Expression, In Vitro Refolding and Characterizing Disulfide Bonds of a Mouse Inhibitory C-Type Lectin-Like Receptor Nkrp1b

    Czech Academy of Sciences Publication Activity Database

    Hernychová, Lucie; Mrázek, Hynek; Ivanova, Ljubina; Kukačka, Zdeněk; Chmelík, Josef; Novák, Petr

    2015-01-01

    Roč. 64, č. 2015 (2015), s. 85-93 ISSN 0862-8408 R&D Projects: GA MŠk(CZ) EE2.3.30.0003; GA MŠk(CZ) ED1.1.00/02.0109; GA MŠk LO1509 Grant - others:OPPC(XE) CZ.2.16/3.1.00/24023 Institutional support: RVO:61388971 Keywords : NK cell * C-type lectin-like receptor (CTLR) * Nkrp1 Subject RIV: CE - Biochemistry Impact factor: 1.643, year: 2015

  8. Synthesis of protected peptides from the human IgG1 hinge region on PEG support using disulfide bond synthons and alkaline or enzymatic detachment

    Czech Academy of Sciences Publication Activity Database

    Niederhafner, Petr; Šafařík, Martin; Šebestík, Jaroslav; Gut, Vladimír; Maloň, Petr; Hlaváček, Jan

    2006-01-01

    Roč. 47, č. 6 (2006), s. 1023-1025 ISSN 0040-4039 R&D Projects: GA ČR(CZ) GA203/03/1362 Institutional research plan: CEZ:AV0Z40550506 Keywords : peptide synthesis * IgG1 hinge peptide * PEG carrier Subject RIV: CC - Organic Chemistry Impact factor: 2.509, year: 2006

  9. Probing the Conformational and Functional Consequences of Disulfide Bond Engineering in Growth Hormone by Hydrogen-Deuterium Exchange Mass Spectrometry Coupled to Electron Transfer Dissociation

    DEFF Research Database (Denmark)

    Seger, Signe T; Breinholt, Jens; Faber, Johan H

    2015-01-01

    Human growth hormone (hGH), and its receptor interaction, is essential for cell growth. To stabilize a flexible loop between helices 3 and 4, while retaining affinity for the hGH receptor, we have engineered a new hGH variant (Q84C/Y143C). Here, we employ hydrogen-deuterium exchange mass spectrom......Human growth hormone (hGH), and its receptor interaction, is essential for cell growth. To stabilize a flexible loop between helices 3 and 4, while retaining affinity for the hGH receptor, we have engineered a new hGH variant (Q84C/Y143C). Here, we employ hydrogen-deuterium exchange mass...

  10. Mimicry of the regulatory role of urokinase in lamellipodia formation by introduction of a non-native interdomain disulfide bond in its receptor

    DEFF Research Database (Denmark)

    Gårdsvoll, Henrik; Kjærgaard, Magnus; Jacobsen, Benedikte

    2011-01-01

    The high-affinity interaction between the urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) plays a regulatory role for both extravascular fibrinolysis and uPAR-mediated adhesion and migration on vitronectin-coated surfaces. We have recently proposed that the ......The high-affinity interaction between the urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) plays a regulatory role for both extravascular fibrinolysis and uPAR-mediated adhesion and migration on vitronectin-coated surfaces. We have recently proposed...... that the adhesive function of uPAR is allosterically regulated via a "tightening" of its three-domain structure elicited by uPA binding. To challenge this proposition, we redesigned the uPAR structure to limit its inherent conformational flexibility by covalently tethering domains DI and DIII via a non...... adhering to vitronectin. In this respect, the engineered constraint in uPAR(H47C-N259C) thus bypasses the regulatory role of uPA binding, resulting in a constitutively active uPAR. In conclusion, our data argue for a biological relevance of the interdomain dynamics of the glycolipid-anchored u...

  11. Diffusionless bonding of aluminum to Zircaloy-2

    International Nuclear Information System (INIS)

    Watson, R.D.

    1965-04-01

    Aluminum can be bonded to zirconium without difficulty even when a thin layer of oxide is present on the surface of the zirconium . No detectable diffusion takes place during the bonding process. The bond layer can be stretched as much. as 8% without affecting the bond. The bond can be heated for 1000 hours at 260 o C (500 o F), and can be water quenched from 260 o C (500 o F) without any noticeable change in the bond strength. An extrusion technique has been devised for making transition sections of aluminum bonded to zirconium which can then be used to join these metals by conventional welding. Welding can be done close to the bond zone without seriously affecting the integrity of the bond. This method of bonding aluminum to Zircaloy-2 is covered by Canadian patent 702,438 January 26, 1965. (author)

  12. Health effect of chronic exposure to carbon disulfide (CS 2 on women employed in viscose industry

    Directory of Open Access Journals (Sweden)

    Krzysztof Sieja

    2018-05-01

    Full Text Available Many women are exposed to carbon disulfide (CS 2 hazards at work every day. Working with CS 2 may cause some women to experience abnormalities in their reproductive health. Until now obtained data is generally concentrated on the health effects of CS 2 observed in the viscose industry. To date, CS 2 has not been studied precisely for its potential to have damaging effects on female reproductive system, especially the frequency of menstrual disturbances and the course of menopause. The aim of the study was to sum up female reproductive health hazards amongst women chronically exposed to CS 2 in their workplace in the viscose industry. In order to study the effect of CS 2 in the contemporary viscose industry, exposure measurements should be collected in prospective or cross-sectional studies. In conclusion, reproductive health hazards for women chronically exposed to CS 2 in the workplace in the viscose industry are the following: 1 menstrual disorders essentially are more frequent than in the case of the healthy women, 2 for women chronically exposed to CS 2 the average menopausal age is statistically earlier, as compared to healthy women, 3 complex disturbances in neurohormonal system for women exposed to CS 2 , resulting from toxic influences of CS 2 , which cause the secretion of estrogens and progesterone in ovaries and dehydroepiandrosterone sulfate in the adrenal gland to diminish. Med Pr 2018;69(3:329–335

  13. Tuning the Schottky rectification in graphene-hexagonal boron nitride-molybdenum disulfide heterostructure.

    Science.gov (United States)

    Liu, Biao; Zhao, Yu-Qing; Yu, Zhuo-Liang; Wang, Lin-Zhi; Cai, Meng-Qiu

    2018-03-01

    It was still a great challenge to design high performance of rectification characteristic for the rectifier diode. Lately, a new approach was proposed experimentally to tune the Schottky barrier height (SBH) by inserting an ultrathin insulated tunneling layer to form metal-insulator-semiconductor (MIS) heterostructures. However, the electronic properties touching off the high performance of these heterostructures and the possibility of designing more efficient applications for the rectifier diode were not presently clear. In this paper, the structural, electronic and interfacial properties of the novel MIS diode with the graphene/hexagonal boron nitride/monolayer molybdenum disulfide (GBM) heterostructure had been investigated by first-principle calculations. The calculated results showed that the intrinsic properties of graphene and MoS 2 were preserved due to the weak van der Waals contact. The height of interfacial Schottky barrier can be tuned by the different thickness of hBN layers. In addition, the GBM Schottky diode showed more excellent rectification characteristic than that of GM Schottky diode due to the interfacial band bending caused by the epitaxial electric field. Based on the electronic band structure, we analyzed the relationship between the electronic structure and the nature of the Schottky rectifier, and revealed the potential of utilizing GBM Schottky diode for the higher rectification characteristic devices. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Phosphorene/rhenium disulfide heterojunction-based negative differential resistance device for multi-valued logic

    Science.gov (United States)

    Shim, Jaewoo; Oh, Seyong; Kang, Dong-Ho; Jo, Seo-Hyeon; Ali, Muhammad Hasnain; Choi, Woo-Young; Heo, Keun; Jeon, Jaeho; Lee, Sungjoo; Kim, Minwoo; Song, Young Jae; Park, Jin-Hong

    2016-11-01

    Recently, negative differential resistance devices have attracted considerable attention due to their folded current-voltage characteristic, which presents multiple threshold voltage values. Because of this remarkable property, studies associated with the negative differential resistance devices have been explored for realizing multi-valued logic applications. Here we demonstrate a negative differential resistance device based on a phosphorene/rhenium disulfide (BP/ReS2) heterojunction that is formed by type-III broken-gap band alignment, showing high peak-to-valley current ratio values of 4.2 and 6.9 at room temperature and 180 K, respectively. Also, the carrier transport mechanism of the BP/ReS2 negative differential resistance device is investigated in detail by analysing the tunnelling and diffusion currents at various temperatures with the proposed analytic negative differential resistance device model. Finally, we demonstrate a ternary inverter as a multi-valued logic application. This study of a two-dimensional material heterojunction is a step forward toward future multi-valued logic device research.

  15. Phosphorene/rhenium disulfide heterojunction-based negative differential resistance device for multi-valued logic

    Science.gov (United States)

    Shim, Jaewoo; Oh, Seyong; Kang, Dong-Ho; Jo, Seo-Hyeon; Ali, Muhammad Hasnain; Choi, Woo-Young; Heo, Keun; Jeon, Jaeho; Lee, Sungjoo; Kim, Minwoo; Song, Young Jae; Park, Jin-Hong

    2016-01-01

    Recently, negative differential resistance devices have attracted considerable attention due to their folded current–voltage characteristic, which presents multiple threshold voltage values. Because of this remarkable property, studies associated with the negative differential resistance devices have been explored for realizing multi-valued logic applications. Here we demonstrate a negative differential resistance device based on a phosphorene/rhenium disulfide (BP/ReS2) heterojunction that is formed by type-III broken-gap band alignment, showing high peak-to-valley current ratio values of 4.2 and 6.9 at room temperature and 180 K, respectively. Also, the carrier transport mechanism of the BP/ReS2 negative differential resistance device is investigated in detail by analysing the tunnelling and diffusion currents at various temperatures with the proposed analytic negative differential resistance device model. Finally, we demonstrate a ternary inverter as a multi-valued logic application. This study of a two-dimensional material heterojunction is a step forward toward future multi-valued logic device research. PMID:27819264

  16. Defect-Mediated Lithium Adsorption and Diffusion on Monolayer Molybdenum Disulfide.

    Science.gov (United States)

    Sun, Xiaoli; Wang, Zhiguo; Fu, Y Q

    2015-12-22

    Monolayer Molybdenum Disulfide (MoS2) is a promising anode material for lithium ion batteries because of its high capacities. In this work, first principle calculations based on spin density functional theory were performed to investigate adsorption and diffusion of lithium on monolayer MoS2 with defects, such as single- and few-atom vacancies, antisite, and grain boundary. The values of adsorption energies on the monolayer MoS2 with the defects were increased compared to those on the pristine MoS2. The presence of defects causes that the Li is strongly bound to the monolayer MoS2 with adsorption energies in the range between 2.81 and 3.80 eV. The donation of Li 2s electron to the defects causes an enhancement of adsorption of Li on the monolayer MoS2. At the same time, the presence of defects does not apparently affect the diffusion of Li, and the energy barriers are in the range of 0.25-0.42 eV. The presence of the defects can enhance the energy storage capacity, suggesting that the monolayer MoS2 with defects is a suitable anode material for the Li-ion batteries.

  17. Optical spectroscopic elucidation of beta-turns in disulfide bridged cyclic tetrapeptides.

    Science.gov (United States)

    Borics, Attila; Murphy, Richard F; Lovas, Sándor

    2007-01-01

    Vibrational circular dichroism (VCD) spectroscopic features of type II beta-turns were characterized previously, but, criteria for differentiation between beta-turn types had not been established yet. Model tetrapeptides, cyclized through a disulfide bridge, were designed on the basis of previous experimental results and the observed incidence of amino acid residues in the i + 1 and i + 2 positions in beta-turns, to determine the features of VCD spectra of type I and II beta-turns. The results were correlated with electronic circular dichroism (ECD) spectra and VCD spectra calculated from conformational data obtained by molecular dynamics (MD) simulations. All cyclic tetrapeptides yielded VCD signals with a higher frequency negative and a lower frequency positive couplet with negative lobes overlapping. MD simulations confirmed the conformational homogeneity of these peptides in solution. Comparison with ECD spectroscopy, MD, and quantum chemical calculation results suggested that the low frequency component of VCD spectra originating from the tertiary amide vibrations could be used to distinguish between types of beta-turn structures. On the basis of this observation, VCD spectroscopic features of type II and VIII beta-turns and ECD spectroscopic properties of a type VIII beta-turn were suggested. The need for independent experimental as well as theoretical investigations to obtain decisive conformational information was recognized. Copyright 2006 Wiley Periodicals, Inc.

  18. Hydrogen-assisted post-growth substitution of tellurium into molybdenum disulfide monolayers with tunable compositions

    Science.gov (United States)

    Yin, Guoli; Zhu, Dancheng; Lv, Danhui; Hashemi, Arsalan; Fei, Zhen; Lin, Fang; Krasheninnikov, Arkady V.; Zhang, Ze; Komsa, Hannu-Pekka; Jin, Chuanhong

    2018-04-01

    Herein we report the successful doping of tellurium (Te) into molybdenum disulfide (MoS2) monolayers to form MoS2x Te2(1-x) alloy with variable compositions via a hydrogen-assisted post-growth chemical vapor deposition process. It is confirmed that H2 plays an indispensable role in the Te substitution into as-grown MoS2 monolayers. Atomic-resolution transmission electron microscopy allows us to determine the lattice sites and the concentration of introduced Te atoms. At a relatively low concentration, tellurium is only substituted in the sulfur sublattice to form monolayer MoS2(1-x)Te2x alloy, while with increasing Te concentration (up to ˜27.6% achieved in this study), local regions with enriched tellurium, large structural distortions, and obvious sulfur deficiency are observed. Statistical analysis of the Te distribution indicates the random substitution. Density functional theory calculations are used to investigate the stability of the alloy structures and their electronic properties. Comparison with experimental results indicate that the samples are unstrained and the Te atoms are predominantly substituted in the top S sublattice. Importantly, such ultimately thin Janus structure of MoS2(1-x)Te2x exhibits properties that are distinct from their constituents. We believe our results will inspire further exploration of the versatile properties of asymmetric 2D TMD alloys.

  19. ReaxFF Reactive Force-Field Study of Molybdenum Disulfide (MoS2).

    Science.gov (United States)

    Ostadhossein, Alireza; Rahnamoun, Ali; Wang, Yuanxi; Zhao, Peng; Zhang, Sulin; Crespi, Vincent H; van Duin, Adri C T

    2017-02-02

    Two-dimensional layers of molybdenum disulfide, MoS 2 , have been recognized as promising materials for nanoelectronics due to their exceptional electronic and optical properties. Here we develop a new ReaxFF reactive potential that can accurately describe the thermodynamic and structural properties of MoS 2 sheets, guided by extensive density functional theory simulations. This potential is then applied to the formation energies of five different types of vacancies, various vacancy migration barriers, and the transition barrier between the semiconducting 2H and metallic 1T phases. The energetics of ripplocations, a recently observed defect in van der Waals layers, is examined, and the interplay between these defects and sulfur vacancies is studied. As strain engineering of MoS 2 sheets is an effective way to manipulate the sheets' electronic and optical properties, the new ReaxFF description can provide valuable insights into morphological changes that occur under various loading conditions and defect distributions, thus allowing one to tailor the electronic properties of these 2D crystals.

  20. Structure-activity studies of vasoactive intestinal peptide (VIP): cyclic disulfide analogs.

    Science.gov (United States)

    Bolin, D R; Cottrell, J; Garippa, R; O'Neill, N; Simko, B; O'Donnell, M

    1993-02-01

    Analogs of vasoactive intestinal peptide with cysteine residues incorporated at selected sites within the sequence were prepared by solid phase methods, oxidized to the corresponding cyclic disulfides and purified to homogeneity by preparative HPLC. The cyclic compounds were assayed as smooth muscle relaxants on isolated guinea pig trachea, as bronchodilators in vivo in guinea pigs, and for binding to VIP receptors in guinea pig lung membranes. Of the analogs prepared at the N-terminus, one compound, Ac-[D-Cys6,D-Cys11,Lys12,Nle17,Val26,Th r28]-VIP, was found to be a full agonist with slightly more than one tenth the potency of native VIP. Most other cyclic analogs in the N-terminal region were found to be inactive. A second analog, Ac-[Lys12,Cys17,Val26,Cys28]-VIP, was also found to be a full agonist with potency about one third that of native VIP. Furthermore, this compound was active as a bronchodilator in vivo in guinea pig, but with somewhat diminished potency as compared to native VIP. Strikingly, this cyclic compound was found to have significantly longer duration of action (> 40 min) when compared to an analogous acyclic compound (5 min). The conformational restrictions imposed by formation of the cyclic ring structures may have stabilized the molecule to degradation, thus enhancing the effective duration of action. Analysis of this series of cyclic analogs has also yielded information about the requirements for the receptor-active conformation of VIP.

  1. First-principles studies of chromium line-ordered alloys in a molybdenum disulfide monolayer

    Science.gov (United States)

    Andriambelaza, N. F.; Mapasha, R. E.; Chetty, N.

    2017-08-01

    Density functional theory calculations have been performed to study the thermodynamic stability, structural and electronic properties of various chromium (Cr) line-ordered alloy configurations in a molybdenum disulfide (MoS2) hexagonal monolayer for band gap engineering. Only the molybdenum (Mo) sites were substituted at each concentration in this study. For comparison purposes, different Cr line-ordered alloy and random alloy configurations were studied and the most thermodynamically stable ones at each concentration were identified. The configurations formed by the nearest neighbor pair of Cr atoms are energetically most favorable. The line-ordered alloys are constantly lower in formation energy than the random alloys at each concentration. An increase in Cr concentration reduces the lattice constant of the MoS2 system following the Vegard’s law. From density of states analysis, we found that the MoS2 band gap is tunable by both the Cr line-ordered alloys and random alloys with the same magnitudes. The reduction of the band gap is mainly due to the hybridization of the Cr 3d and Mo 4d orbitals at the vicinity of the band edges. The band gap engineering and magnitudes (1.65 eV to 0.86 eV) suggest that the Cr alloys in a MoS2 monolayer are good candidates for nanotechnology devices.

  2. Molybdenum disulfide for ultra-low detection of free radicals: electrochemical response and molecular modeling

    Science.gov (United States)

    Gupta, Ankur; Rawal, Takat B.; Neal, Craig J.; Das, Soumen; Rahman, Talat S.; Seal, Sudipta

    2017-06-01

    Two-dimensional (2D) molybdenum disulfide (MoS2) offers attractive properties due to its band gap modulation and has led to significant research-oriented applications (i.e. DNA and protein detection, cell imaging (fluorescent label) etc.). In biology, detection of free radicals (i.e. reactive oxygen species and reactive nitrogen (NO*) species are very important for early discovery and treatment of diseases. Herein, for the first time, we demonstrate the ultra-low (pico-molar) detection of pharmaceutically relevant free radicals using MoS2 for electrochemical sensing. We present pico- to nano- molar level sensitivity in smaller MoS2 with S-deficiency as revealed by x-ray photoelectron spectroscopy. Furthermore, the detection mechanism and size-dependent sensitivity have been investigated by density functional theory (DFT) showing the change in electronic density of states of Mo atoms at edges which lead to the preferred adsorption of H2O2 on Mo edges. The DFT analysis signifies the role of size and S-deficiency in the higher catalytic activity of smaller MoS2 particles and, thus, ultra-low detection.

  3. Metalorganic chemical vapor deposition of iron disulfide and its use for solar energy conversion

    Science.gov (United States)

    Ennaoui, Ahmed; Fiechter, Sebastian; Vogel, Ralf; Giersig, M.; Weller, Horst; Tributsch, Helmut

    1992-12-01

    Thin polycrystalline films of iron disulfide have been grown on different substrates by chemical vapour deposition. The films were characterized using optical absorption and TEM. RBS and EDAX analysis has been used to explore the chemical stoichiometry. XRD and FTIR allowed the identification of both FeS2 phases pyrite and marcasite. A novel method for sensitization of highly porous Ti02 elecrodes with ultra thin (10-20 nm) polycrystalline films of FeS2 (pyrite) is presented. Photoelectrochemical solar cell using the above electrode generated high photovoltage of up to 600mV compared with single crystalline electrode (200 mV). In this device the semiconductor with a small band gap and high absorption coefficient (FeS2 pyrite; EG = 0.9 eV; a = 6 x 105 cm-1) absorbs the light and injects electrons into the conduction band the wide band gap semiconductor (Ti02 anatase; EG = 3.2 eV). Regeneration of holes is taking place by electron transfer from redox system in the electrolyte.

  4. Few-layer molybdenum disulfide transistors and circuits for high-speed flexible electronics.

    Science.gov (United States)

    Cheng, Rui; Jiang, Shan; Chen, Yu; Liu, Yuan; Weiss, Nathan; Cheng, Hung-Chieh; Wu, Hao; Huang, Yu; Duan, Xiangfeng

    2014-10-08

    Two-dimensional layered materials, such as molybdenum disulfide, are emerging as an exciting material system for future electronics due to their unique electronic properties and atomically thin geometry. Here we report a systematic investigation of MoS2 transistors with optimized contact and device geometry, to achieve self-aligned devices with performance including an intrinsic gain over 30, an intrinsic cut-off frequency fT up to 42 GHz and a maximum oscillation frequency fMAX up to 50 GHz, exceeding the reported values for MoS2 transistors to date (fT~0.9 GHz, fMAX~1 GHz). Our results show that logic inverters or radio frequency amplifiers can be formed by integrating multiple MoS2 transistors on quartz or flexible substrates with voltage gain in the gigahertz regime. This study demonstrates the potential of two-dimensional layered semiconductors for high-speed flexible electronics.

  5. Thermal stability and electrical conductivity in polyethers-molybdenum disulfide nanocomposites

    International Nuclear Information System (INIS)

    Mirabal, N.; Aguirre, P.; Santa Ana, M.A.; Benavente, E.; Gonzalez, Guillermo

    2003-01-01

    The intercalation of poly(ethylene oxide) (PEO), into molybdenum disulfide, like that of other electron pair donors, leads to mixed ionic-electronic conductors. At room temperature, intercalates show electrical and lithium-ion conductivities better than MoS 2 and bulk PEO composites, respectively. However, these products are known to be sensitive to temperature; indeed, in the range 80-100 deg. C an irreversible decrease of the electrical conductivity is observed. In order to investigate these features, the thermal behavior of a series of polyethers of different molecular weights (poly(ethylene glycol) (Mw 3400) and PEO with Mw in the range 10 4 -4x10 6 , pure and intercalated in MoS 2 , (Li x (MoS 2 )(polyether) y with x∼0.1 and y=1.1-1.5), was comparatively analyzed. Furthermore, the effect of thermal treatment of the sample on the electrical conductivity was studied for one of the intercalated products. Results indicate that irreversible changes, detected by both loss of weight and a significant conductivity lowering, are occurring in the range from about 100 deg. C to a temperature near to the decomposition point of the organic phase at about 350 deg. C

  6. Rational design of multifunctional devices based on molybdenum disulfide and graphene hybrid nanostructures

    Energy Technology Data Exchange (ETDEWEB)

    Lim, Yi Rang; Lee, Young Bum; Kim, Seong Ku; Kim, Seong Jun [Thin Film Materials Research Center, Korea Research Institute of Chemical Technology, Yuseong, Post Office Box 107, Daejeon 305-600 (Korea, Republic of); Kim, Yooseok; Jeon, Cheolho [Nano-Surface Research Group, Korea Basic Science Institute, Daejeon, 302-333 (Korea, Republic of); Song, Wooseok, E-mail: wssong@krict.re.kr [Thin Film Materials Research Center, Korea Research Institute of Chemical Technology, Yuseong, Post Office Box 107, Daejeon 305-600 (Korea, Republic of); Myung, Sung; Lee, Sun Sook; An, Ki-Seok [Thin Film Materials Research Center, Korea Research Institute of Chemical Technology, Yuseong, Post Office Box 107, Daejeon 305-600 (Korea, Republic of); Lim, Jongsun, E-mail: jslim@krict.re.kr [Thin Film Materials Research Center, Korea Research Institute of Chemical Technology, Yuseong, Post Office Box 107, Daejeon 305-600 (Korea, Republic of)

    2017-01-15

    Highlights: • We fabricated MoS{sub 2}-graphene hybrid thin films for multifunctional applications. • Large-area, uniform multilayer MoS{sub 2} was synthesized on TCVD-grown graphene. • The mobility and photocurrent of the hybrid devices were improved significantly. - Abstract: We rationally designed a new type of hybrid materials, molybdenum disulfide (MoS{sub 2}) synthesized by Mo pre-deposition followed by subsequent sulfurization process directly on thermal chemical vapor deposition (TCVD)-grown graphene, for applications in a multifunctional device. The synthesis of stoichiometric and uniform multilayer MoS{sub 2} and high-crystalline monolayer graphene was evaluated by X-ray photoelectron spectroscopy and Raman spectroscopy. To examine the electrical transport and photoelectrical properties of MoS{sub 2}-graphene hybrid films, field effect transistors (FETs) and visible-light photodetectors based on MoS{sub 2}-graphene were both fabricated. As a result, the extracted mobility for MoS{sub 2}-graphene hybrid FETs was two times higher than that of MoS{sub 2} FETs. In addition, the MoS{sub 2}-graphene photodetectors revealed a significant photocurrent with abrupt switching behavior under periodic illumination.

  7. A rigid disulfide-linked nitroxide side chain simplifies the quantitative analysis of PRE data

    Energy Technology Data Exchange (ETDEWEB)

    Fawzi, Nicolas L. [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States); Fleissner, Mark R. [University of California, Jules Stein Eye Institute and Department of Chemistry and Biochemistry (United States); Anthis, Nicholas J. [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States); Kalai, Tamas; Hideg, Kalman [University of Pecs, Institute of Organic and Medicinal Chemistry (Hungary); Hubbell, Wayne L., E-mail: hubbellw@jsei.ucla.edu [University of California, Jules Stein Eye Institute and Department of Chemistry and Biochemistry (United States); Clore, G. Marius, E-mail: mariusc@mail.nih.gov [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States)

    2011-09-15

    The measurement of {sup 1}H transverse paramagnetic relaxation enhancement (PRE) has been used in biomolecular systems to determine long-range distance restraints and to visualize sparsely-populated transient states. The intrinsic flexibility of most nitroxide and metal-chelating paramagnetic spin-labels, however, complicates the quantitative interpretation of PREs due to delocalization of the paramagnetic center. Here, we present a novel, disulfide-linked nitroxide spin label, R1p, as an alternative to these flexible labels for PRE studies. When introduced at solvent-exposed {alpha}-helical positions in two model proteins, calmodulin (CaM) and T4 lysozyme (T4L), EPR measurements show that the R1p side chain exhibits dramatically reduced internal motion compared to the commonly used R1 spin label (generated by reacting cysteine with the spin labeling compound often referred to as MTSL). Further, only a single nitroxide position is necessary to account for the PREs arising from CaM S17R1p, while an ensemble comprising multiple conformations is necessary for those observed for CaM S17R1. Together, these observations suggest that the nitroxide adopts a single, fixed position when R1p is placed at solvent-exposed {alpha}-helical positions, greatly simplifying the interpretation of PRE data by removing the need to account for the intrinsic flexibility of the spin label.

  8. A rigid disulfide-linked nitroxide side chain simplifies the quantitative analysis of PRE data

    International Nuclear Information System (INIS)

    Fawzi, Nicolas L.; Fleissner, Mark R.; Anthis, Nicholas J.; Kálai, Tamás; Hideg, Kálmán; Hubbell, Wayne L.; Clore, G. Marius

    2011-01-01

    The measurement of 1 H transverse paramagnetic relaxation enhancement (PRE) has been used in biomolecular systems to determine long-range distance restraints and to visualize sparsely-populated transient states. The intrinsic flexibility of most nitroxide and metal-chelating paramagnetic spin-labels, however, complicates the quantitative interpretation of PREs due to delocalization of the paramagnetic center. Here, we present a novel, disulfide-linked nitroxide spin label, R1p, as an alternative to these flexible labels for PRE studies. When introduced at solvent-exposed α-helical positions in two model proteins, calmodulin (CaM) and T4 lysozyme (T4L), EPR measurements show that the R1p side chain exhibits dramatically reduced internal motion compared to the commonly used R1 spin label (generated by reacting cysteine with the spin labeling compound often referred to as MTSL). Further, only a single nitroxide position is necessary to account for the PREs arising from CaM S17R1p, while an ensemble comprising multiple conformations is necessary for those observed for CaM S17R1. Together, these observations suggest that the nitroxide adopts a single, fixed position when R1p is placed at solvent-exposed α-helical positions, greatly simplifying the interpretation of PRE data by removing the need to account for the intrinsic flexibility of the spin label.

  9. Influence of initial sulfur content in precursor solution for the growth of molybdenum disulfide

    Science.gov (United States)

    Tan, A. L.; Ng, S. S.; Abu Hassan, H.

    2018-04-01

    This work investigated the influence of initial sulfur content in the precursor solution for the growth of molybdenum disulfide (MoS2) films by thermal vapour sulfurization (TVS) with sol-gel spin coating as pre-deposition technique. The early introduction of sulfur shows the presence of grains are uniformly distributed and homogeneous on the surface of the film. MoS2 (002) planes are detected for both films with and without initial sulfur conditions, however, the presence of initial sulfur contents gives slightly higher intensity of diffraction peak. Two phonon modes for MoS2, namely the E2g 1 (in-plane) and the A1g (out-of plane), are well detected from which the frequency difference of Raman peaks between E2g 1 and A1g suggest the grown MoS2 consisted of multi-layers. There is a slight shift of E2g 1 which is caused by the carbon impurities but no shift for A1g. Besides, MoS2 film with the presence of initial sulfur content shows better crystal as indicated by its narrower Raman peaks linewidth. Two broad absorption peaks of MoS2 are detected at 614nm and 665nm. Hence, the early introduction of sulfur content in prepared precursor solution is one way of optimizing the growth of MoS2 films.

  10. Mapping Soluble Guanylyl Cyclase and Protein Disulfide Isomerase Regions of Interaction.

    Directory of Open Access Journals (Sweden)

    Erin J Heckler

    Full Text Available Soluble guanylyl cyclase (sGC is a heterodimeric nitric oxide (NO receptor that produces cyclic GMP. This signaling mechanism is a key component in the cardiovascular system. NO binds to heme in the β subunit and stimulates the catalytic conversion of GTP to cGMP several hundred fold. Several endogenous factors have been identified that modulate sGC function in vitro and in vivo. In previous work, we determined that protein disulfide isomerase (PDI interacts with sGC in a redox-dependent manner in vitro and that PDI inhibited NO-stimulated activity in cells. To our knowledge, this was the first report of a physical interaction between sGC and a thiol-redox protein. To characterize this interaction between sGC and PDI, we first identified peptide linkages between sGC and PDI, using a lysine cross-linking reagent and recently developed mass spectrometry analysis. Together with Flag-immunoprecipitation using sGC domain deletions, wild-type (WT and mutated PDI, regions of sGC involved in this interaction were identified. The observed data were further explored with computational modeling to gain insight into the interaction mechanism between sGC and oxidized PDI. Our results indicate that PDI interacts preferentially with the catalytic domain of sGC, thus providing a mechanism for PDI inhibition of sGC. A model in which PDI interacts with either the α or the β catalytic domain is proposed.

  11. Tristetraprolin: A novel target of diallyl disulfide that inhibits the progression of breast cancer.

    Science.gov (United States)

    Xiong, Ting; Liu, Xiao-Wang; Huang, Xue-Long; Xu, Xiong-Feng; Xie, Wei-Quan; Zhang, Su-Jun; Tu, Jian

    2018-05-01

    Diallyl disulfide (DADS), a volatile component of garlic oil, has various biological properties, including antioxidant, antiangiogenic and anticancer effects. The present study aimed to explore novel targets of DADS that may slow or stop the progression of breast cancer. First, xenograft tumor models were created by subcutaneously injecting MCF-7 and MDA-MB-231 breast cancer cells into nude mice. Subsequently, western blot analysis was performed to investigate the expression of tristetraprolin (TTP), urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP-9) in the xenograft tumors, and cell cultures. Tablet cloning, Transwell and wound healing assays revealed that DADS treatment significantly inhibited the proliferation, invasion and migration of breast cancer cells. In addition, DADS treatment led to significant downregulation of uPA and MMP-9 protein expression, but significantly upregulated TTP expression in vivo and in vitro . Knocking down TTP expression using small interfering RNA reversed the aforementioned effects of DADS, which suggests TTP is a key target of DADS in inhibiting the progression of breast cancer.

  12. A high pressure x-ray diffraction study of titanium disulfide

    International Nuclear Information System (INIS)

    Aksoy, Resul; Selvi, Emre; Knudson, Russell; Ma Yanzhang

    2009-01-01

    A high pressure angle dispersive synchrotron x-ray diffraction study of titanium disulfide (TiS 2 ) was carried out to pressures of 45.5 GPa in a diamond-anvil cell. We observed a phase transformation of TiS 2 beginning at about 20.7 GPa. The structure of the high pressure phase needs further identification. By fitting the pressure-volume data to the third-order Birch-Murnaghan equation of state, the bulk modulus, K 0T , was determined to be 45.9 ± 0.7 GPa with its pressure derivative, K' 0T , being 9.5 ± 0.3 at pressures lower than 17.8 GPa. It was found that the compression behavior of TiS 2 is anisotropic along the different axes. The compression ratio of the c-axis is about nine times larger than the a-axis when pressures are lower than 1 GPa. It suddenly decreases to three times larger at pressures of about 3 GPa. This ratio shows a linear decrease with a slope of negative 0.048 at pressures below phase transformation.

  13. Ferroelectric transistors with monolayer molybdenum disulfide and ultra-thin aluminum-doped hafnium oxide

    Science.gov (United States)

    Yap, Wui Chung; Jiang, Hao; Liu, Jialun; Xia, Qiangfei; Zhu, Wenjuan

    2017-07-01

    In this letter, we demonstrate ferroelectric memory devices with monolayer molybdenum disulfide (MoS2) as the channel material and aluminum (Al)-doped hafnium oxide (HfO2) as the ferroelectric gate dielectric. Metal-ferroelectric-metal capacitors with 16 nm thick Al-doped HfO2 are fabricated, and a remnant polarization of 3 μC/cm2 under a program/erase voltage of 5 V is observed. The capability of potential 10 years data retention was estimated using extrapolation of the experimental data. Ferroelectric transistors based on embedded ferroelectric HfO2 and MoS2 grown by chemical vapor deposition are fabricated. Clockwise hysteresis is observed at low program/erase voltages due to slow bulk traps located near the 2D/dielectric interface, while counterclockwise hysteresis is observed at high program/erase voltages due to ferroelectric polarization. In addition, the endurances of the devices are tested, and the effects associated with ferroelectric materials, such as the wake-up effect and polarization fatigue, are observed. Reliable writing/reading in MoS2/Al-doped HfO2 ferroelectric transistors over 2 × 104 cycles is achieved. This research can potentially lead to advances of two-dimensional (2D) materials in low-power logic and memory applications.

  14. Morphological Evolution of Vertically Standing Molybdenum Disulfide Nanosheets by Chemical Vapor Deposition.

    Science.gov (United States)

    Zhang, Song; Liu, Jiajia; Ruiz, Karla Hernandez; Tu, Rong; Yang, Meijun; Li, Qizhong; Shi, Ji; Li, Haiwen; Zhang, Lianmeng; Goto, Takashi

    2018-04-20

    In this study, we demonstrated the chemical vapor deposition (CVD) of vertically standing molybdenum disulfide (MoS₂) nanosheets, with an unconventional combination of molybdenum hexacarbonyl (Mo(CO)₆) and 1,2-ethanedithiol (C₂H₆S₂) as the novel kind of Mo and S precursors respectively. The effect of the distance between the precursor’s outlet and substrates (denoted as d ) on the growth characteristics of MoS₂, including surface morphology and nanosheet structure, was investigated. Meanwhile, the relationship between the structure characteristics of MoS₂ nanosheets and their catalytic performance for hydrogen evolution reaction (HER) was elucidated. The formation of vertically standing nanosheets was analyzed and verified by means of an extrusion growth model. The crystallinity, average length, and average depth between peak and valley ( R z) of MoS₂ nanosheets differed depending on the spatial location of the substrate. Good crystalized MoS₂ nanosheets grown at d = 5.5 cm with the largest average length of 440 nm, and the highest R z of 162 nm contributed to a better HER performance, with a respective Tafel slope and exchange current density of 138.9 mV/decade, and 22.6 μA/cm² for raw data (127.8 mV/decade and 19.3 μA/cm² for iR-corrected data).

  15. Two-dimensional tantalum disulfide: controlling structure and properties via synthesis

    Science.gov (United States)

    Zhao, Rui; Grisafe, Benjamin; Krishna Ghosh, Ram; Holoviak, Stephen; Wang, Baoming; Wang, Ke; Briggs, Natalie; Haque, Aman; Datta, Suman; Robinson, Joshua

    2018-04-01

    Tantalum disulfide (TaS2) is a transition metal dichalcogenide (TMD) that exhibits phase transition induced electronic property modulation at low temperature. However, the appropriate phase must be grown to enable the semiconductor/metal transition that is of interest for next generation electronic applications. In this work, we demonstrate direct and controllable synthesis of ultra-thin 1T-TaS2 and 2H-TaS2 on a variety of substrates (sapphire, SiO2/Si, and graphene) via powder vapor deposition. The synthesis process leads to single crystal domains ranging from 20 to 200 nm thick and 1-10 µm on a side. The TaS2 phase (1T or 2H) is controlled by synthesis temperature, which subsequently is shown to control the electronic properties. Furthermore, this work constitutes the first demonstration of a metal-insulator phase transition in directly synthesized 1T-TaS2 films and domains by electronic means.

  16. Synthesis of novel disulfide and sulfone hybrid scaffolds as potent β-glucuronidase inhibitor.

    Science.gov (United States)

    Taha, Muhammad; Ismail, Nor Hadiani; Imran, Syahrul; Wadood, Abdul; Rahim, Fazal; Al Muqarrabin, Laode Muhammad Ramadhan; Zaki, Hamizah Mohd; Ahmat, Norizan; Nasir, Abdul; Khan, Fahad

    2016-10-01

    Novel series of disulfide and sulfone hybrid analogs (1-20) were synthesized and characterized through EI-MS and (1)H NMR and evaluated for β-glucuronidase inhibitory potential. All synthesized analogs except 13 and 15 showed excellent β-glucuronidase inhibitory potential with IC50 value ranging in between 2.20-88.16μM as compared to standard d-saccharic acid 1,4 lactone (48.4±1.25μM). Analogs 19, 16, 4, 1, 17, 6, 10, 3, 18, 2, 11, 14 and 5 showed many fold potent activity against β-glucuronidase inhibitor. Structure activity relationship showed that substitution of electron withdrawing groups at ortho as well as para position on phenyl ring increase potency. Electron withdrawing groups at meta position on phenyl ring showed slightly low potency as compared to ortho and para position. The binding interactions were confirmed through molecular docking studies. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Cytotoxicity and Efflux Pump Inhibition Induced by Molybdenum Disulfide and Boron Nitride Nanomaterials with Sheetlike Structure.

    Science.gov (United States)

    Liu, Su; Shen, Zhuoyan; Wu, Bing; Yu, Yue; Hou, Hui; Zhang, Xu-Xiang; Ren, Hong-Qiang

    2017-09-19

    Sheetlike molybdenum disulfide (MoS 2 ) and boron nitride (BN) nanomaterials have attracted attention in the past few years due to their unique material properties. However, information on adverse effects and their underlying mechanisms for sheetlike MoS 2 and BN nanomaterials is rare. In this study, cytotoxicities of sheetlike MoS 2 and BN nanomaterials on human hepatoma HepG2 cells were systematically investigated at different toxic end points. Results showed that MoS 2 and BN nanomaterials decreased cell viability at 30 μg/mL and induced adverse effects on intracellular ROS generation (≥2 μg/mL), mitochondrial depolarization (≥4 μg/mL), and membrane integrity (≥8 μg/mL for MoS 2 and ≥2 μg/mL for BN). Furthermore, this study first found that low exposure concentrations (0.2-2 μg/mL) of MoS 2 and BN nanomaterials could increase plasma membrane fluidity and inhibit transmembrane ATP binding cassette (ABC) efflux transporter activity, which make both nanomaterials act as a chemosensitizer (increasing arsenic toxicity). Damage to plasma membrane and release of soluble Mo or B species might be two reasons that both nanomaterials inhibit efflux pump activities. This study provides a systematic understanding of the cytotoxicity of sheetlike MoS 2 and BN nanomaterials at different exposure levels, which is important for their safe use.

  18. Contact research strategy for emerging molybdenum disulfide and other two-dimensional field-effect transistors

    Directory of Open Access Journals (Sweden)

    Yuchen Du

    2014-09-01

    Full Text Available Layered two-dimensional (2D semiconducting transition metal dichalcogenides (TMDs have been widely isolated, synthesized, and characterized recently. Numerous 2D materials are identified as the potential candidates as channel materials for future thin film technology due to their high mobility and the exhibiting bandgaps. While many TMD filed-effect transistors (FETs have been widely demonstrated along with a significant progress to clearly understand the device physics, large contact resistance at metal/semiconductor interface still remain a challenge. From 2D device research point of view, how to minimize the Schottky barrier effects on contacts thus reduce the contact resistance of metals on 2D materials is very critical for the further development of the field. Here, we present a review of contact research on molybdenum disulfide and other TMD FETs from the fundamental understanding of metal-semiconductor interfaces on 2D materials. A clear contact research strategy on 2D semiconducting materials is developed for future high-performance 2D FETs with aggressively scaled dimensions.

  19. In-plane and cross-plane thermal conductivities of molybdenum disulfide

    International Nuclear Information System (INIS)

    Ding, Zhiwei; Pei, Qing-Xiang; Zhang, Yong-Wei; Jiang, Jin-Wu

    2015-01-01

    We investigate the in-plane and cross-plane thermal conductivities of molybdenum disulfide (MoS 2 ) using non-equilibrium molecular dynamics simulations. We find that the in-plane thermal conductivity of monolayer MoS 2 is about 19.76 W mK −1 . Interestingly, the in-plane thermal conductivity of multilayer MoS 2 is insensitive to the number of layers, which is in strong contrast to the in-plane thermal conductivity of graphene where the interlayer interaction strongly affects the in-plane thermal conductivity. This layer number insensitivity is attributable to the finite energy gap in the phonon spectrum of MoS 2 , which makes the phonon–phonon scattering channel almost unchanged with increasing layer number. For the cross-plane thermal transport, we find that the cross-plane thermal conductivity of multilayer MoS 2 can be effectively tuned by applying cross-plane strain. More specifically, a 10% cross-plane compressive strain can enhance the thermal conductivity by a factor of 10, while a 5% cross-plane tensile strain can reduce the thermal conductivity by 90%. Our findings are important for thermal management in MoS 2 based nanodevices and for thermoelectric applications of MoS 2 . (paper)

  20. High-damage-resistant tungsten disulfide saturable absorber mirror for passively Q-switched fiber laser.

    Science.gov (United States)

    Chen, Hao; Chen, YuShan; Yin, Jinde; Zhang, Xuejun; Guo, Tuan; Yan, Peiguang

    2016-07-25

    In this paper, we demonstrate a high-damage-resistant tungsten disulfide saturable absorber mirror (WS2-SAM) fabricated by magnetron sputtering technique. The WS2-SAM has an all-fiber-integrated configuration and high-damage-resistant merit because the WS2 layer is protected by gold film so as to avoid being oxidized and destroyed at high pump power. Employing the WS2-SAM in an Erbium-doped fiber laser (EDFL) with linear cavity, the stable Q-switching operation is achieved at central wavelength of 1560 nm, with the repetition rates ranging from 29.5 kHz to 367.8 kHz and the pulse duration ranging from 1.269 μs to 154.9 ns. For the condition of the maximum pump power of 600 mW, the WS2-SAM still works stably with an output power of 25.2 mW, pulse energy of 68.5 nJ, and signal-noise-ratio of 42 dB. The proposed WS2-SAM configuration provides a promising solution for advanced pulsed fiber lasers with the characteristics of high damage resistance, high output energy, and wide tunable frequency.