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Sample records for additional disulfide bond

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

    PubMed Central

    Vinther, Tine N; Norrman, Mathias; Ribel, Ulla; Huus, Kasper; Schlein, Morten; Steensgaard, Dorte B; Pedersen, Thomas Å; Pettersson, Ingrid; Ludvigsen, Svend; Kjeldsen, Thomas; Jensen, Knud J; Hubálek, František

    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 under high physical stress even though the C-terminus of the B-chain thought to be directly involved in fibril formation was not modified. Importantly, this analog was capable of forming hexamer upon Zn addition as typical for wild-type insulin and its crystal structure showed only minor deviations from the classical insulin structure. Furthermore, the additional disulfide bond prevented this insulin analog from adopting the R-state conformation and thus showing that the R-state conformation is not a prerequisite for binding to insulin receptor as previously suggested. In summary, this is the first example of an insulin analog featuring a fourth disulfide bond with increased structural stability and retained function. PMID:23281053

  2. Chemical methods for producing disulfide bonds in peptides and proteins to study folding regulation.

    PubMed

    Okumura, Masaki; Shimamoto, Shigeru; Hidaka, Yuji

    2014-04-01

    Disulfide bonds play a critical role in the folding of secretory and membrane proteins. Oxidative folding reactions of disulfide bond-containing proteins typically require several hours or days, and numerous misbridged disulfide isomers are often observed as intermediates. The rate-determining step in refolding is thought to be the disulfide-exchange reaction from nonnative to native disulfide bonds in folding intermediates, which often precipitate during the refolding process because of their hydrophobic properties. To overcome this, chemical additives or a disulfide catalyst, protein disulfide isomerase (PDI), are generally used in refolding experiments to regulate disulfide-coupled peptide and protein folding. This unit describes such methods in the context of the thermodynamic and kinetic control of peptide and protein folding, including (1) regulation of disulfide-coupled peptides and protein folding assisted by chemical additives, (2) reductive unfolding of disulfide-containing peptides and proteins, and (3) regulation of disulfide-coupled peptide and protein folding using PDI.

  3. Assigning Peptide Disulfide Linkage Pattern Among Regio-Isomers via Methoxy Addition to Disulfide and Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Durand, Kirt L.; Tan, Lei; Stinson, Craig A.; Love-Nkansah, Chasity B.; Ma, Xiaoxiao; Xia, Yu

    2017-02-01

    Pinpointing disulfide linkage pattern is critical in the characterization of proteins and peptides consisting of multiple disulfide bonds. Herein, we report a method based on coupling online disulfide modification and tandem mass spectrometry (MS/MS) to distinguish peptide disulfide regio-isomers. Such a method relies on a new disulfide bond cleavage reaction in solution, involving methanol as a reactant and 254 nm ultraviolet (UV) irradiation. This reaction leads to selective cleavage of a disulfide bond and formation of sulfenic methyl ester (-SOCH3) at one cysteine residue and a thiol (-SH) at the other. Under low energy collision-induced dissociation (CID), cysteine sulfenic methyl ester motif produces a signature methanol loss (-32 Da), allowing its identification from other possible isomeric structures such as S-hydroxylmethyl (-SCH2OH) and methyl sulfoxide (-S(O)-CH3). Since disulfide bond can be selectively cleaved and modified upon methoxy addition, subsequent MS2 CID of the methoxy addition product provides enhanced sequence coverage as demonstrated by the analysis of bovine insulin. More importantly, this reaction does not induce disulfide scrambling, likely due to the fact that radical intermediates are not involved in the process. An approach based on methoxy addition followed by MS3 CID has been developed for assigning disulfide linkage patterns in peptide disulfide regio-isomers. This methodology was successfully applied to characterizing peptide systems having two disulfide bonds and three disulfide linkage isomers: side-by-side, overlapped, and looped-within-a-loop configurations.

  4. Cellular disulfide bond formation in bioactive peptides and proteins.

    PubMed

    Patil, Nitin A; Tailhades, Julien; Hughes, Richard Anthony; Separovic, Frances; Wade, John D; Hossain, Mohammed Akhter

    2015-01-14

    Bioactive peptides play important roles in metabolic regulation and modulation and many are used as therapeutics. These peptides often possess disulfide bonds, which are important for their structure, function and stability. A systematic network of enzymes--a disulfide bond generating enzyme, a disulfide bond donor enzyme and a redox cofactor--that function inside the cell dictates the formation and maintenance of disulfide bonds. The main pathways that catalyze disulfide bond formation in peptides and proteins in prokaryotes and eukaryotes are remarkably similar and share several mechanistic features. This review summarizes the formation of disulfide bonds in peptides and proteins by cellular and recombinant machinery.

  5. Disulfide Bond Requirements for Active Wnt Ligands*

    PubMed Central

    MacDonald, Bryan T.; Hien, Annie; Zhang, Xinjun; Iranloye, Oladoyin; Virshup, David M.; Waterman, Marian L.; He, Xi

    2014-01-01

    Secreted Wnt lipoproteins are cysteine-rich and lipid-modified morphogens that bind to the Frizzled (FZD) receptor and LDL receptor-related protein 6 (LRP6). Wnt engages FZD through protruding thumb and index finger domains, which are each assembled from paired β strands secured by disulfide bonds and grasp two sides of the FZD ectodomain. The importance of Wnt disulfide bonds has been assumed but uncharacterized. We systematically analyzed cysteines and associated disulfide bonds in the prototypic Wnt3a. Our data show that mutation of any individual cysteine of Wnt3a results in covalent Wnt oligomers through ectopic intermolecular disulfide bond formation and diminishes/abolishes Wnt signaling. Although individual cysteine mutations in the amino part of the saposin-like domain and in the base of the index finger are better tolerated and permit residual Wnt3a secretion/activity, those in the amino terminus, the thumb, and at the tip of the index finger are incompatible with secretion and/or activity. A few select double cysteine mutants based on the disulfide bond pattern restore Wnt secretion/activity. Further, a double cysteine mutation at the index finger tip results in a Wnt3a with normal secretion but minimal FZD binding and dominant negative properties. Our results experimentally validate predictions from the Wnt crystal structure and highlight critical but different roles of the saposin-like and cytokine-like domains, including the thumb and the index finger in Wnt folding/secretion and FZD binding. Finally, we modified existing expression vectors for 19 epitope-tagged human WNT proteins by removal of a tag-supplied ectopic cysteine, thereby generating tagged WNT ligands active in canonical and non-canonical signaling. PMID:24841207

  6. Assigning Peptide Disulfide Linkage Pattern Among Regio-Isomers via Methoxy Addition to Disulfide and Tandem Mass Spectrometry.

    PubMed

    Durand, Kirt L; Tan, Lei; Stinson, Craig A; Love-Nkansah, Chasity B; Ma, Xiaoxiao; Xia, Yu

    2017-02-13

    Pinpointing disulfide linkage pattern is critical in the characterization of proteins and peptides consisting of multiple disulfide bonds. Herein, we report a method based on coupling online disulfide modification and tandem mass spectrometry (MS/MS) to distinguish peptide disulfide regio-isomers. Such a method relies on a new disulfide bond cleavage reaction in solution, involving methanol as a reactant and 254 nm ultraviolet (UV) irradiation. This reaction leads to selective cleavage of a disulfide bond and formation of sulfenic methyl ester (-SOCH3) at one cysteine residue and a thiol (-SH) at the other. Under low energy collision-induced dissociation (CID), cysteine sulfenic methyl ester motif produces a signature methanol loss (-32 Da), allowing its identification from other possible isomeric structures such as S-hydroxylmethyl (-SCH2OH) and methyl sulfoxide (-S(O)-CH3). Since disulfide bond can be selectively cleaved and modified upon methoxy addition, subsequent MS(2) CID of the methoxy addition product provides enhanced sequence coverage as demonstrated by the analysis of bovine insulin. More importantly, this reaction does not induce disulfide scrambling, likely due to the fact that radical intermediates are not involved in the process. An approach based on methoxy addition followed by MS(3) CID has been developed for assigning disulfide linkage patterns in peptide disulfide regio-isomers. This methodology was successfully applied to characterizing peptide systems having two disulfide bonds and three disulfide linkage isomers: side-by-side, overlapped, and looped-within-a-loop configurations. Graphical Abstract ᅟ.

  7. Converting a Sulfenic Acid Reductase into a Disulfide Bond Isomerase

    PubMed Central

    Chatelle, Claire; Kraemer, Stéphanie; Ren, Guoping; Chmura, Hannah; Marechal, Nils; Boyd, Dana; Roggemans, Caroline; Ke, Na; Riggs, Paul; Bardwell, James

    2015-01-01

    Abstract Aims: Posttranslational formation of disulfide bonds is essential for the folding of many secreted proteins. Formation of disulfide bonds in a protein with more than two cysteines is inherently fraught with error and can result in incorrect disulfide bond pairing and, consequently, misfolded protein. Protein disulfide bond isomerases, such as DsbC of Escherichia coli, can recognize mis-oxidized proteins and shuffle the disulfide bonds of the substrate protein into their native folded state. Results: We have developed a simple blue/white screen that can detect disulfide bond isomerization in vivo, using a mutant alkaline phosphatase (PhoA*) in E. coli. We utilized this screen to isolate mutants of the sulfenic acid reductase (DsbG) that allowed this protein to act as a disulfide bond isomerase. Characterization of the isolated mutants in vivo and in vitro allowed us to identify key amino acid residues responsible for oxidoreductase properties of thioredoxin-like proteins such as DsbC or DsbG. Innovation and Conclusions: Using these key residues, we also identified and characterized interesting environmental homologs of DsbG with novel properties, thus demonstrating the capacity of this screen to discover and elucidate mechanistic details of in vivo disulfide bond isomerization. Antioxid. Redox Signal. 23, 945–957. PMID:26191605

  8. Mechanism of SN2 disulfide bond cleavage by phosphorus nucleophiles. Implications for biochemical disulfide reducing agents.

    PubMed

    Dmitrenko, Olga; Thorpe, Colin; Bach, Robert D

    2007-10-26

    The B3LYP variant of DFT has been used to study the mechanism of S-S bond scission in dimethyl disulfide by a phosphorus nucleophile, trimethylphospine (TMP). The reaction is highly endothermic in the gas phase and requires significant external stabilization of the charged products. DFT calculations (B3LYP) were performed with explicit (water molecules added) and implicit solvent corrections (COSMO model). The transition structures for this SN2 displacement reaction in a number of model systems have been located and fully characterized. The reaction barriers calculated with different approaches for different systems are quite close (around 11 kcal/mol). Remarkably, the calculations suggest that the reaction is almost barrierless with respect to the preorganized reaction complex and that most of the activation energy is required to rearrange the disulfide and TMP to its most effective orientation for the SMe group transfer way. Different reactivities of different phosphorus nucleophiles were suggested to be the result of steric effects, as manifested largely by varying amounts of hindrance to solvation of the initial product phosphonium ion. These data indicate that the gas-phase addition of a phosphine to the disulfide moiety will most likely form a phosphonium cation-thiolate anion salt, in the presence of four or more water molecules, that provide sufficient H-bonding stabilization to allow displacement of the thiolate anion, a normal uncomplicated SN2 transition state is to be expected.

  9. Catalysis of Protein Disulfide Bond Isomerization in a Homogeneous Substrate†

    PubMed Central

    Kersteen, Elizabeth A.; Barrows, Seth R.; Raines, Ronald T.

    2008-01-01

    Protein disulfide isomerase (PDI) catalyzes the rearrangement of nonnative disulfide bonds in the endoplasmic reticulum of eukaryotic cells, a process that often limits the rate at which polypeptide chains fold into a native protein conformation. The mechanism of the reaction catalyzed by PDI is unclear. In assays involving protein substrates, the reaction appears to involve the complete reduction of some or all of its nonnative disulfide bonds followed by oxidation of the resulting dithiols. The substrates in these assays are, however, heterogeneous, which complicates mechanistic analyses. Here, we report the first analysis of disulfide bond isomerization in a homogeneous substrate. Our substrate is based on tachyplesin I, a 17-mer peptide that folds into a _-hairpin stabilized by two disulfide bonds. We describe the chemical synthesis of a variant of tachyplesin I in which its two disulfide bonds are in a nonnative state and side chains near its N-and C-terminus contain a fluorescence donor (tryptophan) and acceptor (N_-dansyllysine). Fluorescence resonance energy transfer from 280 to 465 nm increases by 28-fold upon isomerization of the disulfide bonds into their native state (which has a lower E°_ = -0.313 V than does PDI). We use this continuous assay to analyze catalysis by wild-type human PDI and a variant in which the C-terminal cysteine residue within each Cys—Gly—His—Cys active site is replaced with alanine. We find that wild-type PDI catalyzes the isomerization of the substrate with kcat/KM = 1.7 _ 105 M–1M s–1, which is the largest value yet reported for catalysis of disulfide bond isomerization. The variant, which is a poor catalyst of disulfide bond reduction and dithiol oxidation, retains virtually all of the activity of wild-type PDI in catalysis of disulfide bond isomerization. Thus, the C-terminal cysteine residues play an insignificant role in the isomerization of the disulfide bonds in nonnative tachyplesin I. We conclude that

  10. An in vivo pathway for disulfide bond isomerization in Escherichia coli.

    PubMed

    Rietsch, A; Belin, D; Martin, N; Beckwith, J

    1996-11-12

    Biochemical studies have shown that the periplasmic protein disulfide oxidoreductase DsbC can isomerize aberrant disulfide bonds. Here we present the first evidence for an in vivo role of DsbC in disulfide bond isomerization. Furthermore, our data suggest that the enzymes DsbA and DsbC play distinct roles in the cell in disulfide bond formation and isomerization, respectively. We have shown that mutants in dsbC display a defect in disulfide bond formation specific for proteins with multiple disulfide bonds. The defect can be complemented by the addition of reduced dithiothreitol to the medium, suggesting that absence of DsbC results in accumulation of misoxidized proteins. Mutations in the dipZ and trxA genes have similar phenotypes. We propose that DipZ, a cytoplasmic membrane protein with a thioredoxin-like domain, and thioredoxin, the product of the trxA gene, are components of a pathway for maintaining DsbC active as a protein disulfide bond isomerase.

  11. Characterization of disulfide bonds by planned digestion and tandem mass spectrometry.

    PubMed

    Na, Seungjin; Paek, Eunok; Choi, Jong-Soon; Kim, Duwoon; Lee, Seung Jae; Kwon, Joseph

    2015-04-01

    The identification of disulfide bonds provides critical information regarding the structure and function of a protein and is a key aspect in understanding signaling cascades in biological systems. Recent proteomic approaches using digestion enzymes have facilitated the characterization of disulfide-bonds and/or oxidized products from cysteine residues, although these methods have limitations in the application of MS/MS. For example, protein digestion to obtain the native form of disulfide bonds results in short lengths of amino acids, which can cause ambiguous MS/MS analysis due to false positive identifications. In this study we propose a new approach, termed planned digestion, to obtain sufficient amino acid lengths after cleavage for proteomic approaches. Application of the DBond software to planned digestion of specific proteins accurately identified disulfide-linked peptides. RNase A was used as a model protein in this study because the disulfide bonds of this protein have been well characterized. Application of this approach to peptides digested with Asp-N/C (chemical digestion) and trypsin under acid hydrolysis conditions identified the four native disulfide bonds of RNase A. Missed cleavages introduced by trypsin treatment for only 3 hours generated sufficient lengths of amino acids for identification of the disulfide bonds. Analysis using MS/MS successfully showed additional fragmentation patterns that are cleavage products of S-S and C-S bonds of disulfide-linkage peptides. These fragmentation patterns generate thioaldehydes, persulfide, and dehydroalanine. This approach of planned digestion with missed cleavages using the DBond algorithm could be applied to other proteins to determine their disulfide linkage and the oxidation patterns of cysteine residues.

  12. The disulfide bond pattern of catrocollastatin C, a disintegrin-like/cysteine-rich protein isolated from Crotalus atrox venom.

    PubMed Central

    Calvete, J. J.; Moreno-Murciano, M. P.; Sanz, L.; Jürgens, M.; Schrader, M.; Raida, M.; Benjamin, D. C.; Fox, J. W.

    2000-01-01

    The disulfide bond pattern of catrocollastatin-C was determined by N-terminal sequencing and mass spectrometry. The N-terminal disintegrin-like domain is a compact structure including eight disulfide bonds, seven of them in the same pattern as the disintegrin bitistatin. The protein has two extra cysteine residues (XIII and XVI) that form an additional disulfide bond that is characteristically found in the disintegrin-like domains of cellular metalloproteinases (ADAMs) and PIII snake venom Zn-metalloproteinases (SVMPs). The C-terminal cysteine-rich domain of catrocollastatin-C contains five disulfide bonds between nearest-neighbor cysteines and a long range disulfide bridge between CysV and CysX. These results provide structural evidence for a redefinition of the disintegrin-like and cysteine-rich domain boundaries. An evolutionary pathway for ADAMs, PIII, and PII SVMPs based on disulfide bond engineering is also proposed. PMID:10933502

  13. Disulfide bond formation in the Escherichia coli cytoplasm: an in vivo role reversal for the thioredoxins.

    PubMed Central

    Stewart, E J; Aslund, F; Beckwith, J

    1998-01-01

    Cytoplasmic proteins do not generally contain structural disulfide bonds, although certain cytoplasmic enzymes form such bonds as part of their catalytic cycles. The disulfide bonds in these latter enzymes are reduced in Escherichia coli by two systems; the thioredoxin pathway and the glutathione/glutaredoxin pathway. However, structural disulfide bonds can form in proteins in the cytoplasm when the gene (trxB) for the enzyme thioredoxin reductase is inactivated by mutation. This disulfide bond formation can be detected by assessing the state of the normally periplasmic enzyme alkaline phosphatase (AP) when it is localized to the cytoplasm. Here we show that the formation of disulfide bonds in cytoplasmic AP in the trxB mutant is dependent on the presence of two thioredoxins in the cell, thioredoxins 1 and 2, the products of the genes trxA and trxC, respectively. Our evidence supports a model in which the oxidized forms of these thioredoxins directly catalyze disulfide bond formation in cytoplasmic AP, a reversal of their normal role. In addition, we show that the recently discovered thioredoxin 2 can perform many of the roles of thioredoxin 1 in vivo, and thus is able to reduce certain essential cytoplasmic enzymes. Our results suggest that the three most effective cytoplasmic disulfide-reducing proteins are thioredoxin 1, thioredoxin 2 and glutaredoxin 1; expression of any one of these is sufficient to support aerobic growth. Our results help to explain how the reducing environment in the cytoplasm is maintained so that disulfide bonds do not normally occur. PMID:9755155

  14. Structural basis of protein disulfide bond generation in the cell.

    PubMed

    Inaba, Kenji

    2010-09-01

    The formation of protein disulfide bonds is an oxidative reaction that is crucial for the folding and maturation of many secreted and membrane proteins. Both prokaryotic and eukaryotic cells possess various disulfide oxidoreductases and redox-active cofactors to accelerate this oxidative reaction in a correct manner. Crystal or solution structures have been solved for some of the oxidoreductases in the past 10 years, leading to remarkable progress in the field of thiol-based redox cell biology. Consequently, structural and mechanistic similarities in the disulfide bond formation pathways have been uncovered. This review highlights the molecular basis of the elaborate oxidative systems operating in the Escherichia coli periplasm, the endoplasmic reticulum lumen and the mitochondrial intermembrane space. The accumulated knowledge provides important insights into how protein and redox homeostasis are maintained in the cell.

  15. Dual Beneficial Effect of Interloop Disulfide Bond for Single Domain Antibody Fragments*

    PubMed Central

    Govaert, Jochen; Pellis, Mireille; Deschacht, Nick; Vincke, Cécile; Conrath, Katja; Muyldermans, Serge; Saerens, Dirk

    2012-01-01

    The antigen-binding fragment of functional heavy chain antibodies (HCAbs) in camelids comprises a single domain, named the variable domain of heavy chain of HCAbs (VHH). The VHH harbors remarkable amino acid substitutions in the framework region-2 to generate an antigen-binding domain that functions in the absence of a light chain partner. The substitutions provide a more hydrophilic, hence more soluble, character to the VHH but decrease the intrinsic stability of the domain. Here we investigate the functional role of an additional hallmark of dromedary VHHs, i.e. the extra disulfide bond between the first and third antigen-binding loops. After substituting the cysteines forming this interloop cystine by all 20 amino acids, we selected and characterized several VHHs that retain antigen binding capacity. Although VHH domains can function in the absence of an interloop disulfide bond, we demonstrate that its presence constitutes a net advantage. First, the disulfide bond stabilizes the domain and counteracts the destabilization by the framework region-2 hallmark amino acids. Second, the disulfide bond rigidifies the long third antigen-binding loop, leading to a stronger antigen interaction. This dual beneficial effect explains the in vivo antibody maturation process favoring VHH domains with an interloop disulfide bond. PMID:22128183

  16. Disulfide Bond Formation and Activation of Escherichia coli β-Galactosidase under Oxidizing Conditions

    PubMed Central

    Seras-Franzoso, Joaquin; Affentranger, Roman; Ferrer-Navarro, Mario; Daura, Xavier; Villaverde, Antonio

    2012-01-01

    Escherichia coli β-galactosidase is probably the most widely used reporter enzyme in molecular biology, cell biology, and biotechnology because of the easy detection of its activity. Its large size and tetrameric structure make this bacterial protein an interesting model for crystallographic studies and atomic mapping. In the present study, we investigate a version of Escherichia coli β-galactosidase produced under oxidizing conditions, in the cytoplasm of an Origami strain. Our data prove the activation of this microbial enzyme under oxidizing conditions and clearly show the occurrence of a disulfide bond in the β-galactosidase structure. Additionally, the formation of this disulfide bond is supported by the analysis of a homology model of the protein that indicates that two cysteines located in the vicinity of the catalytic center are sufficiently close for disulfide bond formation. PMID:22286993

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

    PubMed

    Takeda, Mitsuhiro; Terauchi, Tsutomu; Kainosho, Masatsune

    2012-02-01

    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.

  18. Topology of the disulfide bonds in the antiviral lectin scytovirin

    PubMed Central

    Moulaei, Tinoush; Stuchlik, Olga; Reed, Matthew; Yuan, Weirong; Pohl, Jan; Lu, Wuyuan; Haugh-Krumpe, Lauren; O'Keefe, Barry R; Wlodawer, Alexander

    2010-01-01

    The antiviral lectin scytovirin (SVN) contains a total of five disulfide bonds in two structurally similar domains. Previous reports provided contradictory results on the disulfide pairing in each individual domain, and we have now re-examined the disulfide topology. N-terminal sequencing and mass spectrometry were used to analyze proteolytic fragments of native SVN obtained at acidic pH, yielding the assignment as Cys7–Cys55, Cys20–Cys32, Cys26–Cys38, Cys68–Cys80, and Cys74–Cys86. We also analyzed the N-terminal domain of SVN (SD1, residues 1–48) prepared by expression/oxidative folding of the recombinant protein and by chemical synthesis. The disulfide pairing in the chemically synthesized SD1 was forced into predetermined topologies: SD1A (Cys20–Cys26, Cys32–Cys38) or SD1B (Cys20–Cys32, Cys26–Cys38). The topology of native SVN was found to be in agreement with the SD1B and the one determined for the recombinant SD1 domain. Although the two synthetic forms of SD1 were distinct when subjected to chromatography, their antiviral properties were indistinguishable, having low nM activity against HIV. Tryptic fragments, the “cystine clusters” [Cys20–Cys32/Cys26–Cys38; SD1] and [Cys68–Cys80/Cys74–C-86; SD2], were found to undergo rapid disulfide interchange at pH 8. This interchange resulted in accumulation of artifactual fragments in alkaline pH digests that are structurally unrelated to the original topology, providing a rational explanation for the differences between the topology reported herein and the one reported earlier (Bokesh et al., Biochemistry 2003;42:2578–2584). Our observations emphasize the fact that proteins such as SVN, with disulfide bonds in close proximity, require considerable precautions when being fragmented for the purpose of disulfide assignment. PMID:20572021

  19. Dissecting the Machinery That Introduces Disulfide Bonds in Pseudomonas aeruginosa

    PubMed Central

    Arts, Isabelle S.; Ball, Geneviève; Leverrier, Pauline; Garvis, Steven; Nicolaes, Valérie; Vertommen, Didier; Ize, Bérengère; Tamu Dufe, Veronica; Messens, Joris; Voulhoux, Romé; Collet, Jean-François

    2013-01-01

    ABSTRACT Disulfide bond formation is required for the folding of many bacterial virulence factors. However, whereas the Escherichia coli disulfide bond-forming system is well characterized, not much is known on the pathways that oxidatively fold proteins in pathogenic bacteria. Here, we report the detailed unraveling of the pathway that introduces disulfide bonds in the periplasm of the human pathogen Pseudomonas aeruginosa. The genome of P. aeruginosa uniquely encodes two DsbA proteins (P. aeruginosa DsbA1 [PaDsbA1] and PaDsbA2) and two DsbB proteins (PaDsbB1 and PaDsbB2). We found that PaDsbA1, the primary donor of disulfide bonds to secreted proteins, is maintained oxidized in vivo by both PaDsbB1 and PaDsbB2. In vitro reconstitution of the pathway confirms that both PaDsbB1 and PaDsbB2 shuttle electrons from PaDsbA1 to membrane-bound quinones. Accordingly, deletion of both P. aeruginosa dsbB1 (PadsbB1) and PadsbB2 is required to prevent the folding of several P. aeruginosa virulence factors and to lead to a significant decrease in pathogenicity. Using a high-throughput proteomic approach, we also analyzed the impact of PadsbA1 deletion on the global periplasmic proteome of P. aeruginosa, which allowed us to identify more than 20 new potential substrates of this major oxidoreductase. Finally, we report the biochemical and structural characterization of PaDsbA2, a highly oxidizing oxidoreductase, which seems to be expressed under specific conditions. By fully dissecting the machinery that introduces disulfide bonds in P. aeruginosa, our work opens the way to the design of novel antibacterial molecules able to disarm this pathogen by preventing the proper assembly of its arsenal of virulence factors. PMID:24327342

  20. Radical induced disulfide bond cleavage within peptides via ultraviolet irradiation of an electrospray plume.

    PubMed

    Stinson, Craig A; Xia, Yu

    2013-05-21

    Radical induced disulfide bond cleavage in peptides was demonstrated by ultraviolet (UV) radiation of the electrospray ionization (ESI) plume using a low pressure mercury (LP-Hg) lamp. Tandem mass spectrometry and accurate mass measurements confirmed that the primary reaction products were due to disulfide bond cleavage to form thiol (-SH) and sulfinyl radical (-SO˙). Mechanistic studies showed that the 185 nm emission from a LP-Hg lamp was responsible for UV photolysis of atmospheric O2, which further initiated secondary radical formation and subsequent disulfide bond cleavage by radical attack. The radical induced disulfide bond cleavage was found to be analytically useful in providing rich sequence information for naturally occurring peptides containing intrachain disulfide bonds. The utility of this method was also demonstrated for facile disulfide peptide identification and characterization from protein digests.

  1. Peptide Bond Formation in Water Mediated by Carbon Disulfide.

    PubMed

    Leman, Luke J; Huang, Zheng-Zheng; Ghadiri, M Reza

    2015-09-01

    Demonstrating plausible nonenzymatic polymerization mechanisms for prebiotic monomers represents a fundamental goal in prebiotic chemistry. While a great deal is now known about the potentially prebiotic synthesis of amino acids, our understanding of abiogenic polymerization processes to form polypeptides is less well developed. Here, we show that carbon disulfide (CS2), a component of volcanic emission and sulfide mineral weathering, and a widely used synthetic reagent and solvent, promotes peptide bond formation in modest yields (up to ∼20%) from α-amino acids under mild aqueous conditions. Exposure of a variety of α-amino acids to CS2 initially yields aminoacyl dithiocarbamates, which in turn generate reactive 2-thiono-5-oxazolidone intermediates, the thio analogues of N-carboxyanhydrides. Along with peptides, thiourea and thiohydantoin species are produced. Amino acid stereochemistry was preserved in the formation of peptides. Our findings reveal that CS2 could contribute to peptide bond formation, and possibly other condensation reactions, in abiogenic settings.

  2. DBCP: a web server for disulfide bonding connectivity pattern prediction without the prior knowledge of the bonding state of cysteines

    PubMed Central

    Lin, Hsuan-Hung; Tseng, Lin-Yu

    2010-01-01

    The proper prediction of the location of disulfide bridges is efficient in helping to solve the protein folding problem. Most of the previous works on the prediction of disulfide connectivity pattern use the prior knowledge of the bonding state of cysteines. The DBCP web server provides prediction of disulfide bonding connectivity pattern without the prior knowledge of the bonding state of cysteines. The method used in this server improves the accuracy of disulfide connectivity pattern prediction (Qp) over the previous studies reported in the literature. This DBCP server can be accessed at http://120.107.8.16/dbcp or http://140.120.14.136/dbcp. PMID:20530534

  3. Diversity of the Epsilonproteobacteria Dsb (disulfide bond) systems.

    PubMed

    Bocian-Ostrzycka, Katarzyna M; Grzeszczuk, Magdalena J; Dziewit, Lukasz; Jagusztyn-Krynicka, Elżbieta K

    2015-01-01

    The bacterial proteins of the Dsb family-important components of the post-translational protein modification system-catalyze the formation of disulfide bridges, a process that is crucial for protein structure stabilization and activity. Dsb systems play an essential role in the assembly of many virulence factors. Recent rapid advances in global analysis of bacteria have thrown light on the enormous diversity among bacterial Dsb systems. While the Escherichia coli disulfide bond-forming system is quite well understood, the mechanisms of action of Dsb systems in other bacteria, including members of class Epsilonproteobacteria that contain pathogenic and non-pathogenic bacteria colonizing extremely diverse ecological niches, are poorly characterized. Here we present a review of current knowledge on Epsilonproteobacteria Dsb systems. We have focused on the Dsb systems of Campylobacter spp. and Helicobacter spp. because our knowledge about Dsb proteins of Wolinella and Arcobacter spp. is still scarce and comes mainly from bioinformatic studies. Helicobacter pylori is a common human pathogen that colonizes the gastric epithelium of humans with severe consequences. Campylobacter spp. is a leading cause of zoonotic enteric bacterial infections in most developed and developing nations. We focus on various aspects of the diversity of the Dsb systems and their influence on pathogenicity, particularly because Dsb proteins are considered as potential targets for a new class of anti-virulence drugs to treat human infections by Campylobacter or Helicobacter spp.

  4. Disulfide bond structure determination and biochemical analysis of glycoprotein C from herpes simplex virus.

    PubMed Central

    Rux, A H; Moore, W T; Lambris, J D; Abrams, W R; Peng, C; Friedman, H M; Cohen, G H; Eisenberg, R J

    1996-01-01

    A biochemical analysis of glycoprotein C (gC of herpes simplex virus was undertaken to further characterize the structure of the glycoprotein and to determine its disulfide bond arrangement. We used three recombinant forms of gC, gC1(457t), gC1(delta33-123t), and gC2(426t), each truncated prior to the transmembrane region. The proteins were expressed and secreted by using a baculovirus expression system and have been shown to bind to monoclonal antibodies which recognize discontinuous epitopes and to complement component C3b in a dose-dependent manner. We confirmed the N-terminal residues of each mature protein by Edman degradation and confirmed the internal deletion in gC1(delta33-123t). The molecular weight and extent of glycosylation of gC1 (457t), gC1(delta33-123t), and gC2(426t) were determined by treating each protein with endoglycosidases and then subjecting it to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometric analysis. The data indicate that eight to nine of the predicted N-linked oligosaccharide sites on gC1(457t) are occupied by glycans of approximately 1,000 Da. In addition, O-linked oligosaccharides are present on gC1(457t), primarily localized to the N-terminal region (amino acids [aa] 33 to 123) of the protein. gC2(426t) contains N-linked oligosaccharides, but no O-linked oligosaccharides were detected. To determine the disulfide bond arrangement of the eight cysteines of gC1(457t),the protein was cleaved with cyanogen bromide. SDS-PAGE analysis followed by Edman degradation identified three cysteine-containing fragments which are not connected by disulfide linkages. Chemical modification of cysteines combined with matrix-assisted laser desorption ionization mass spectrometry identified disulfide bonds between cysteine 1 (aa 127) and cysteine 2 (aa 144) and between cysteine 3 (aa 286) and cysteine 4 (aa 347). Further proteolysis of the cyanogen bromide-generated fragment containing cysteine 5 through

  5. Intramolecular disulfide bonds between conserved cysteines in wheat gliadins control their deposition into protein bodies.

    PubMed

    Shimoni, Y; Galili, G

    1996-08-02

    Following synthesis, wheat gliadin storage proteins are deposited into protein bodies inside the endomembrane system in a way that enables not only their efficient accumulation and dehydration during seed maturation, but also their rapid rehydration and degradation during germination. In the present report, we studied the mechanism of gliadin deposition and whether it was controlled by the conformation of these proteins. Although gliadins are generally known to be insoluble in aqueous solutions, sucrose gradient analysis showed that a considerable amount of these proteins appeared as relatively soluble monomers in developing grains. In vitro reduction of the intramolecular disulfide bonds that are present in natural monomeric gliadins caused their precipitation into insoluble aggregates. In addition, pulse-chase experiments in the absence or presence of reducing agents showed that formation of intramolecular disulfide bonds also played a major role in folding and deposition of the gliadins in vivo. Our results imply that following sequestration into the endoplasmic reticulum, the gliadins fold into relatively soluble monomers, which are incompetent for rapid aggregation and gradually assemble into protein bodies. This pattern of deposition apparently depends on the conformation of the gliadins, which is stabilized by intramolecular disulfide bonds formed between the conserved cysteines. The contribution of this study to the understanding of the evolution and function of gliadins is discussed.

  6. Legionella pneumophila utilizes a single-player disulfide-bond oxidoreductase system to manage disulfide bond formation and isomerization.

    PubMed

    Kpadeh, Zegbeh Z; Day, Shandra R; Mills, Brandy W; Hoffman, Paul S

    2015-03-01

    Legionella pneumophila uses a single homodimeric disulfide bond (DSB) oxidoreductase DsbA2 to catalyze extracytoplasmic protein folding and to correct DSB errors through protein-disulfide isomerase (PDI) activity. In Escherichia coli, these functions are separated to avoid futile cycling. In L. pneumophila, DsbA2 is maintained as a mixture of disulfides (S-S) and free thiols (SH), but when expressed in E. coli, only the SH form is observed. We provide evidence to suggest that structural differences in DsbB oxidases (LpDsbB1 and LpDsbB2) and DsbD reductases (LpDsbD1 and LpDsbD2) (compared with E. coli) permit bifunctional activities without creating a futile cycle. LpdsbB1 and LpdsbB2 partially complemented an EcdsbB mutant while neither LpdsbD1 nor LpdsbD2 complemented an EcdsbD mutant unless DsbA2 was also expressed. When the dsb genes of E. coli were replaced with those of L. pneumophila, motility was restored and DsbA2 was present as a mixture of redox forms. A dominant-negative approach to interfere with DsbA2 function in L. pneumophila determined that DSB oxidase activity was necessary for intracellular multiplication and assembly/function of the Dot/Icm Type IVb secretion system. Our studies show that a single-player system may escape the futile cycle trap by limiting transfer of reducing equivalents from LpDsbDs to DsbA2.

  7. Global disulfide bond profiling for crude snake venom using dimethyl labeling coupled with mass spectrometry and RADAR algorithm.

    PubMed

    Huang, Sheng Yu; Chen, Sung Fang; Chen, Chun Hao; Huang, Hsuan Wei; Wu, Wen Guey; Sung, Wang Chou

    2014-09-02

    Snake venom consists of toxin proteins with multiple disulfide linkages to generate unique structures and biological functions. Determination of these cysteine connections usually requires the purification of each protein followed by structural analysis. In this study, dimethyl labeling coupled with LC-MS/MS and RADAR algorithm was developed to identify the disulfide bonds in crude snake venom. Without any protein separation, the disulfide linkages of several cytotoxins and PLA2 could be solved, including more than 20 disulfide bonds. The results show that this method is capable of analyzing protein mixture. In addition, the approach was also used to compare native cytotoxin 3 (CTX III) and its scrambled isomer, another category of protein mixture, for unknown disulfide bonds. Two disulfide-linked peptides were observed in the native CTX III, and 10 in its scrambled form, X-CTX III. This is the first study that reports a platform for the global cysteine connection analysis on a protein mixture. The proposed method is simple and automatic, offering an efficient tool for structural and functional studies of venom proteins.

  8. Overview of the regulation of disulfide bond formation in Peptide and protein folding.

    PubMed

    Hidaka, Yuji

    2014-04-01

    Disulfide bonds play a critical role in the maintenance of the native conformation of proteins under thermodynamic control. In general, disulfide bond formation is associated with protein folding, and this restricts the formation of folding intermediates such as misbridged disulfide isomers or kinetically trapped conformations, which provide important information related to how proteins fold into their native conformation. Therefore, numerous studies have focused on the structural analysis of folding intermediates in vitro. However, isolating or trapping folding intermediates, as well as the entire proteins, including mutant proteins, is not an easy task. Several chemical methods have recently been developed for examining peptide and protein folding and for producing, e.g., intact, post-translationally modified, or kinetically trapped proteins, or proteins with misbridged disulfide bonds. This overview introduces chemical methods for regulating the formation of disulfide bonds of peptides and proteins in the context of the thermodynamic and kinetic control of peptide and protein folding.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  11. Ion mobility mass spectrometry as a potential tool to assign disulfide bonds arrangements in peptides with multiple disulfide bridges.

    PubMed

    Echterbille, Julien; Quinton, Loïc; Gilles, Nicolas; De Pauw, Edwin

    2013-05-07

    Disulfide bridges play a major role in defining the structural properties of peptides and proteins. However, the determination of the cysteine pairing is still challenging. Peptide sequences are usually achieved using tandem mass spectrometry (MS/MS) spectra of the totally reduced unfolded species, but the cysteine pairing information is lost. On the other hand, MS/MS experiments performed on native folded species show complex spectra composed of nonclassical ions. MS/MS alone does not allow either the cysteine pairing or the full sequence of an unknown peptide to be determined. The major goal of this work is to set up a strategy for the full structural characterization of peptides including disulfide bridges annotation in the sequence. This strategy was developed by combining ion mobility spectrometry (IMS) and collision-induced dissociation (CID). It is assumed that the opening of one S-S bridge in a peptide leads to a structural evolution which results in a modification of IMS drift time. In the presence of multiple S-S bridges, the shift in arrival time will depend on which disulfide(s) has (have) been reduced and on the shape adopted by the generated species. Due to specific fragmentations observed for each species, CID experiments performed after the mobility separation could provide not only information on peptide sequence but also on the localization of the disulfide bridges. To achieve this goal, synthetic peptides containing two disulfides were studied. The openings of the bridges were carried out following different experimental conditions such as reduction, reduction/alkylation, or oxidation. Due to disulfide scrambling highlighted with the reduction approaches, oxidation of S-S bonds into cysteic acids appeared to be the best strategy. Cysteine connectivity was then unambiguously determined for the two peptides, without any disulfide scrambling interference.

  12. Ion Mobility-Mass Spectrometry as a Tool for the Structural Characterization of Peptides Bearing Intramolecular Disulfide Bond(s)

    NASA Astrophysics Data System (ADS)

    Massonnet, Philippe; Haler, Jean R. N.; Upert, Gregory; Degueldre, Michel; Morsa, Denis; Smargiasso, Nicolas; Mourier, Gilles; Gilles, Nicolas; Quinton, Loïc; De Pauw, Edwin

    2016-10-01

    Disulfide bonds are post-translationnal modifications that can be crucial for the stability and the biological activities of natural peptides. Considering the importance of these disulfide bond-containing peptides, the development of new techniques in order to characterize these modifications is of great interest. For this purpose, collision cross cections (CCS) of a large data set of 118 peptides (displaying various sequences) bearing zero, one, two, or three disulfide bond(s) have been measured in this study at different charge states using ion mobility-mass spectrometry. From an experimental point of view, CCS differences (ΔCCS) between peptides bearing various numbers of disulfide bonds and peptides having no disulfide bonds have been calculated. The ΔCCS calculations have also been applied to peptides bearing two disulfide bonds but different cysteine connectivities (Cys1-Cys2/Cys3-Cys4; Cys1-Cys3/Cys2-Cys4; Cys1-Cys4/Cys2-Cys3). The effect of the replacement of a proton by a potassium adduct on a peptidic structure has also been investigated.

  13. Soluble expression of disulfide bond containing proteins FGF15 and FGF19 in the cytoplasm of Escherichia coli.

    PubMed

    Kong, Bo; Guo, Grace L

    2014-01-01

    Fibroblast growth factor 19 (FGF19) is the human ortholog of mouse FGF15, and both proteins function as an endocrine signal to regulate various liver functions. FGF15/FGF19 protein contains two disulfide bonds. It is unfavorable to form disulfide bonds in Escherichia coli (E. coli) cytoplasm because of the bacterial cytoplasmic reducing environment. Modification of the cytoplasmic reducing environment and/or co-expression of protein chaperones are common strategies to express disulfide bond containing proteins in E. coli. In the current study, we report a method to produce soluble FGF15/FGF19 protein in cytoplasm of E. coli. Several commercial available strains with the disruption of thiol-redox pathways, and/or co-expression of redoxase or refolding chaperones were used to develop this novel method for expression of FGF15/FGF19 in E. coli. Mutation of the thiol-disulfide bond reducing pathway in E. coli or N-terminal fusion of thioredox (TRX) alone is not enough to support disulfide bond formation in FGF15/19 proteins. However, TRX fusion protein improved FGF19 solubility in strains of thiol-redox system mutants. In addition, DsbC co-expressed in thiol-redox system mutants alone improved and further enhanced FGF19 solubility with combination of TRX fusion tag. The soluble FGF19 proteins were easily purified through Ni-NTA affinity chromatography and anion exchange chromatography, and the purified protein maintained its biological activities, confirmed by suppressing hepatic Cyp7a1 gene transcription in mice and by activating ERK1/2 signaling pathway in HepG2 cells. In contrast, soluble FGF15 protein in cytoplasm remained very low using these strategies. In summary, we have successfully developed a method to express functional FGF19 protein in prokaryotic cells, and this strategy may be adapted for the expression of other disulfide-containing proteins.

  14. Heterologous expression of five disulfide-bonded insecticidal spider peptides.

    PubMed

    Estrada, Georgina; Silva, Anita O; Villegas, Elba; Ortiz, Ernesto; Beirão, Paulo S L; Corzo, Gerardo

    2016-09-01

    The genes of the five disulfide-bonded peptide toxins 1 and 2 (named Oxytoxins or Oxotoxins) from the spider Oxyopes lineatus were cloned into the expression vector pQE30 containing a 6His-tag and a Factor Xa proteolytic cleavage region. These two recombinant vectors were transfected into Escherichia coli BL21 cells and expressed under induction with isopropyl thiogalactoside (IPTG). The product of each gene was named HisrOxyTx1 or HisrOxyTx2, and the protein expression was ca 14 and 6 mg/L of culture medium, respectively. Either recombinant toxin HisrOxyTx1 or HisrOxyTx2 were found exclusively in inclusion bodies, which were solubilized using a chaotropic agent, and then, purified using affinity chromatography and reverse-phase HPLC (RP-HPLC). The HisrOxyTx1 and HisrOxyTx2 products, obtained from the affinity chromatographic step, showed several peptide fractions having the same molecular mass of 9913.1 and 8030.1 Da, respectively, indicating that both HisrOxyTx1 and HisrOxyTx2 were oxidized forming several distinct disulfide bridge arrangements. The isoforms of both HisrOxyTx1 and HisrOxyTx2 after DTT reduction eluted from the column as a single protein component of 9923 and 8040 Da, respectively. In vitro folding of either HisrOxyTx1 or HisrOxyTx2 yielded single oxidized components, which were cleaved independently by the proteolytic enzyme Factor Xa to give the recombinant peptides rOxyTx1 and rOxyTx2. The experimental molecular masses of rOxyTx1 and rOxyTx2 were 8059.0 and 6176.4 Da, respectively, which agree with their expected theoretical masses. The recombinant peptides rOxyTx1 and rOxyTx2 showed lower but comparable toxicity to the native toxins when injected into lepidopteran larvae; furthermore, rOxyTx1 was able to inhibit calcium ion currents on dorsal unpaired median (DUM) neurons from Periplaneta americana.

  15. Role of disulfide bonds in modulating internal motions of proteins to tune their function: molecular dynamics simulation of scorpion toxin Lqh III.

    PubMed

    Moghaddam, Majid Erfani; Naderi-Manesh, Hossein

    2006-04-01

    A series of 1-ns MD simulations were performed on the scorpion toxin Lqh III in native and disulfide bond broken states. The removal of disulfide bonds has caused hydrogen bond network alteration in the five-residue turn, the long loop, the alpha-helix, the loop connecting strands II and III, and the C-terminal region. In addition and more importantly, it has influenced the amplitude of the fluctuations of five-residue turn, loops, and C-terminal region with a minor effect on the fluctuations of the cysteines in the broken bond sites. These findings suggest that disulfide bonds are not the most important factors in rigidifying their own locations, while they have more important effects at a global scale. Furthermore, our results reveal that disulfide bonds have considerable influence on the functionally important essential modes of motions and the correlations between the motions of the binding site residues. Therefore, we can conclude that disulfide bonds have a crucial role in modulating the function via adjusting the dynamics of scorpion toxin molecules. Although this conclusion cannot be generalized to all peptides and proteins, it demonstrates the importance of more investigations on this aspect of disulfide bond efficacy.

  16. Chemical methods and approaches to the regioselective formation of multiple disulfide bonds.

    PubMed

    Shimamoto, Shigeru; Katayama, Hidekazu; Okumura, Masaki; Hidaka, Yuji

    2014-04-01

    Disulfide-bond formation plays an important role in the stabilization of the native conformation of peptides and proteins. In the case of multidisulfide-containing peptides and proteins, numerous folding intermediates are produced, including molecules that contain non-native and native disulfide bonds during in vitro folding. These intermediates can frequently be trapped covalently during folding and subsequently analyzed. The structural characterization of these kinetically trapped disulfide intermediates provides a clue to understanding the oxidative folding pathway. To investigate the folding of disulfide-containing peptides and proteins, in this unit, chemical methods are described for regulating regioselective disulfide formation (1) by using a combination of several types of thiol protecting groups, (2) by incorporating unique SeCys residues into a protein or peptide molecule, and (3) by combining with post-translational modification.

  17. Stabilization of cyclohexanone monooxygenase by a computationally designed disulfide bond spanning only one residue

    PubMed Central

    van Beek, Hugo L.; Wijma, Hein J.; Fromont, Lucie; Janssen, Dick B.; Fraaije, Marco W.

    2014-01-01

    Enzyme stability is an important parameter in biocatalytic applications, and there is a strong need for efficient methods to generate robust enzymes. We investigated whether stabilizing disulfide bonds can be computationally designed based on a model structure. In our approach, unlike in previous disulfide engineering studies, short bonds spanning only a few residues were included. We used cyclohexanone monooxygenase (CHMO), a Baeyer–Villiger monooxygenase (BVMO) from Acinetobacter sp. NCIMB9871 as the target enzyme. This enzyme has been the prototype BVMO for many biocatalytic studies even though it is notoriously labile. After creating a small library of mutant enzymes with introduced cysteine pairs and subsequent screening for improved thermostability, three stabilizing disulfide bonds were identified. The introduced disulfide bonds are all within 12 Å of each other, suggesting this particular region is critical for unfolding. This study shows that stabilizing disulfide bonds do not have to span many residues, as the most stabilizing disulfide bond, L323C–A325C, spans only one residue while it stabilizes the enzyme, as shown by a 6 °C increase in its apparent melting temperature. PMID:24649397

  18. Stabilization of cyclohexanone monooxygenase by a computationally designed disulfide bond spanning only one residue.

    PubMed

    van Beek, Hugo L; Wijma, Hein J; Fromont, Lucie; Janssen, Dick B; Fraaije, Marco W

    2014-01-01

    Enzyme stability is an important parameter in biocatalytic applications, and there is a strong need for efficient methods to generate robust enzymes. We investigated whether stabilizing disulfide bonds can be computationally designed based on a model structure. In our approach, unlike in previous disulfide engineering studies, short bonds spanning only a few residues were included. We used cyclohexanone monooxygenase (CHMO), a Baeyer-Villiger monooxygenase (BVMO) from Acinetobacter sp. NCIMB9871 as the target enzyme. This enzyme has been the prototype BVMO for many biocatalytic studies even though it is notoriously labile. After creating a small library of mutant enzymes with introduced cysteine pairs and subsequent screening for improved thermostability, three stabilizing disulfide bonds were identified. The introduced disulfide bonds are all within 12 Å of each other, suggesting this particular region is critical for unfolding. This study shows that stabilizing disulfide bonds do not have to span many residues, as the most stabilizing disulfide bond, L323C-A325C, spans only one residue while it stabilizes the enzyme, as shown by a 6 °C increase in its apparent melting temperature.

  19. The presence of disulfide bonds reveals an evolutionarily conserved mechanism involved in mitochondrial protein translocase assembly

    PubMed Central

    Wrobel, Lidia; Sokol, Anna M.; Chojnacka, Magdalena; Chacinska, Agnieszka

    2016-01-01

    Disulfide bond formation is crucial for the biogenesis and structure of many proteins that are localized in the intermembrane space of mitochondria. The importance of disulfide bond formation within mitochondrial proteins was extended beyond soluble intermembrane space proteins. Tim22, a membrane protein and core component of the mitochondrial translocase TIM22, forms an intramolecular disulfide bond in yeast. Tim22 belongs to the Tim17/Tim22/Tim23 family of protein translocases. Here, we present evidence of the high evolutionary conservation of disulfide bond formation in Tim17 and Tim22 among fungi and metazoa. Topological models are proposed that include the location of disulfide bonds relative to the predicted transmembrane regions. Yeast and human Tim22 variants that are not oxidized do not properly integrate into the membrane complex. Moreover, the lack of Tim17 oxidation disrupts the TIM23 translocase complex. This underlines the importance of disulfide bond formation for mature translocase assembly through membrane stabilization of weak transmembrane domains. PMID:27265872

  20. Engineering de novo disulfide bond in bacterial α-type carbonic anhydrase for thermostable carbon sequestration

    NASA Astrophysics Data System (ADS)

    Jo, Byung Hoon; Park, Tae Yoon; Park, Hyun June; Yeon, Young Joo; Yoo, Young Je; Cha, Hyung Joon

    2016-07-01

    Exploiting carbonic anhydrase (CA), an enzyme that rapidly catalyzes carbon dioxide hydration, is an attractive biomimetic route for carbon sequestration due to its environmental compatibility and potential economic viability. However, the industrial applications of CA are strongly hampered by the unstable nature of enzymes. In this work, we introduced in silico designed, de novo disulfide bond in a bacterial α-type CA to enhance thermostability. Three variants were selected and expressed in Escherichia coli with an additional disulfide bridge. One of the variants showed great enhancement in terms of both kinetic and thermodynamic stabilities. This improvement could be attributed to the loss of conformational entropy of the unfolded state, showing increased rigidity. The variant showed an upward-shifted optimal temperature and appeared to be thermoactivated, which compensated for the lowered activity at 25 °C. Collectively, the variant constructed by the rapid and effective de novo disulfide engineering can be used as an efficient biocatalyst for carbon sequestration under high temperature conditions.

  1. Engineering de novo disulfide bond in bacterial α-type carbonic anhydrase for thermostable carbon sequestration

    PubMed Central

    Jo, Byung Hoon; Park, Tae Yoon; Park, Hyun June; Yeon, Young Joo; Yoo, Young Je; Cha, Hyung Joon

    2016-01-01

    Exploiting carbonic anhydrase (CA), an enzyme that rapidly catalyzes carbon dioxide hydration, is an attractive biomimetic route for carbon sequestration due to its environmental compatibility and potential economic viability. However, the industrial applications of CA are strongly hampered by the unstable nature of enzymes. In this work, we introduced in silico designed, de novo disulfide bond in a bacterial α-type CA to enhance thermostability. Three variants were selected and expressed in Escherichia coli with an additional disulfide bridge. One of the variants showed great enhancement in terms of both kinetic and thermodynamic stabilities. This improvement could be attributed to the loss of conformational entropy of the unfolded state, showing increased rigidity. The variant showed an upward-shifted optimal temperature and appeared to be thermoactivated, which compensated for the lowered activity at 25 °C. Collectively, the variant constructed by the rapid and effective de novo disulfide engineering can be used as an efficient biocatalyst for carbon sequestration under high temperature conditions. PMID:27385052

  2. Simple, Automated, High Resolution Mass Spectrometry Method to Determine the Disulfide Bond and Glycosylation Patterns of a Complex Protein

    PubMed Central

    Pike, Gennett M.; Madden, Benjamin J.; Melder, Deborah C.; Charlesworth, M. Cristine; Federspiel, Mark J.

    2011-01-01

    Enveloped viruses must fuse the viral and cellular membranes to enter the cell. Understanding how viral fusion proteins mediate entry will provide valuable information for antiviral intervention to combat associated disease. The avian sarcoma and leukosis virus envelope glycoproteins, trimers composed of surface (SU) and transmembrane heterodimers, break the fusion process into several steps. First, interactions between SU and a cell surface receptor at neutral pH trigger an initial conformational change in the viral glycoprotein trimer followed by exposure to low pH enabling additional conformational changes to complete the fusion of the viral and cellular membranes. Here, we describe the structural characterization of the extracellular region of the subgroup A avian sarcoma and leukosis viruses envelope glycoproteins, SUATM129 produced in chicken DF-1 cells. We developed a simple, automated method for acquiring high resolution mass spectrometry data using electron capture dissociation conditions that preferentially cleave the disulfide bond more readily than the peptide backbone amide bonds that enabled the identification of disulfide-linked peptides. Seven of nine disulfide bonds were definitively assigned; the remaining two bonds were assigned to an adjacent pair of cysteine residues. The first cysteine of surface and the last cysteine of the transmembrane form a disulfide bond linking the heterodimer. The surface glycoprotein contains a free cysteine at residue 38 previously reported to be critical for virus entry. Eleven of 13 possible SUATM129 N-linked glycosylation sites were modified with carbohydrate. This study demonstrates the utility of this simple yet powerful method for assigning disulfide bonds in a complex glycoprotein. PMID:21454567

  3. The role of intra-domain disulfide bonds in heat-induced irreversible denaturation of camelid single domain VHH antibodies

    PubMed Central

    Akazawa-Ogawa, Yoko; Uegaki, Koichi; Hagihara, Yoshihisa

    2016-01-01

    Camelid-derived single domain VHH antibodies are highly heat resistant, and the mechanism of heat-induced VHH denaturation predominantly relies on the chemical modification of amino acids. Although chemical modification of disulfide bonds has been recognized as a cause for heat-induced denaturation of many proteins, there have been no mutagenesis studies, in which the number of disulfide bonds was controlled. In this article, we examined a series of mutants of two different VHHs with single, double or no disulfide bonds, and scrutinized the effects of these disulfide bond modifications on VHH denaturation. With the exception of one mutant, the heat resistance of VHHs decreased when the number of disulfide bonds increased. The effect of disulfide bonds on heat denaturation was more striking if the VHH had a second disulfide bond, suggesting that the contribution of disulfide shuffling is significant in proteins with multiple disulfide bonds. Furthermore, our results directly indicate that removal of a disulfide bond can indeed increase the heat resistance of a protein, irrespective of the negative impact on equilibrium thermodynamic stability. PMID:26289739

  4. The role of intra-domain disulfide bonds in heat-induced irreversible denaturation of camelid single domain VHH antibodies.

    PubMed

    Akazawa-Ogawa, Yoko; Uegaki, Koichi; Hagihara, Yoshihisa

    2016-01-01

    Camelid-derived single domain VHH antibodies are highly heat resistant, and the mechanism of heat-induced VHH denaturation predominantly relies on the chemical modification of amino acids. Although chemical modification of disulfide bonds has been recognized as a cause for heat-induced denaturation of many proteins, there have been no mutagenesis studies, in which the number of disulfide bonds was controlled. In this article, we examined a series of mutants of two different VHHs with single, double or no disulfide bonds, and scrutinized the effects of these disulfide bond modifications on VHH denaturation. With the exception of one mutant, the heat resistance of VHHs decreased when the number of disulfide bonds increased. The effect of disulfide bonds on heat denaturation was more striking if the VHH had a second disulfide bond, suggesting that the contribution of disulfide shuffling is significant in proteins with multiple disulfide bonds. Furthermore, our results directly indicate that removal of a disulfide bond can indeed increase the heat resistance of a protein, irrespective of the negative impact on equilibrium thermodynamic stability.

  5. Selective Gas-Phase Ion/Ion Reactions: Enabling Disulfide Mapping via Oxidation and Cleavage of Disulfide Bonds in Intermolecularly-Linked Polypeptide Ions.

    PubMed

    Pilo, Alice L; McLuckey, Scott A

    2016-09-20

    The selective gas-phase oxidation of disulfide bonds to their thiosulfinate form using ion/ion reactions and subsequent cleavage is demonstrated here. Oxidizing reagent anions are observed to attach to all polypeptides, regardless of amino acid composition. Direct proton transfer yielding a charge-reduced peptide is also frequently observed. Activation of the ion/ion complex between an oxidizing reagent anion and a disulfide-containing peptide cation results in oxygen transfer from the reagent anion to the peptide cation to form the [M+H+O](+) species. This thiosulfinate derivative can undergo one of several rearrangements that result in cleavage of the disulfide bond. Species containing an intermolecular disulfide bond undergo separation of the two chains upon activation. Further activation can be used to generate more sequence information from each chain. These oxidation ion/ion reactions have been used to illustrate the identification of S-glutathionylated and S-cysteinylated peptides, in which low molecular weight thiols are attached to cysteine residues in peptides via disulfide bonds. The oxidation chemistry effectively labels peptide ions with readily oxidized groups, such as disulfide bonds. This enables a screening approach for the identification of disulfide-linked peptides in a disulfide mapping application involving enzymatic digestion. The mixtures of ions generated by tryptic and peptic digestions of lysozyme and insulin, respectively, without prior separation or isolation were subjected both to oxidation and proton transfer ion/ion chemistry to illustrate the identification of peptides in the mixtures with readily oxidized groups.

  6. Structure-based approach to the prediction of disulfide bonds in proteins.

    PubMed

    Salam, Noeris K; Adzhigirey, Matvey; Sherman, Woody; Pearlman, David A

    2014-10-01

    Protein engineering remains an area of growing importance in pharmaceutical and biotechnology research. Stabilization of a folded protein conformation is a frequent goal in projects that deal with affinity optimization, enzyme design, protein construct design, and reducing the size of functional proteins. Indeed, it can be desirable to assess and improve protein stability in order to avoid liabilities such as aggregation, degradation, and immunogenic response that may arise during development. One way to stabilize a protein is through the introduction of disulfide bonds. Here, we describe a method to predict pairs of protein residues that can be mutated to form a disulfide bond. We combine a physics-based approach that incorporates implicit solvent molecular mechanics with a knowledge-based approach. We first assign relative weights to the terms that comprise our scoring function using a genetic algorithm applied to a set of 75 wild-type structures that each contains a disulfide bond. The method is then tested on a separate set of 13 engineered proteins comprising 15 artificial stabilizing disulfides introduced via site-directed mutagenesis. We find that the native disulfide in the wild-type proteins is scored well, on average (within the top 6% of the reasonable pairs of residues that could form a disulfide bond) while 6 out of the 15 artificial stabilizing disulfides scored within the top 13% of ranked predictions. Overall, this suggests that the physics-based approach presented here can be useful for triaging possible pairs of mutations for disulfide bond formation to improve protein stability.

  7. Defining the disulfide bonds of insulin-like growth factor-binding protein-5 by tandem mass spectrometry with electron transfer dissociation and collision-induced dissociation.

    PubMed

    Nili, Mahta; Mukherjee, Aditi; Shinde, Ujwal; David, Larry; Rotwein, Peter

    2012-01-06

    The six high-affinity insulin-like growth factor-binding proteins (IGFBPs) comprise a conserved family of secreted molecules that modulate IGF actions by regulating their half-life and access to signaling receptors, and also exert biological effects that are independent of IGF binding. IGFBPs are composed of cysteine-rich amino- (N-) and carboxyl- (C-) terminal domains, along with a cysteine-poor central linker segment. IGFBP-5 is the most conserved IGFBP, and contains 18 cysteines, but only 2 of 9 putative disulfide bonds have been mapped to date. Using a mass spectrometry (MS)-based strategy combining sequential electron transfer dissociation (ETD) and collision-induced dissociation (CID) steps, in which ETD fragmentation preferentially induces cleavage of disulfide bonds, and CID provides exact disulfide linkage assignments between liberated peptides, we now have definitively mapped 5 disulfide bonds in IGFBP-5. In addition, in conjunction with ab initio molecular modeling we are able to assign the other 4 disulfide linkages to within a GCGCCXXC motif that is conserved in five IGFBPs. Because of the nature of ETD fragmentation MS experiments were performed without chemical reduction of IGFBP-5. Our results not only establish a disulfide bond map of IGFBP-5 but also define a general approach that takes advantage of the specificity of ETD and the scalability of tandem MS, and the predictive power of ab initio molecular modeling to characterize unknown disulfide linkages in proteins.

  8. The interdomain disulfide bond of a homogeneous rabbit pneumococcal antibody light chain.

    PubMed

    Strosberg, A D; Margolies, M N; Haber, E

    1975-11-01

    Rabbit light chain 3315, prepared from a homogeneous antipneumococcal antibody, was subjected to hydrolysis by pepsin without prior reduction and alkylation of the intrachain disulfide bonds. Gel filtration of the hydrolysate on Sephadex G-10, G-15, and G-25 and ion exchange chromatography on SP-Sephadex yielded several disulfide bridge peptides. These were fully reduced and alkulated and sequenced by Edman degradation. The peptides were located in the light chain sequence determined in independent studies from our laboratory. The half-cystine residues in this KB rabbit chain are located at positions 23, 80, 88, 134, 171, 194, and 214. The extra disulfide bridge extends between residues 80 and 171, thus joining the variable and constant domains. This is consistent with x-ray diffraction crystallographic studies showing that the corresponding residues in human light chains are separated by a distance compatible with disulfide bond formation.

  9. CHANGES IN DISULFIDE BOND CONTENT OF PROTEINS IN A YEAST STRAIN LACKING MAJOR SOURCES OF NADPH

    PubMed Central

    Minard, Karyl I.; Carroll, Christopher A.; Weintraub, Susan T.; Mc-Alister-Henn, Lee

    2006-01-01

    A yeast mutant lacking the two major cytosolic sources of NADPH, glucose-6-phosphate dehydrogenase (Zwf1p) and NADP+-specific isocitrate dehydrogenase (Idp2p), has been demonstrated to lose viability when shifted to medium with acetate or oleate as the carbon source. This loss in viability was found to correlate with an accumulation of endogenous oxidative byproducts of respiration and peroxisomal β-oxidation. To assess effects on cellular protein of endogenous versus exogenous oxidative stress, a proteomics approach was used to compare disulfide bond-containing proteins in the idp2Δzwf1Δ strain following shifts to acetate and oleate media with those in the parental strain following similar shifts to media containing hydrogen peroxide. Among prominent disulfide bond-containing proteins were several with known antioxidant functions. These and several other proteins were detected as multiple electrophoretic isoforms, with some isoforms containing disulfide bonds under all conditions and other isoforms exhibiting a redox-sensitive content of disulfide bonds, i.e., in the idp2Δzwf1Δ strain and in the hydrogen peroxide-challenged parental strain. The disulfide bond content of some isoforms of these proteins was also elevated in the parental strain grown on glucose, possibly suggesting a redirection of NADPH reducing equivalents to support rapid growth. Further examination of protein carbonylation in the idp2Δzwf1Δ strain shifted to oleate medium also led to identification of common and unique protein targets of endogenous oxidative stress. PMID:17157197

  10. Size and conformation limits to secretion of disulfide-bonded loops in autotransporter proteins.

    PubMed

    Leyton, Denisse L; Sevastsyanovich, Yanina R; Browning, Douglas F; Rossiter, Amanda E; Wells, Timothy J; Fitzpatrick, Rebecca E; Overduin, Michael; Cunningham, Adam F; Henderson, Ian R

    2011-12-09

    Autotransporters are a superfamily of virulence factors typified by a channel-forming C terminus that facilitates translocation of the functional N-terminal passenger domain across the outer membrane of Gram-negative bacteria. This final step in the secretion of autotransporters requires a translocation-competent conformation for the passenger domain that differs markedly from the structure of the fully folded secreted protein. The nature of the translocation-competent conformation remains controversial, in particular whether the passenger domain can adopt secondary structural motifs, such as disulfide-bonded segments, while maintaining a secretion-competent state. Here, we used the endogenous and closely spaced cysteine residues of the plasmid-encoded toxin (Pet) from enteroaggregative Escherichia coli to investigate the effect of disulfide bond-induced folding on translocation of an autotransporter passenger domain. We reveal that rigid structural elements within disulfide-bonded segments are resistant to autotransporter-mediated secretion. We define the size limit of disulfide-bonded segments tolerated by the autotransporter system demonstrating that, when present, cysteine pairs are intrinsically closely spaced to prevent congestion of the translocator pore by large disulfide-bonded regions. These latter data strongly support the hairpin mode of autotransporter biogenesis.

  11. 46 CFR 151.50-40 - Additional requirements for carbon disulfide (carbon bisulfide) and ethyl ether.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 5 2012-10-01 2012-10-01 false Additional requirements for carbon disulfide (carbon... Special Requirements § 151.50-40 Additional requirements for carbon disulfide (carbon bisulfide) and ethyl... waterways at the loading and unloading points. (f) The special requirements of § 151.50-41 for...

  12. 46 CFR 151.50-40 - Additional requirements for carbon disulfide (carbon bisulfide) and ethyl ether.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 5 2011-10-01 2011-10-01 false Additional requirements for carbon disulfide (carbon... Special Requirements § 151.50-40 Additional requirements for carbon disulfide (carbon bisulfide) and ethyl... waterways at the loading and unloading points. (f) The special requirements of § 151.50-41 for...

  13. 46 CFR 151.50-40 - Additional requirements for carbon disulfide (carbon bisulfide) and ethyl ether.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 5 2010-10-01 2010-10-01 false Additional requirements for carbon disulfide (carbon... Special Requirements § 151.50-40 Additional requirements for carbon disulfide (carbon bisulfide) and ethyl... waterways at the loading and unloading points. (f) The special requirements of § 151.50-41 for...

  14. 46 CFR 151.50-40 - Additional requirements for carbon disulfide (carbon bisulfide) and ethyl ether.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 5 2013-10-01 2013-10-01 false Additional requirements for carbon disulfide (carbon... Special Requirements § 151.50-40 Additional requirements for carbon disulfide (carbon bisulfide) and ethyl... waterways at the loading and unloading points. (f) The special requirements of § 151.50-41 for...

  15. 46 CFR 151.50-40 - Additional requirements for carbon disulfide (carbon bisulfide) and ethyl ether.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 5 2014-10-01 2014-10-01 false Additional requirements for carbon disulfide (carbon... Special Requirements § 151.50-40 Additional requirements for carbon disulfide (carbon bisulfide) and ethyl... waterways at the loading and unloading points. (f) The special requirements of § 151.50-41 for...

  16. Identification of disulfide bond formation between MitoNEET and glutamate dehydrogenase 1.

    PubMed

    Roberts, Morgan E; Crail, Jacquelyn P; Laffoon, Megan M; Fernandez, William G; Menze, Michael A; Konkle, Mary E

    2013-12-17

    MitoNEET is a protein that was identified as a drug target for diabetes, but its cellular function as well as its role in diabetes remains elusive. Protein pull-down experiments identified glutamate dehydrogenase 1 (GDH1) as a potential binding partner. GDH1 is a key metabolic enzyme with emerging roles in insulin regulation. MitoNEET forms a covalent complex with GDH1 through disulfide bond formation and acts as an activator. Proteomic analysis identified the specific cysteine residues that participate in the disulfide bond. This is the first report that effectively links mitoNEET to activation of the insulin regulator GDH1.

  17. Disulfide bond cleavage in TEMPO-free radical initiated peptide sequencing mass spectrometry.

    PubMed

    Lee, Minhee; Lee, Younjin; Kang, Minhyuk; Park, Hyeyeon; Seong, Yeonmi; Sung, Bong June; Moon, Bongjin; Oh, Han Bin

    2011-08-01

    The gas-phase free radical initiated peptide sequencing (FRIPS) fragmentation behavior of o-TEMPO-Bz-conjugated peptides with an intra- and intermolecular disulfide bond was investigated using MS(n) tandem mass spectrometry experiments. Investigated peptides included four peptides with an intramolecular cyclic disulfide bond, Bactenecin (RLCRIVVIRVCR), TGF-α (CHSGYVGVRC), MCH (DFDMLRCMLGRVFRPCWQY) and Adrenomedullin (16-31) (CRFGTCTVQKLAHQIY), and two peptides with an intermolecular disulfide bond. Collisional activation of the benzyl radical conjugated peptide cation, which was generated through the release of a TEMPO radical from o-TEMPO-Bz-conjugated peptides upon initial collisional activation, produced a large number of peptide backbone fragments in which the S-S or C-S bond was readily cleaved. The observed peptide backbone fragments included a-, c-, x- or z-types, which indicates that the radical-driven peptide fragmentation mechanism plays an important role in TEMPO-FRIPS mass spectrometry. FRIPS application of the linearly linked disulfide peptides further showed that the S-S or C-S bond was selectively and preferentially cleaved, followed by peptide backbone dissociations. In the FRIPS mass spectra, the loss of •SH or •SSH was also abundantly found. On the basis of these findings, FRIPS fragmentation pathways for peptides with a disulfide bond are proposed. For the cleavage of the S-S bond, the abstraction of a hydrogen atom at C(β) by the benzyl radical is proposed to be the initial radical abstraction/transfer reaction. On the other hand, H-abstraction at C(α) is suggested to lead to C-S bond cleavage, which yields [ion ± S] fragments or the loss of •SH or •SSH.

  18. Cytosolic disulfide bond formation in cells infected with large nucleocytoplasmic DNA viruses.

    PubMed

    Hakim, Motti; Fass, Deborah

    2010-10-01

    Proteins that have evolved to contain stabilizing disulfide bonds generally fold in a membrane-delimited compartment in the cell [i.e., the endoplasmic reticulum (ER) or the mitochondrial intermembrane space (IMS)]. These compartments contain sulfhydryl oxidase enzymes that catalyze the pairing and oxidation of cysteine residues. In contrast, most proteins in a healthy cytosol are maintained in reduced form through surveillance by NADPH-dependent reductases and the lack of sulfhydryl oxidases. Nevertheless, one of the core functionalities that unify the broad and diverse set of nucleocytoplasmic large DNA viruses (NCLDVs) is the ability to catalyze disulfide formation in the cytosol. The substrates of this activity are proteins that contribute to the assembly, structure, and infectivity of the virions. If the last common ancestor of NCLDVs was present during eukaryogenesis as has been proposed, it is interesting to speculate that viral disulfide bond formation pathways may have predated oxidative protein folding in intracellular organelles.

  19. The biologically active form of the sea urchin egg receptor for sperm is a disulfide-bonded homo-multimer

    PubMed Central

    1994-01-01

    Since many cell surface receptors exist in their active form as oligomeric complexes, we have investigated the subunit composition of the biologically active sperm receptor in egg plasma membranes from Strongylocentrotus purpuratus. Electrophoretic analysis of the receptor without prior reduction of disulfide bonds revealed that the surface receptor exists in the form of a disulfide-bonded multimer, estimated to be a tetramer. These findings are in excellent agreement with the fact that the NH2-terminus of the extracellular domain of the sperm receptor is rich in cysteine residues. Studies with cross-linking agents of various length and hydrophobicity suggest that no other major protein is tightly associated with the receptor. Given the multimeric structure of the receptor, we investigated the effect of disulfide bond reduction on its biological activity. Because in quantitative bioassays fertilization was found to be inhibited by treatment of eggs with 5 mM dithiothreitol, we undertook more direct studies of the effect of reduction on properties of the receptor. First, we studied the effect of addition of isolated, pure receptor on fertilization. Whereas the non-reduced, native receptor complex inhibited fertilization in a dose- dependent manner, the reduced and alkylated receptor was inactive. Second, we tested the ability of the isolated receptor to mediate binding of acrosome-reacted sperm to polystyrene beads. Whereas beads coated with native receptor bound sperm, those containing reduced and alkylated receptor did not. Thus, these results demonstrate that the biologically active form of the sea urchin sperm receptor consists only of 350 kD subunits and that these must be linked as a multimer via disulfide bonds to produce a complex that is functional in sperm recognition and binding. PMID:8188748

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

    PubMed

    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 (<200 amino acids) this correlation is negative. Medium-sized 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. Direct evidence that two cysteines in the dopamine transporter form a disulfide bond.

    PubMed

    Chen, Rong; Wei, Hua; Hill, Erik R; Chen, Lucy; Jiang, Liying; Han, Dawn D; Gu, Howard H

    2007-04-01

    We have generated a fully functional dopamine transporter (DAT) mutant (dmDATx7) with all cysteines removed except the two cysteines in extracellular loop 2 (EL2). Random mutagenesis at either or both EL2 cysteines did not produce any functional transporter mutants, suggesting that the two cysteines cannot be replaced by any other amino acids. The cysteine-specific reagent MTSEA-biotin labeled dmDATx7 only after a DTT treatment which reduces disulfide bond. Since there are no other cysteines in dmDATx7, the MTSEA-biotin labeling must be on the EL2 cysteines made available by the DTT treatment. This result provides the first direct evidence that the EL2 cysteines form a disulfide bond. Interestingly, the DTT treatment had little effect on transport activity suggesting that the disulfide bond is not necessary for the uptake function of DAT. Our results and previous results are consistent with the notion that the disulfide bond between EL2 cysteines is required for DAT biosynthesis and/or its delivery to the cell surface.

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

    PubMed

    Yu, Xiao-Wei; Tan, Nian-Jiang; Xiao, Rong; Xu, Yan

    2012-01-01

    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.

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

    SciTech Connect

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

    2008-10-31

    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{sub 108/121}, HB-EGF-Cys/Ser{sub 116/132}, and HB-EGF-Cys/Ser{sub 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{sub 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{sub 108/121} and HB-EGF-Cys/Ser{sub 116/132} stable cell lines which exhibited accumulated expression immediately outside the nucleus. rHB-EGF, HB-EGF, and HB-EGF{sub 134/143} proteins competed with {sup 125}I-EGF in an A431 competitive binding assay, whereas HB-EGF-Cys/Ser{sub 108/121} and HB-EGF-Cys/Ser{sub 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{sub 134/143} antagonizes EGFRs.

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

    SciTech Connect

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

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer We dissect how individual disulfide bond affects the amyloidogenicity of insulin. Black-Right-Pointing-Pointer A controlled reduction system for insulin is established in this study. Black-Right-Pointing-Pointer Disulfide breakage is associated with unfolding and increased amyloidogenicity. Black-Right-Pointing-Pointer Breakage of A6-A11 is associated with significantly increased cytotoxicity. Black-Right-Pointing-Pointer 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

  5. Reactivity of disulfide bonds is markedly affected by structure and environment: implications for protein modification and stability

    PubMed Central

    Karimi, Maryam; Ignasiak, Marta T.; Chan, Bun; Croft, Anna K.; Radom, Leo; Schiesser, Carl H.; Pattison, David I.; Davies, Michael J.

    2016-01-01

    Disulfide bonds play a key role in stabilizing protein structures, with disruption strongly associated with loss of protein function and activity. Previous data have suggested that disulfides show only modest reactivity with oxidants. In the current study, we report kinetic data indicating that selected disulfides react extremely rapidly, with a variation of 104 in rate constants. Five-membered ring disulfides are particularly reactive compared with acyclic (linear) disulfides or six-membered rings. Particular disulfides in proteins also show enhanced reactivity. This variation occurs with multiple oxidants and is shown to arise from favorable electrostatic stabilization of the incipient positive charge on the sulfur reaction center by remote groups, or by the neighboring sulfur for conformations in which the orbitals are suitably aligned. Controlling these factors should allow the design of efficient scavengers and high-stability proteins. These data are consistent with selective oxidative damage to particular disulfides, including those in some proteins. PMID:27941824

  6. Reactivity of disulfide bonds is markedly affected by structure and environment: implications for protein modification and stability

    NASA Astrophysics Data System (ADS)

    Karimi, Maryam; Ignasiak, Marta T.; Chan, Bun; Croft, Anna K.; Radom, Leo; Schiesser, Carl H.; Pattison, David I.; Davies, Michael J.

    2016-12-01

    Disulfide bonds play a key role in stabilizing protein structures, with disruption strongly associated with loss of protein function and activity. Previous data have suggested that disulfides show only modest reactivity with oxidants. In the current study, we report kinetic data indicating that selected disulfides react extremely rapidly, with a variation of 104 in rate constants. Five-membered ring disulfides are particularly reactive compared with acyclic (linear) disulfides or six-membered rings. Particular disulfides in proteins also show enhanced reactivity. This variation occurs with multiple oxidants and is shown to arise from favorable electrostatic stabilization of the incipient positive charge on the sulfur reaction center by remote groups, or by the neighboring sulfur for conformations in which the orbitals are suitably aligned. Controlling these factors should allow the design of efficient scavengers and high-stability proteins. These data are consistent with selective oxidative damage to particular disulfides, including those in some proteins.

  7. Reactivity of disulfide bonds is markedly affected by structure and environment: implications for protein modification and stability.

    PubMed

    Karimi, Maryam; Ignasiak, Marta T; Chan, Bun; Croft, Anna K; Radom, Leo; Schiesser, Carl H; Pattison, David I; Davies, Michael J

    2016-12-12

    Disulfide bonds play a key role in stabilizing protein structures, with disruption strongly associated with loss of protein function and activity. Previous data have suggested that disulfides show only modest reactivity with oxidants. In the current study, we report kinetic data indicating that selected disulfides react extremely rapidly, with a variation of 10(4) in rate constants. Five-membered ring disulfides are particularly reactive compared with acyclic (linear) disulfides or six-membered rings. Particular disulfides in proteins also show enhanced reactivity. This variation occurs with multiple oxidants and is shown to arise from favorable electrostatic stabilization of the incipient positive charge on the sulfur reaction center by remote groups, or by the neighboring sulfur for conformations in which the orbitals are suitably aligned. Controlling these factors should allow the design of efficient scavengers and high-stability proteins. These data are consistent with selective oxidative damage to particular disulfides, including those in some proteins.

  8. Solution structure and activity of the synthetic four-disulfide bond Mediterranean mussel defensin (MGD-1).

    PubMed

    Yang, Y S; Mitta, G; Chavanieu, A; Calas, B; Sanchez, J F; Roch, P; Aumelas, A

    2000-11-28

    MGD-1 is a 39-residue defensin-like peptide isolated from the edible Mediterranean mussel, Mytilus galloprovincialis. This peptide is characterized by the presence of four disulfide bonds. We report here its solid-phase synthesis and an easy way to improve the yield of the four native disulfide bonds. Synthetic and native MGD-1 display similar antibacterial activity, suggesting that the hydroxylation of Trp28 observed in native MGD-1 is not involved in the antimicrobial effect. The three-dimensional solution structure of MGD-1 has been established using (1)H NMR and mainly consists of a helical part (Asn7-Ser16) and two antiparallel beta-strands (Arg20-Cys25 and Cys33-Arg37), together giving rise to the common cystine-stabilized alpha-beta motif frequently observed in scorpion toxins. In MGD-1, the cystine-stabilized alpha-beta motif is stabilized by four disulfide bonds (Cys4-Cys25, Cys10-Cys33, Cys14-Cys35, and Cys21-Cys38), instead of by the three disulfide bonds commonly found in arthropod defensins. Except for the Cys21-Cys38 disulfide bond which is solvent-exposed, the three others belong to the particularly hydrophobic core of the highly constrained structure. Moreover, the C4-P5 amide bond in the cis conformation characterizes the MGD-1 structure. MGD-1 and insect defensin A possess similar bactericidal anti-Gram-positive activity, suggesting that the fourth disulfide bond of MGD-1 is not essential for the biological activity. In agreement with the general features of antibacterial peptides, the MGD-1 and defensin A structures display a typical distribution of positively charged and hydrophobic side chains. The positively charged residues of MGD-1 are located in three clusters. For these two defensin peptides isolated from insects and mollusks, it appears that the rather well conserved location of certain positively charged residues and of the large hydrophobic cluster are enough to generate the bactericidal potency and the Gram-positive specificity.

  9. Different dynamics and pathway of disulfide bonds reduction of two human defensins, a molecular dynamics simulation study.

    PubMed

    Zhang, Liqun

    2017-04-01

    Human defensins are a class of antimicrobial peptides that are crucial components of the innate immune system. Both human α defensin type 5 (HD5) and human β defensin type 3 (hBD-3) have 6 cysteine residues which form 3 pairs of disulfide bonds in oxidizing condition. Disulfide bond linking is important to the protein structure stabilization, and the disulfide bond linking and breaking order have been shown to influence protein function. In this project, microsecond long molecular dynamics simulations were performed to study the structure and dynamics of HD5 and hBD-3 wildtype and analogs which have all 3 disulfide bonds released in reducing condition. The structure of hBD-3 was found to be more dynamic and flexible than HD5, based on RMSD, RMSF, and radius of gyration calculations. The disulfide bridge breaking order of HD5 and hBD-3 in reducing condition was predicted by two kinds of methods, which gave consistent results. It was found that the disulfide bonds breaking pathways for HD5 and hBD-3 are very different. The breaking of disulfide bonds can influence the dimer interface by making the dimer structure less stable for both kinds of defensin. In order to understand the difference in dynamics and disulfide bond breaking pathway, hydrophilic and hydrophobic accessible surface areas (ASA), buried surface area between cysteine pairs, entropy of cysteine pairs, and internal energy were calculated. Comparing to the wildtype, hBD-3 analog is more hydrophobic, while HD5 is more hydrophilic. For hBD-3, the disulfide breaking is mainly entropy driven, while other factors such as the solvation effects may take the major role in controlling HD5 disulfide breaking pathway. Proteins 2017; 85:665-681. © 2016 Wiley Periodicals, Inc.

  10. The effect of disulfide bond introduction and related Cys/Ser mutations on the stability of a cyclohexanone monooxygenase.

    PubMed

    Schmidt, Sandy; Genz, Maika; Balke, Kathleen; Bornscheuer, Uwe T

    2015-11-20

    Baeyer-Villiger monooxygenases (BVMO) belong to the class B of flavin-dependent monooxygenases (type I BVMOs) and catalyze the oxidation of (cyclic) ketones into esters and lactones. The prototype BVMO is the cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871. This enzyme shows an impressive substrate scope with a high chemo-, regio- and/or enantioselectivity. BVMO reactions are often difficult, if not impossible to achieve by chemical approaches and this makes these enzymes thus highly desired candidates for industrial applications. Unfortunately, the industrial use is hampered by several factors related to the lack of stability of these biocatalysts. Thus, the aim of this study was to improve the CHMO's long-term stability, one of the most relevant parameter for biocatalytic processes, and additionally its stability against oxidation. We used an easy computational method for the prediction of stabilizing disulfide bonds in the CHMO-scaffold. The three most promising predicted disulfide pairs were created and biochemically characterized. The most oxidatively stable variant (Y411C-A463C) retained nearly 60% activity after incubation with 25 mM H2O2 whereas the wild type retained only 16%. In addition, one extra disulfide pair (T415C-A463C) was created and tested for increased stability. The melting temperature (Tm) of this variant was increased by 5°C with simultaneous improved long-term stability. After verification by ABD-F labeling that this mutant does not form a disulfide bond, single and double Cys/Ser mutants were prepared and investigated. Subsequent analysis revealed that the T415C single point variant is the most stable variant with a 30-fold increased long-term stability (33% residual activity after 24h incubation at 25°C) showcasing a great achievement for practical applications.

  11. Negative effects of a disulfide bond mismatch in anti-rabies G protein single-chain antibody variable fragment FV57.

    PubMed

    Duan, Ye; Gu, Tiejun; Zhang, Xizhen; Jiang, Chunlai; Yuan, Ruosen; Li, Zhuang; Wang, Dandan; Chen, Xiaoxu; Wu, Chunlai; Chen, Yan; Wu, Yongge; Kong, Wei

    2014-06-01

    Rabies virus (RV) causes a fatal infectious disease requiring efficient post-exposure prophylaxis (PEP), which includes a rabies vaccine and rabies immunoglobulin (RIG). The single-chain antibody variable fragment (scFv), a small engineered antibody fragment derived from an antibody variable heavy chain and light chain, has the potential to replace the current application of RIG. In previous studies, we constructed and evaluated an anti-rabies virus G protein scFv (FV57) based on the monoclonal antibody CR57. Of the five cysteines in FV57, four are linked in intra-chain disulfide bonds (Cys-VH28/Cys-VH98 and Cys-VL16/Cys-VL84), and one is free (Cys-VL85). However, the thiol in Cys-VL85 neighboring Cys-VL84 in the CDR3 of the light chain is likely to mismatch with the thiol in Cys-VL16 during the renaturing process. In order to study effects of the mismatched disulfide bond, Cys-VL85 and Cys-VL84 of FV57 were mutated to serine to construct mutants FV57(VL85S) and FV57(VL84S). Furthermore, the disulfide bonds in the light chain of FV57, FV57(VL85S) and FV57(VL84S) were deleted by mutating Cys-VL16 to serine. All mutants were prepared and evaluated along with the original FV57. The results indicated that the mismatched disulfide bond of FV57 linking the light chain FR1 and CDR3 would confer deleterious negative effects on its activity against RV, likely due to spatial hindrance in the light chain CDR3. Moreover, avoidance of the disulfide bond mismatch provided an additional 30% protective efficacy against RV infection in the mouse RV challenge model. Thus, modifications of FV57 to eliminate the disulfide bond mismatch may provide a candidate therapeutic agent for effective PEP against rabies.

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

    PubMed Central

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

    2014-01-01

    Recombinant wild-pyrazinamidase from H37Rv M. 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. PMID:25199451

  13. Prediction of the disulfide-bonding state of cysteines in proteins based on dipeptide composition.

    PubMed

    Song, Jiang-Ning; Wang, Ming-Lei; Li, Wei-Jiang; Xu, Wen-Bo

    2004-05-21

    In this paper, a novel approach has been introduced to predict the disulfide-bonding state of cysteines in proteins by means of a linear discriminator based on their dipeptide composition. The prediction is performed with a newly enlarged dataset with 8114 cysteine-containing segments extracted from 1856 non-homologous proteins of well-resolved three-dimensional structures. The oxidation of cysteines exhibits obvious cooperativity: almost all cysteines in disulfide-bond-containing proteins are in the oxidized form. This cooperativity can be well described by protein's dipeptide composition, based on which the prediction accuracy of the oxidation form of cysteines scores as high as 89.1% and 85.2%, when measured on cysteine and protein basis using the rigorous jack-knife procedure, respectively. The result demonstrates the applicability of this new relatively simple method and provides superior prediction performance compared with existing methods for the prediction of the oxidation states of cysteines in proteins.

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

    SciTech Connect

    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

    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.

  15. Air sparging for prevention of antibody disulfide bond reduction in harvested CHO cell culture fluid.

    PubMed

    Mun, Melissa; Khoo, Stefanie; Do Minh, Aline; Dvornicky, James; Trexler-Schmidt, Melody; Kao, Yung-Hsiang; Laird, Michael W

    2015-04-01

    During the scale-up of several Chinese Hamster Ovary (CHO) cell monoclonal antibody production processes, significant reduction of the antibody interchain disulfide bonds was observed. The reduction was correlated with excessive mechanical cell shear during the harvest operations. These antibody reduction events resulted in failed product specifications and the subsequent loss of the drug substance batches. Several methods were recently developed to prevent antibody reduction, including modifying the cell culture media, using pre- and post-harvest chemical additions to the cell culture fluid (CCF), lowering the pH, and air sparging of the harvested CCF (HCCF). The work described in this paper further explores the option of HCCF air sparging for preventing antibody reduction. Here, a small-scale model was developed using a 3-L bioreactor to mimic the conditions of a manufacturing-scale harvest vessel and was subsequently employed to evaluate several air sparging strategies. In addition, these studies enabled further understanding of the relationships between cell lysis levels, oxygen consumption, and antibody reduction. Finally, the effectiveness of air sparging for several CHO cell lines and the potential impact on product quality were assessed to demonstrate that air sparging is an effective method in preventing antibody reduction.

  16. Identification of disulfide bonds in wheat gluten proteins by means of mass spectrometry/electron transfer dissociation.

    PubMed

    Lutz, Elena; Wieser, Herbert; Koehler, Peter

    2012-04-11

    Disulfide bonds within gluten proteins play a key role in the breadmaking performance of wheat flour. In the present study, disulfide bonds of wheat gluten proteins were identified by using a new liquid chromatography-mass spectrometry (LC-MS) technique with alternating electron transfer dissociation (ETD)/collision-induced dissociation (CID). Wheat flour was partially hydrolyzed with thermolysin (pH 6.5, 37 °C, 16 h), and the digest was subjected to LC-MS with alternating ETD/CID fragmentation. Whereas CID provided peptide fragments with intact disulfide bonds, cleavage of disulfide bonds was preferred over peptide backbone fragmentations in ETD. The simultaneous observation of disulfide-linked and disulfide-cleaved peptide ions in the mass spectra not only provided distinct interpretation with high confidence but also simplified the conventional approach for determination of disulfide bonds, which often requires two separate experiments with and without chemical reduction. By application of the new method 14 cystine peptides were identified. Eight peptides confirmed previously established disulfide bonds within gluten proteins, and the other six cystine peptides were identified for the first time. One of the newly identified cystine peptides represented a "head-to-tail" cross-link between high molecular weight glutenin subunits. This type of cross-link, which has been postulated as an integral part of glutenin models published previously, has now been proven experimentally for the first time. From the six remaining cystine peptides interchain disulfide bonds between α-gliadins, γ-gliadins, and low molecular weight glutenin subunits were established.

  17. Localization of the labile disulfide bond between SU and TM of the murine leukemia virus envelope protein complex to a highly conserved CWLC motif in SU that resembles the active-site sequence of thiol-disulfide exchange enzymes.

    PubMed Central

    Pinter, A; Kopelman, R; Li, Z; Kayman, S C; Sanders, D A

    1997-01-01

    Previous studies have indicated that the surface (SU) and transmembrane (TM) subunits of the envelope protein (Env) of murine leukemia viruses (MuLVs) are joined by a labile disulfide bond that can be stabilized by treatment of virions with thiol-specific reagents. In the present study this observation was extended to the Envs of additional classes of MuLV, and the cysteines of SU involved in this linkage were mapped by proteolytic fragmentation analyses to the CWLC sequence present at the beginning of the C-terminal domain of SU. This sequence is highly conserved across a broad range of distantly related retroviruses and resembles the CXXC motif present at the active site of thiol-disulfide exchange enzymes. A model is proposed in which rearrangements of the SU-TM intersubunit disulfide linkage, mediated by the CWLC sequence, play roles in the assembly and function of the Env complex. PMID:9311907

  18. 43 CFR 3154.2 - Additional bonding.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 43 Public Lands: Interior 2 2011-10-01 2011-10-01 false Additional bonding. 3154.2 Section 3154.2 Public Lands: Interior Regulations Relating to Public Lands (Continued) BUREAU OF LAND MANAGEMENT... Requirements § 3154.2 Additional bonding. The authorized officer may increase the amount of any bond that...

  19. Electron capture by the thiyl radical and disulfide bond: ligand effects on the reduction potential.

    PubMed

    Roos, Goedele; De Proft, Frank; Geerlings, Paul

    2013-04-15

    The effect of non-polar and polar ligands and of monovalent cations on the one-electron reduction potential of the thiyl radical and the disulfide bond was evaluated. The reduction potentials E° for the CH3S(.)-nL/CH3S(-)-nL and CH3SSCH3-L/CH3SSCH3(.-)-L redox couples were calculated at the B3LYP, M06-2X and MP2 levels of theory, with n=1, 2 and L=CH4, C2H4, H2O, CH3OH, NH3, CH3COOH, CH3CONH2, NH4(+), Na(+), K(+) and Li(+). Non-polar ligands decrease the E° value of the thiyl radical and disulfide bond, while neutral polar ligands favour electron uptake. Charged polar ligands and cations favour electron capture by the thiyl radical while disfavouring electron uptake by the disulfide bond. Thus, the same type of ligand can have a different effect on E° depending on the redox couple. Therefore, properties of an isolated ligand cannot uniquely determine E°. The ligand effects on E° are discussed in terms of the vertical electron affinity and reorganization energy, as well as molecular orbital theory. For a given redox couple, the ligand type influences the nature of the anion formed upon electron capture and the corresponding reorganization process towards the reduced geometry.

  20. Blood-Stable, Tumor-Adaptable Disulfide Bonded mPEG-(Cys)4-PDLLA Micelles for Chemotherapy

    PubMed Central

    Lee, Seung-Young; Kim, Sungwon; Tyler, Jacqueline; Park, Kinam; Cheng, Ji-Xin

    2012-01-01

    Although targeted delivery mediated by ligand modified or tumor microenvironment sensitive nanocarriers has been extensively pursued for cancer chemotherapy, the efficiency is still limited by premature drug release after systemic administration. Herein we report a highly blood-stable, tumor-adaptable drug carrier made of disulfide (DS) bonded mPEG-(Cys)4-PDLLA micelles. Intravenously injected disulfide bonded micelles stably retained doxorubicin in the bloodstream and efficiently delivered the drug to a tumor, with a 7-fold increase of the drug in the tumor and 1.9-fold decrease in the heart, as compared with self-assembled (SA), non-crosslinked mPEG-PDLLA micelles. In vivo administration of disulfide bonded micelles led to doxorubicin accumulation in cancer cell nuclei, which was not observed after administration of self-assembled micelles. With a doxorubicin dose as low as 2 mg/kg, disulfide bonded micelles almost completely suppressed tumor growth in mice. PMID:23079665

  1. Interplay between disulfide bonding and N-glycosylation defines SLC4 Na+-coupled transporter extracellular topography.

    PubMed

    Zhu, Quansheng; Kao, Liyo; Azimov, Rustam; Abuladze, Natalia; Newman, Debra; Kurtz, Ira

    2015-02-27

    The extracellular loop 3 (EL-3) of SLC4 Na(+)-coupled transporters contains 4 highly conserved cysteines and multiple N-glycosylation consensus sites. In the electrogenic Na(+)-HCO3(-) cotransporter NBCe1-A, EL-3 is the largest extracellular loop and is predicted to consist of 82 amino acids. To determine the structural-functional importance of the conserved cysteines and the N-glycosylation sites in NBCe1-A EL-3, we analyzed the potential interplay between EL-3 disulfide bonding and N-glycosylation and their roles in EL-3 topological folding. Our results demonstrate that the 4 highly conserved cysteines form two intramolecular disulfide bonds, Cys(583)-Cys(585) and Cys(617)-Cys(642), respectively, that constrain EL-3 in a folded conformation. The formation of the second disulfide bond is spontaneous and unaffected by the N-glycosylation state of EL-3 or the first disulfide bond, whereas formation of the first disulfide bond relies on the presence of the second disulfide bond and is affected by N-glycosylation. Importantly, EL-3 from each monomer is adjacently located at the NBCe1-A dimeric interface. When the two disulfide bonds are missing, EL-3 adopts an extended conformation highly accessible to protease digestion. This unique adjacent parallel location of two symmetrically folded EL-3 loops from each monomer resembles a domain-like structure that is potentially important for NBCe1-A function in vivo. Moreover, the formation of this unique structure is critically dependent on the finely tuned interplay between disulfide bonding and N-glycosylation in the membrane processed NBCe1-A dimer.

  2. Dynamical roles of metal ions and the disulfide bond in Cu, Zn superoxide dismutase folding and aggregation.

    PubMed

    Ding, Feng; Dokholyan, Nikolay V

    2008-12-16

    Misfolding and aggregation of Cu, Zn superoxide dismutase (SOD1) is implicated in neuronal death in amyotrophic lateral sclerosis. Each SOD1 monomer binds to 1 copper and 1 zinc ion and maintains its disulfide bond (Cys-57-Cys-146) in the reducing cytoplasm of cell. Mounting experimental evidence suggests that metal loss and/or disulfide reduction are important for initiating misfolding and aggregation of SOD1. To uncover the role of metals and the disulfide bond in the SOD1 folding, we systemically study the folding thermodynamics and structural dynamics of SOD1 monomer and dimer with and without metal binding and under disulfide-intact or disulfide-reduced environments in computational simulations. We use all-atom discrete molecular dynamics for sampling. Our simulation results provide dynamical evidence to the stabilizing role of metal ions in both dimer and monomer SOD1. The disulfide bond anchors a loop (Glu-49 to Asn-53) that contributes to the dimer interface. The reduction of the disulfide bond in SOD1 with metal ions depleted results in a flexible Glu-49-Asn-53 loop, which, in turn, disrupts dimer formation. Interestingly, the disulfide bond reduction does not affect the thermostability of monomer SOD1 as significantly as the metal ions do. We further study the structural dynamics of metal-free SOD1 monomers, the precursor for aggregation, in simulations and find inhomogeneous local unfolding of beta-strands. The strands protected by the metal-binding and electrostatic loops are found to unfold first after metal loss, leading to a partially unfolded beta-sheet prone to aggregation. Our simulation study sheds light on the critical role of metals and disulfide bond in SOD1 folding and aggregation.

  3. Overexpression of Protein Disulfide Isomerase DsbC Stabilizes Multiple-Disulfide-Bonded Recombinant Protein Produced and Transported to the Periplasm in Escherichia coli

    PubMed Central

    Kurokawa, Yoichi; Yanagi, Hideki; Yura, Takashi

    2000-01-01

    Dsb proteins (DsbA, DsbB, DsbC, and DsbD) catalyze formation and isomerization of protein disulfide bonds in the periplasm of Escherichia coli. By using a set of Dsb coexpression plasmids constructed recently, we analyzed the effects of Dsb overexpression on production of horseradish peroxidase (HRP) isozyme C that contains complex disulfide bonds and tends to aggregate when produced in E. coli. When transported to the periplasm, HRP was unstable but was markedly stabilized upon simultaneous overexpression of the set of Dsb proteins (DsbABCD). Whereas total HRP production increased severalfold upon overexpression of at least disulfide-bonded isomerase DsbC, maximum transport of HRP to the periplasm seemed to require overexpression of all DsbABCD proteins, suggesting that excess Dsb proteins exert synergistic effects in assisting folding and transport of HRP. Periplasmic production of HRP also increased when calcium, thought to play an essential role in folding of nascent HRP polypeptide, was added to the medium with or without Dsb overexpression. These results suggest that Dsb proteins and calcium play distinct roles in periplasmic production of HRP, presumably through facilitating correct folding. The present Dsb expression plasmids should be useful in assessing and dissecting periplasmic production of proteins that contain multiple disulfide bonds in E. coli. PMID:10966415

  4. Crystal Structure of Reduced and of Oxidized Peroxiredoxin IV Enzyme Reveals a Stable Oxidized Decamer and a Non-disulfide-bonded Intermediate in the Catalytic Cycle*

    PubMed Central

    Cao, Zhenbo; Tavender, Timothy J.; Roszak, Aleksander W.; Cogdell, Richard J.; Bulleid, Neil J.

    2011-01-01

    Peroxiredoxin IV (PrxIV) is an endoplasmic reticulum-localized enzyme that metabolizes the hydrogen peroxide produced by endoplasmic reticulum oxidase 1 (Ero1). It has been shown to play a role in de novo disulfide formation, oxidizing members of the protein disulfide isomerase family of enzymes, and is a member of the typical 2-Cys peroxiredoxin family. We have determined the crystal structure of both reduced and disulfide-bonded, as well as a resolving cysteine mutant of human PrxIV. We show that PrxIV has a similar structure to other typical 2-Cys peroxiredoxins and undergoes a conformational change from a fully folded to a locally unfolded form following the formation of a disulfide between the peroxidatic and resolving cysteine residues. Unlike other mammalian typical 2-Cys peroxiredoxins, we show that human PrxIV forms a stable decameric structure even in its disulfide-bonded state. In addition, the structure of a resolving cysteine mutant reveals an intermediate in the reaction cycle that adopts the locally unfolded conformation. Interestingly the peroxidatic cysteine in the crystal structure is sulfenylated rather than sulfinylated or sulfonylated. In addition, the peroxidatic cysteine in the resolving cysteine mutant is resistant to hyper-oxidation following incubation with high concentrations of hydrogen peroxide. These results highlight some unique properties of PrxIV and suggest that the equilibrium between the fully folded and locally unfolded forms favors the locally unfolded conformation upon sulfenylation of the peroxidatic cysteine residue. PMID:21994946

  5. Proteolysis approach without chemical modification for a simple and rapid analysis of disulfide bonds using thermostable protease-immobilized microreactors.

    PubMed

    Yamaguchi, Hiroshi; Miyazaki, Masaya; Maeda, Hideaki

    2010-08-01

    Disulfide bonds in proteins are important not only for the conformational stability of the protein but also for the regulation of oxidation-reduction in signal transduction. The conventional method for the assignment of disulfide bond by chemical cleavage and/or proteolysis is a time-consuming multi-step procedure. In this study, we report a simple and rapid analysis of disulfide bond from protein digests that were prepared by the thermostable protease-immobilized microreactors. The feasibility and performance of this approach were evaluated by digesting lysozyme and BSA at several temperatures. The proteins which stabilize their conformations by disulfide bonds were thermally denatured during proteolysis and were efficiently digested by the immobilized protease but not by free protease. The digests were directly analyzed by ESI-TOF MS without any purification or concentration step. All four disulfide bonds on lysozyme and 10 of 17 on BSA were assigned from the digests by the trypsin-immobilized microreactor at 50 degrees C. The procedure for proteolysis and the assignment were achieved within 2 h without any reduction and alkylation procedure. From the present results, the proteolysis approach by the thermostable protease-immobilized microreactor provides a strategy for the high-throughput analysis of disulfide bond in proteomics.

  6. Combined use of ion mobility and collision-induced dissociation to investigate the opening of disulfide bridges by electron-transfer dissociation in peptides bearing two disulfide bonds.

    PubMed

    Massonnet, Philippe; Upert, Gregory; Smargiasso, Nicolas; Gilles, Nicolas; Quinton, Loïc; De Pauw, Edwin

    2015-01-01

    Disulfide bonds are post-translational modifications (PTMs) often found in peptides and proteins. They increase their stability toward enzymatic degradations and provide the structure and (consequently) the activity of such folded proteins. The characterization of disulfide patterns, i.e., the cysteine connectivity, is crucial to achieve a global picture of the active conformation of the protein of interest. Electron-transfer dissociation (ETD) constitutes a valuable tool to cleave the disulfide bonds in the gas phase, avoiding chemical reduction/alkylation in solution. To characterize the cysteine pairing, the present work proposes (i) to reduce by ETD one of the two disulfide bridges of model peptides, resulting in the opening of the cyclic structures, (ii) to separate the generated species by ion mobility, and (iii) to characterize the species using collision-induced dissociation (CID). Results of this strategy applied to several peptides show different behaviors depending on the connectivity. The loss of SH· radical species, observed for all the peptides, confirms the cleavage of the disulfides during the ETD process.

  7. Maternal nicotine exposure leads to impaired disulfide bond formation and augmented endoplasmic reticulum stress in the rat placenta.

    PubMed

    Wong, Michael K; Nicholson, Catherine J; Holloway, Alison C; Hardy, Daniel B

    2015-01-01

    Maternal nicotine exposure has been associated with many adverse fetal and placental outcomes. Although underlying mechanisms remain elusive, recent studies have identified that augmented endoplasmic reticulum (ER) stress is linked to placental insufficiency. Moreover, ER function depends on proper disulfide bond formation--a partially oxygen-dependent process mediated by protein disulfide isomerase (PDI) and ER oxidoreductases. Given that nicotine compromised placental development in the rat, and placental insufficiency has been associated with poor disulfide bond formation and ER stress, we hypothesized that maternal nicotine exposure leads to both placental ER stress and impaired disulfide bond formation. To test this hypothesis, female Wistar rats received daily subcutaneous injections of either saline (vehicle) or nicotine bitartrate (1 mg/kg) for 14 days prior to mating and during pregnancy. Placentas were harvested on embryonic day 15 for analysis. Protein and mRNA expression of markers involved in ER stress (e.g., phosphorylated eIF2α, Grp78, Atf4, and CHOP), disulfide bond formation (e.g., PDI, QSOX1, VKORC1), hypoxia (Hif1α), and amino acid deprivation (GCN2) were quantified via Western blot and/or Real-time PCR. Maternal nicotine exposure led to increased expression of Grp78, phosphorylated eIF2α, Atf4, and CHOP (p<0.05) in the rat placenta, demonstrating the presence of augmented ER stress. Decreased expression of PDI and QSOX1 (p<0.05) reveal an impaired disulfide bond formation pathway, which may underlie nicotine-induced ER stress. Finally, elevated expression of Hif1α and GCN2 (p<0.05) indicate hypoxia and amino acid deprivation in nicotine-exposed placentas, respectively, which may also cause impaired disulfide bond formation and augmented ER stress. This study is the first to link maternal nicotine exposure with both placental ER stress and disulfide bond impairment in vivo, providing novel insight into the mechanisms underlying nicotine

  8. Disulfide bond bridge insertion turns hydrophobic anticancer prodrugs into self-assembled nanomedicines.

    PubMed

    Wang, Yongjun; Liu, Dan; Zheng, Qingchuan; Zhao, Qiang; Zhang, Hongjuan; Ma, Yan; Fallon, John K; Fu, Qiang; Haynes, Matthew T; Lin, Guimei; Zhang, Rong; Wang, Dun; Yang, Xinggang; Zhao, Linxiang; He, Zhonggui; Liu, Feng

    2014-10-08

    It is commonly observed that hydrophobic molecules alone cannot self-assemble into stable nanoparticles, requiring amphiphilic or ionic materials to support nanoparticle stability and function in vivo. We report herein newly self-assembled nanomedicines through entirely different mechanisms. We present proof-of-concept methodology and results in support of our hypothesis that disulfide-induced nanomedicines (DSINMs) are promoted and stabilized by the insertion of a single disulfide bond into hydrophobic molecules, in order to balance the competition between intermolecular forces involved in the self-assembly of nanomedicines. This hypothesis has been explored through diverse synthetic compounds, which include four first-line chemotherapy drugs (paclitaxel, doxorubicin, fluorouracil, and gemcitabine), two small-molecule natural products and their derivatives, as well as a fluorescent probe. Such an unprecedented and highly reproducible system has the potential to serve as a synthetic platform for a wide array of safe and effective therapeutic and diagnostic nanomedicine strategies.

  9. Disulfide Bond Bridge Insertion Turns Hydrophobic Anticancer Prodrugs into Self-Assembled Nanomedicines

    PubMed Central

    2015-01-01

    It is commonly observed that hydrophobic molecules alone cannot self-assemble into stable nanoparticles, requiring amphiphilic or ionic materials to support nanoparticle stability and function in vivo. We report herein newly self-assembled nanomedicines through entirely different mechanisms. We present proof-of-concept methodology and results in support of our hypothesis that disulfide-induced nanomedicines (DSINMs) are promoted and stabilized by the insertion of a single disulfide bond into hydrophobic molecules, in order to balance the competition between intermolecular forces involved in the self-assembly of nanomedicines. This hypothesis has been explored through diverse synthetic compounds, which include four first-line chemotherapy drugs (paclitaxel, doxorubicin, fluorouracil, and gemcitabine), two small-molecule natural products and their derivatives, as well as a fluorescent probe. Such an unprecedented and highly reproducible system has the potential to serve as a synthetic platform for a wide array of safe and effective therapeutic and diagnostic nanomedicine strategies. PMID:25188744

  10. Organic Matter Polymerization by Disulfide Bonding Near the Chemocline in Cariaco Basin

    NASA Astrophysics Data System (ADS)

    Raven, M. R.; Adkins, J. F.; Sessions, A. L.

    2013-12-01

    The preservation of organic carbon in sediments as kerogen is an essential pathway in the global carbon cycle, but the chemical reactions involved in kerogen formation remain poorly understood. Previous researchers have found that many sediments deposited under euxinic conditions contain sulfur-bearing non-polar lipids as well as disulfide bonds among lipid and carbohydrate monomers. It remains unclear, however, when during organic matter decomposition and diagenesis these different sulfur-bearing structures form, and how different environmental conditions affect the extent of organic matter sulfurization. We investigate organic sulfurization processes armed with a technique for measuring the sulfur-isotopic compositions of individual organosulfur compounds by coupled gas chromatography - inductively coupled plasma mass spectrometry. Organic compounds were extracted from sediments and water column sediment traps from Cariaco Basin, a euxinic basin in the Caribbean Sea. We measured the sulfur-isotopic compositions of both non-polar lipids and of derivatized disulfide-bound compounds from eight sediment trap profiles and a six-meter-long sediment core. In Cariaco Basin, lipid sulfurization processes appear to begin near the chemocline and continue in sediments on timescales of thousands of years. Slow diagenetic sulfurization in sediments produces lipid monomers with sulfur atoms in ring structures that are 34S-depleted relative to coexisting dissolved sulfide. Lipid monomers become progressively enriched in 34S over time, indicating ongoing formation coinciding with an increase in the amount of total sulfur in bulk kerogen. One of the most abundant monomers observed in Cariaco sediments, a phytol-related thiophene, is also produced intermittently near the chemocline. Phytol thiophene δ34S values in sediment traps are similar to those observed in shallow Cariaco sediments except during occasional ';enrichment events,' when phytol thiophene δ34S values increase to

  11. LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity.

    PubMed

    Krause, A; Neitz, S; Mägert, H J; Schulz, A; Forssmann, W G; Schulz-Knappe, P; Adermann, K

    2000-09-01

    We report the isolation and characterization of a novel human peptide with antimicrobial activity, termed LEAP-1 (liver-expressed antimicrobial peptide). Using a mass spectrometric assay detecting cysteine-rich peptides, a 25-residue peptide containing four disulfide bonds was identified in human blood ultrafiltrate. LEAP-1 expression was predominantly detected in the liver, and, to a much lower extent, in the heart. In radial diffusion assays, Gram-positive Bacillus megaterium, Bacillus subtilis, Micrococcus luteus, Staphylococcus carnosus, and Gram-negative Neisseria cinerea as well as the yeast Saccharomyces cerevisiae dose-dependently exhibited sensitivity upon treatment with synthetic LEAP-1. The discovery of LEAP-1 extends the known families of mammalian peptides with antimicrobial activity by its novel disulfide motif and distinct expression pattern.

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

    PubMed Central

    Kellett-Clarke, Helena; Stegmann, Monika; Barclay, A. Neil; Metcalfe, Clive

    2015-01-01

    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. PMID:26379032

  13. Collision-activated cleavage of a peptide/antibiotic disulfide linkage: possible evidence for intramolecular disulfide bond rearrangement upon collisional activation.

    PubMed

    Fagerquist, Clifton K

    2004-01-01

    Ceftiofur is an important veterinary beta-lactam antibiotic whose bioactive metabolite, desfuroylceftiofur, has a free thiol group. Desfuroylceftiofur (DFC) was reacted with two peptides, [Arg8]-vasopressin and reduced glutathione, both of which have cysteine residues to form disulfide-linked peptide/antibiotic complexes. The products of the reaction, [vasopressin + (DFC-H) + (DFC-H) + H]+, [(vasopressin+H) + (DFC-H) + H]+ and [(glutathione-H) + (DFC-H) + H]+, were analyzed using collision-activated dissociation (CAD) with a quadrupole ion trap tandem mass spectrometer. MS/MS of [vasopressin + (DFC-H) + (DFC-H) + H]+ resulted in facile dissociative loss of one and two covalently bound DFC moieties. Loss of one DFC resulted from either homolytic or heterolytic dissociation of the peptide/antibiotic disulfide bond with equal or unequal partitioning of the two sulfur atoms between the fragment ion and neutral loss. Hydrogen migration preceded heterolytic dissociation. Loss of two DFC moieties from [vasopressin + (DFC-H) + (DFC-H) + H]+ appears to result from collision-activated intramolecular disulfide bond rearrangement (IDBR) to produce cyclic [vasopressin + H]+ (at m/z 1084) as well as other cyclic fragment ions at m/z 1084 +/- 32 and +64. The cyclic structure of these ions could only be inferred as MS/MS may result in rearrangement to non-cyclic structures prior to dissociative loss. IDBR was also detected from MS(3) experiments of [vasopressin + (DFC-H) + (DFC-H) + H]+ fragment ions. MS/MS of [(glutathione-H) + (DFC-H) + H]+ resulted in cleavage of the peptide backbone with retention of the DFC moiety as well as heterolytic cleavage of the peptide/antibiotic disulfide bond to produce the fragment ion: [(DFC-2H) + H]+. These results demonstrate the facile dissociative loss by CAD of DFC moieties covalently attached to peptides through disulfide bonds. Published in 2004 by John Wiley & Sons, Ltd.

  14. Stability constraints and protein evolution: the role of chain length, composition and disulfide bonds.

    PubMed

    Bastolla, U; Demetrius, Lloyd

    2005-09-01

    Stability of the native state is an essential requirement in protein evolution and design. Here we investigated the interplay between chain length and stability constraints using a simple model of protein folding and a statistical study of the Protein Data Bank. We distinguish two types of stability of the native state: with respect to the unfolded state (unfolding stability) and with respect to misfolded configurations (misfolding stability). Several contributions to stability are evaluated and their correlations are disentangled through principal components analysis, with the following main results. (1) We show that longer proteins can fulfil more easily the requirements of unfolding and misfolding stability, because they have a higher number of native interactions per residue. Consistently, in longer proteins native interactions are weaker and they are less optimized with respect to non-native interactions. (2) Stability against misfolding is negatively correlated with the strength of native interactions, which is related to hydrophobicity. Hence there is a trade-off between unfolding and misfolding stability. This trade-off is influenced by protein length: less hydrophobic sequences are observed in very long proteins. (3) The number of disulfide bonds is positively correlated with the deficit of free energy stabilizing the native state. Chain length and the number of disulfide bonds per residue are negatively correlated in proteins with short chains and uncorrelated in proteins with long chains. (4) The number of salt bridges per residue and per native contact increases with chain length. We interpret these observations as an indication that the constraints imposed by unfolding stability are less demanding in long proteins and they are further reduced by the competing requirement for stability against misfolding. In particular, disulfide bonds appear to be positively selected in short proteins, whereas they evolve in an effectively neutral way in long proteins.

  15. The Pediocin PA-1 Accessory Protein Ensures Correct Disulfide Bond Formation in the Antimicrobial Peptide Pediocin PA-1.

    PubMed

    Oppegård, Camilla; Fimland, Gunnar; Anonsen, Jan Haug; Nissen-Meyer, Jon

    2015-05-19

    Peptides, in contrast to proteins, are generally not large enough to form stable and well-defined three-dimensional structures. However, peptides are still able to form correct disulfide bonds. Using pediocin-like bacteriocins, we have examined how this may be achieved. Some pediocin-like bacteriocins, such as pediocin PA-1 and sakacin P[N24C+44C], have four cysteines. There are three possible ways by which the four cysteines may combine to form two disulfide bonds, and the three variants are expected to be produced in approximately equal amounts if their formation is random. Pediocin PA-1 and sakacin P[N24C+44C] with correct disulfide bonds were the main products when they were secreted by the pediocin PA-1 ABC transporter and accessory protein, but when they were secreted by the corresponding secretion machinery for sakacin A, a pediocin-like bacteriocin with one disulfide bond (two cysteines), peptides with all three possible disulfide bonds were produced in approximately equal amounts. All five cysteines in the pediocin PA-1 ABC transporter and the two cysteines (that form a CxxC motif) in the accessory protein were individually replaced with serines to examine their involvement in disulfide bond formation in pediocin PA-1. The Cys86Ser mutation in the accessory protein caused a 2-fold decrease in the amount of pediocin PA-1 with correct disulfide bonds, while the Cys83Ser mutation nearly abolished the production of pediocin PA-1 and resulted in the production of all three disufide bond variants in equal amounts. The Cys19Ser mutation in the ABC transporter completely abolished secretion of pediocin PA-1, suggesting that Cys19 is in the proteolytic active site and involved in cleaving the prebacteriocin. Replacing the other four cysteines in the ABC transporter with serines caused a slight reduction in the overall amount of secreted pediocin PA-1, but the relative amount with the correct disulfide bonds remained large. These results indicate that the pediocin

  16. Overexpression of the rhodanese PspE, a single cysteine-containing protein, restores disulfide bond formation to an Escherichia coli strain lacking DsbA

    PubMed Central

    Chng, Shu-Sin; Dutton, Rachel J.; Denoncin, Katleen; Vertommen, Didier; Collet, Jean-Francois; Kadokura, Hiroshi; Beckwith, Jonathan

    2012-01-01

    Summary Escherichia coli uses the DsbA/DsbB system for introducing disulfide bonds into proteins in the cell envelope. Deleting either dsbA or dsbB or both reduces disulfide bond formation but does not entirely eliminate it. Whether such background disulfide bond forming activity is enzyme-catalyzed is not known. To identify possible cellular factors that might contribute to the background activity, we studied the effects of overexpressing endogenous proteins on disulfide bond formation in the periplasm. We find that overexpressing PspE, a periplasmic rhodanese, partially restores substantial disulfide bond formation to a dsbA strain. This activity depends on DsbC, the bacterial disulfide bond isomerase, but not on DsbB. We show that overexpressed PspE is oxidized to the sulfenic acid form and reacts with substrate proteins to form mixed disulfide adducts. DsbC either prevents the formation of these mixed disulfides or resolves these adducts subsequently. In the process, DsbC itself gets oxidized and proceeds to catalyze disulfide bond formation. Although this PspE/DsbC system is not responsible for the background disulfide bond forming activity, we suggest that it might be utilized in other organisms lacking the DsbA/DsbB system. PMID:22809289

  17. Chitosan films with improved tensile strength and toughness from N-acetyl-cysteine mediated disulfide bonds.

    PubMed

    Miles, Kevin Barrett; Ball, Rebecca Lee; Matthew, Howard William Trevor

    2016-03-30

    To improve the mechanical properties of chitosan (Ct) materials without the use of cytotoxic crosslinkers, disulfide cross-linkable Ct was synthesized by grafting N-acetyl-cysteine (NAC) to Ct using carbodiimide chemistry. Cast films of NAC-Ct conjugates were prepared with degrees of substitution (DS) of 0%, 6%, 15%, and 20%, and the disulfide bond formation was induced by increasing the reaction media pH to 11. The tensile strength, breaking strain, elastic moduli and toughness of disulfide cross-linked polymers were analyzed by monotonic tensile testing of hydrated NAC-Ct films. Crystallinity was determined via XRD. Results demonstrated that NAC incorporation and crosslinking in chitosan produced tougher polymer films with 4-fold higher tensile strength (10 MPa) and 6-fold greater elongation (365%), but reduced crystallinity, compared to unmodified chitosan. The resilience of NAC-Ct films was evaluated by cyclic testing, and results demonstrate that increasing NAC content produced a more resilient material that dissipated less energy when deformed. These improved mechanical properties broaden chitosan's applicability towards the construction of mechanically robust implantable scaffolds for tissue regeneration.

  18. Coupling of disulfide bond and distal histidine dissociation in human ferrous cytoglobin regulates ligand binding.

    PubMed

    Beckerson, Penny; Reeder, Brandon J; Wilson, Michael T

    2015-02-13

    Earlier kinetics studies on cytoglobin did not assign functional properties to specific structural forms. Here, we used defined monomeric and dimeric forms and cysteine mutants to show that an intramolecular disulfide bond (C38-C83) alters the dissociation rate constant of the intrinsic histidine (H81) (∼1000 fold), thus controlling binding of extrinsic ligands. Through time-resolved spectra we have unequivocally assigned CO binding to hexa- and penta-coordinate forms and have made direct measurement of histidine rebinding following photolysis. We present a model that describes how the cysteine redox state of the monomer controls histidine dissociation rate constants and hence extrinsic ligand binding.

  19. Additive for iron disulfide cathodes used in thermal batteries

    DOEpatents

    Not Available

    1982-03-23

    The invention comprises thermal batteries employing an FeS/sub 2/ depolarizer itself. A minor amount of CaSi/sub 2/ preferably 1-3% by weight is provided as an additive in the FeS/sub 2/ depolarizer to eliminate the voltage transient (spike) which normally occurs upon activation of batteries of this type. The amount of FeS/sub 2/ by weight generally comprises 64 to 90%.

  20. Additive for iron disulfide cathodes used in thermal batteries

    DOEpatents

    Armijo, James R.; Searcy, Jimmie Q.

    1983-01-01

    The invention comprises thermal batteries employing an FeS.sub.2 depolarizer, i.e. cathode material, and the depolarizer itself. A minor amount of CaSi.sub.2 preferably, 1-3% by weight is provided as an additive in the FeS.sub.2 depolarizer to eliminate the voltage transient (spike) which normally occurs upon activation of batteries of this type. The amount of FeS.sub.2 by weight generally comprises 64-90%.

  1. Enhancement of gene delivery using novel homodimeric tat peptide formed by disulfide bond.

    PubMed

    Lee, Soo-Jin; Yoon, Sung-Hwa; Doh, Kyung-Oh

    2011-08-01

    Cationic liposomes have been actively used as gene delivery vehicle because of their minimal toxicity, but their relatively low efficiency of gene delivery is the major disadvantage of these vectors. Recently, cysteine residue incorporation to HIV-1 Tat peptide increased liposomemediated transfection compared with unmodified Tat peptide. Therefore, we designed a novel modified Tat peptide having a homodimeric (Tat-CTHD, Tat-NTHD) and closed structure (cyclic Tat) simply by using the disulfide bond between cysteines to develop a more efficient and safe nonviral gene delivery system. The mixing of Tat-CTHD and Tat-NTHD with DNA before mixing with lipofectamine increased the transfection efficiency compared with unmodified Tat peptide and lipofectamine only in MCF-7 breast cancer cells and rat vascular smooth muscle cells. However, cyclic Tat did not show any improvement in the transfection efficiency. In the gel retardation assay, Tat-CTHD and Tat-NTHD showed more strong binding with DNA than unmodified Tat and cyclic Tat peptide. This enhancement was only shown when Tat-CTHD and Tat-NTHD were mixed with DNA before mixing with lipofectamine. The effects of Tat- CTHD and Tat-NTHD were also valid in the experiment using DOTAP and DMRIE instead of lipofectamine. We could not find any significant cytotoxicity in the working concentration and more usage of these peptides. In conclusion, we have designed a novel transfection-enhancing peptide by easy homodimerization of Tat peptide, and the simple mix of these novel peptides with DNA increased the gene transfer of cationic lipids more efficiently with no additional cytotoxicity.

  2. Native disulfide bonds in plasma retinol-binding protein are not essential for all-trans-retinol-binding activity.

    PubMed

    Reznik, Gabriel O; Yu, Yong; Tarr, George E; Cantor, Charles R

    2003-01-01

    A human plasma retinol-binding protein (RBP) mutant, named RBP-S, has been designed and produced in which the six native cysteine residues, involved in the formation of three disulfide bonds, have been replaced with serine. A hexa-histidine tag was also added to the C-terminus of RBP for ease of purification. The removal of the disulfide bonds led to a decrease in the affinity of RBP for all trans-retinol. Data indicates all-trans-retinol binds RBP and RBP-S with Kd = 4 x 10(-8) M and 1 x 10(-7) M, respectively, at approximately 20 degrees C. RBP-S has reduced stability as compared to natural RBP below pH 8.0 and at room temperature. Circular dichroism in the far-UV shows that there is a relaxation of the RBP structure upon the removal of its disulfide bonds. Circular dichroism in the near-UV shows that in the absence of the disulfide bonds, the optical activity of RBP is higher in the 310-330 nm than in the 280-290 nm range. This work suggests that the three native disulfide bonds aid in the folding of RBP but are not essential to produce a soluble, active protein.

  3. Characterization of Sviceucin from Streptomyces Provides Insight into Enzyme Exchangeability and Disulfide Bond Formation in Lasso Peptides.

    PubMed

    Li, Yanyan; Ducasse, Rémi; Zirah, Séverine; Blond, Alain; Goulard, Christophe; Lescop, Ewen; Giraud, Caroline; Hartke, Axel; Guittet, Eric; Pernodet, Jean-Luc; Rebuffat, Sylvie

    2015-11-20

    Lasso peptides are bacterial ribosomally synthesized and post-translationally modified peptides. They have sparked increasing interest in peptide-based drug development because of their compact, interlocked structure, which offers superior stability and protein-binding capacity. Disulfide bond-containing lasso peptides are rare and exhibit highly sought-after activities. In an effort to expand the repertoire of such molecules, we heterologously expressed, in Streptomyces coelicolor, the gene cluster encoding sviceucin, a type I lasso peptide with two disulfide bridges originating from Streptomyces sviceus, which allowed it to be fully characterized. Sviceucin and its reduced forms were characterized by mass spectrometry and peptidase digestion. The three-dimensional structure of sviceucin was determined using NMR. Sviceucin displayed antimicrobial activity selectively against Gram-positive bacteria and inhibition of fsr quorum sensing in Enterococcus faecalis. This study adds sviceucin to the type I lasso peptide family as a new representative. Moreover, new clusters encoding disulfide-bond containing lasso peptides from Actinobacteria were identified by genome mining. Genetic and functional analyses revealed that the formation of disulfide bonds in sviceucin does not require a pathway-encoded thiol-disulfide oxidoreductase. Most importantly, we demonstrated the functional exchangeability of the sviceucin and microcin J25 (a non-disulfide-bridged lasso peptide) macrolactam synthetases in vitro, highlighting the potential of hybrid lasso synthetases in lasso peptide engineering.

  4. Disulfide-Dependent Self-Assembly of Adiponectin Octadecamers from Trimers and Presence of Stable Octadecameric Adiponectin Lacking Disulfide Bonds In Vitro†

    PubMed Central

    Briggs, David B.; Jones, Christopher M.; Mashalidis, Ellene H.; Nuñez, Martha; Hausrath, Andrew C.; Wysocki, Vicki H.; Tsao, Tsu-Shuen

    2009-01-01

    Adiponectin is a circulating insulin-sensitizing hormone that homo-oligomerizes into trimers, hexamers, and higher molecular weight (HMW) species. Low levels of circulating HMW adiponectin appear to increase the risk for insulin resistance. Currently, assembly of adiponectin oligomers, and consequently mechanisms responsible for decreased HMW adiponectin in insulin resistance, are not well understood. In the work reported here, we analyzed the re-assembly of the most abundant HMW adiponectin species, the octadecamer, following its collapse to smaller oligomers in vitro. Purified bovine serum adiponectin octadecamer was treated with reducing agents at pH 5 to obtain trimers. These reduced trimers partially and spontaneously reassembled into octadecamers upon oxidative formation of disulfide bonds. Disulfide bonds appear to occupy a greater role in the process of oligomerization than in the structural stabilization of mature octadecamer. Stable octadecamers lacking virtually all disulfide bonds could be observed in abundance using native gel electrophoresis, dynamic light scattering, and collision-induced dissociation nano-electrospray ionization mass spectrometry. These findings indicate that while disulfide bonds help to maintain the mature octadecameric adiponectin structure, their more important function is to stabilize intermediates during the assembly of octadecamer. Adiponectin oligomerization must proceed through intermediates that are at least partially reduced. Accordingly, fully oxidized adiponectin hexamers failed to reassemble into octadecamers at a rate comparable to that of reduced trimers. As the findings from the present study are based on in vitro experiments, their in vivo relevance remains unclear. Nevertheless, they describe a redox environment-dependent model of adiponectin oligomerization that can be tested using cell-based approaches. PMID:19943704

  5. Challenges in the expression of disulfide bonded, threonine-rich antifreeze proteins in bacteria and yeast.

    PubMed

    Tyshenko, Michael G; d'Anjou, Marc; Davies, Peter L; Daugulis, Andrew J; Walker, Virginia K

    2006-05-01

    Certain freeze-intolerant insects produce antifreeze proteins (AFPs) during overwintering including the spruce budworm (Choristoneura fumiferana) and yellow mealworm (Tenebrio molitor) AFP gene families. However, only a few of the isoforms, encoded by their multiple-copy gene families, have been characterized. When expressed in bacterial systems the insect AFPs have to be denatured and refolded in vitro, a procedure that is not uniformly successful, presumably due to the beta-helix structure and the requirement for disulfide bonds. In an attempt to overcome these difficulties, bacterial vectors and hosts that have been developed to produce soluble, folded proteins, as well as a yeast expression system (Pichia pastoris) were employed. Bacterial expression resulted in low quantities of active recombinant protein for certain isoforms. In contrast, both small and large-scale fermentation of recombinant AFP in Pichia yielded substantial protein production (100 mg/L) but functional ice binding activity of protein produced in three different transformed yeast strains (KM71, X33 or GS115) was low. Inappropriate O-linked glycosylation of the Thr-rich AFPs appeared to be partially reversed by mild chemical deglycosylation, but activity remained low. Substantial quantities, as well as activity were recovered when a fish AFP, with disulfide bonds, but without potential Thr glycosylation sites was expressed in the yeast system.

  6. Thermal unfolding of nucleoside hydrolases from the hyperthermophilic archaeon Sulfolobus solfataricus: role of disulfide bonds.

    PubMed

    Porcelli, Marina; De Leo, Ester; Del Vecchio, Pompea; Fuccio, Francesca; Cacciapuoti, Giovanna

    2012-03-01

    Nucleoside hydrolases are metalloproteins that hydrolyze the N-glycosidic bond of β-ribonucleosides, forming the free purine/pyrimidine base and ribose. We report the stability of the two hyperthermophilic enzymes Sulfolobus solfataricus pyrimidine-specific nucleoside hydrolase (SsCU-NH) and Sulfolobus solfataricus purine-specific inosineadenosine- guanosine nucleoside hydrolase (SsIAG-NH) against the denaturing action of temperature and guanidine hydrochloride by means of circular dichroism and fluorescence spectroscopy. The guanidine hydrochloride-induced unfolding is reversible for both enzymes as demonstrated by the analysis of the refolding process by activity assays and fluorescence measurements. The evidence that the denaturation of SsIAG-NH carried out in the presence of reducing agents proved to be reversible indicates that the presence of disulfide bonds interferes with the refolding process of this enzyme. Both enzymes are highly thermostable and no thermal unfolding transition can be obtained up to 108°C. SsIAG-NH is thermally denatured under reducing conditions (T(m)=93°C) demonstrating the contribution of disulfide bridges to enzyme thermostability.

  7. A disulfide bond in the TIM23 complex is crucial for voltage gating and mitochondrial protein import

    PubMed Central

    Peleh, Valentina; Martinez-Caballero, Sonia; Sommer, Frederik; van der Laan, Martin; Schroda, Michael

    2016-01-01

    Tim17 is a central, membrane-embedded subunit of the mitochondrial protein import machinery. In this study, we show that Tim17 contains a pair of highly conserved cysteine residues that form a structural disulfide bond exposed to the intermembrane space (IMS). This disulfide bond is critical for efficient protein translocation through the TIM23 complex and for dynamic gating of its preprotein-conducting channel. The disulfide bond in Tim17 is formed during insertion of the protein into the inner membrane. Whereas the import of Tim17 depends on the binding to the IMS protein Mia40, the oxidoreductase activity of Mia40 is surprisingly dispensable for Tim17 oxidation. Our observations suggest that Tim17 can be directly oxidized by the sulfhydryl oxidase Erv1. Thus, import and oxidation of Tim17 are mediated by the mitochondrial disulfide relay, though the mechanism by which the disulfide bond in Tim17 is formed differs considerably from that of soluble IMS proteins. PMID:27502485

  8. A disulfide bond in the TIM23 complex is crucial for voltage gating and mitochondrial protein import.

    PubMed

    Ramesh, Ajay; Peleh, Valentina; Martinez-Caballero, Sonia; Wollweber, Florian; Sommer, Frederik; van der Laan, Martin; Schroda, Michael; Alexander, R Todd; Campo, María Luisa; Herrmann, Johannes M

    2016-08-15

    Tim17 is a central, membrane-embedded subunit of the mitochondrial protein import machinery. In this study, we show that Tim17 contains a pair of highly conserved cysteine residues that form a structural disulfide bond exposed to the intermembrane space (IMS). This disulfide bond is critical for efficient protein translocation through the TIM23 complex and for dynamic gating of its preprotein-conducting channel. The disulfide bond in Tim17 is formed during insertion of the protein into the inner membrane. Whereas the import of Tim17 depends on the binding to the IMS protein Mia40, the oxidoreductase activity of Mia40 is surprisingly dispensable for Tim17 oxidation. Our observations suggest that Tim17 can be directly oxidized by the sulfhydryl oxidase Erv1. Thus, import and oxidation of Tim17 are mediated by the mitochondrial disulfide relay, though the mechanism by which the disulfide bond in Tim17 is formed differs considerably from that of soluble IMS proteins.

  9. On-Line Electrochemical Reduction of Disulfide Bonds: Improved FTICR-CID and -ETD Coverage of Oxytocin and Hepcidin

    NASA Astrophysics Data System (ADS)

    Nicolardi, Simone; Giera, Martin; Kooijman, Pieter; Kraj, Agnieszka; Chervet, Jean-Pierre; Deelder, André M.; van der Burgt, Yuri E. M.

    2013-12-01

    Particularly in the field of middle- and top-down peptide and protein analysis, disulfide bridges can severely hinder fragmentation and thus impede sequence analysis (coverage). Here we present an on-line/electrochemistry/ESI-FTICR-MS approach, which was applied to the analysis of the primary structure of oxytocin, containing one disulfide bridge, and of hepcidin, containing four disulfide bridges. The presented workflow provided up to 80 % (on-line) conversion of disulfide bonds in both peptides. With minimal sample preparation, such reduction resulted in a higher number of peptide backbone cleavages upon CID or ETD fragmentation, and thus yielded improved sequence coverage. The cycle times, including electrode recovery, were rapid and, therefore, might very well be coupled with liquid chromatography for protein or peptide separation, which has great potential for high-throughput analysis.

  10. Glutathione and thioredoxin systems contribute to recombinant monoclonal antibody interchain disulfide bond reduction during bioprocessing.

    PubMed

    Handlogten, Michael W; Zhu, Min; Ahuja, Sanjeev

    2017-03-06

    Antibody interchain disulfide bond reduction during biopharmaceutical manufacturing has received increased attention since it was first reported in 2010. Antibody reduction leads to loss of product and reduced product stability. It is therefore critical to understand the underlying mechanisms of reduction. To date, the thioredoxin system has been reported as the sole contributor to antibody reduction during bioprocessing. In this work, we show that the glutathione system, in addition to the thioredoxin system, is involved in reducing antibody molecules and the contributions of the two systems can vary depending upon the cell culture process. The roles of the glutathione and thioredoxin systems were evaluated for three molecules with different IgG subclass where reduction was observed during manufacturing: mAb A, mAb B, and mAb C representing an IgG1 , IgG2 , and IgG4, respectively. The expression of enzymes for both the thioredoxin and glutathione systems were confirmed in all three cell lines. Inhibitors were evaluated using purified mammalian reductases to evaluate their specificity. The optimized experimental conditions enabled both the determination of reductase activity contributed from as well as the amount of antibody reduced by each enzymatic system. Our results demonstrate that the underlying enzymatic mechanisms are different depending upon the cell culture process; one of the two systems may be the dominant mechanism, or both enzymatic systems may be involved. Specifically, the glutathione system was found to be the major contributor to mAb A reduction while the thioredoxin system was the major contributor to mAb C reduction. Intriguingly, mAb B experienced significant reduction from both enzymatic systems. In summary, we have demonstrated that in addition to the thioredoxin pathway, the glutathione system is a second major pathway contributing to antibody reduction and this knowledge can be leveraged to develop more specific antibody reduction

  11. Radically different thioredoxin domain arrangement of ERp46, an efficient disulfide bond introducer of the mammalian PDI family.

    PubMed

    Kojima, Rieko; Okumura, Masaki; Masui, Shoji; Kanemura, Shingo; Inoue, Michio; Saiki, Masatoshi; Yamaguchi, Hiroshi; Hikima, Takaaki; Suzuki, Mamoru; Akiyama, Shuji; Inaba, Kenji

    2014-03-04

    The mammalian endoplasmic reticulum (ER) contains a diverse oxidative protein folding network in which ERp46, a member of the protein disulfide isomerase (PDI) family, serves as an efficient disulfide bond introducer together with Peroxiredoxin-4 (Prx4). We revealed a radically different molecular architecture of ERp46, in which the N-terminal two thioredoxin (Trx) domains with positively charged patches near their peptide-binding site and the C-terminal Trx are linked by unusually long loops and arranged extendedly, forming an opened V-shape. Whereas PDI catalyzes native disulfide bond formation by the cooperative action of two mutually facing redox-active sites on folding intermediates bound to the central cleft, ERp46 Trx domains are separated, act independently, and engage in rapid but promiscuous disulfide bond formation during early oxidative protein folding. Thus, multiple PDI family members likely contribute to different stages of oxidative folding and work cooperatively to ensure the efficient production of multi-disulfide proteins in the ER.

  12. Characterization of an alternative low energy fold for bovine α-lactalbumin formed by disulfide bond shuffling.

    PubMed

    Lewney, Sarah; Smith, Lorna J

    2012-03-01

    Bovine α-lactalbumin (αLA) forms a misfolded disulfide bond shuffled isomer, X-αLA. This X-αLA isomer contains two native disulfide bridges (Cys 6-Cys 120 and Cys 28-Cys 111) and two non-native disulfide bridges (Cys 61-Cys 73 and Cys 77-Cys 91). MD simulations have been used to characterize the X-αLA isomer and its formation via disulfide bond shuffling and to compare it with the native fold of αLA. In the simulations of the X-αLA isomer the structure of the α-domain of native αLA is largely retained in agreement with experimental data. However, there are significant rearrangements in the β-domain, including the loss of the native β-sheet and calcium binding site. Interestingly, the energies of X-αLA and native αLA in simulations in the absence of calcium are closely similar. Thus, the X-αLA isomer represents a different low energy fold for the protein. Calcium binding to native αLA is shown to help preserve the structure of the β-domain of the protein limiting possibilities for disulfide bond shuffling. Hence, binding calcium plays an important role in both maintaining the native structure of αLA and providing a mechanism for distinguishing between folded and misfolded species.

  13. Structure-activity relationships of the intramolecular disulfide bonds in coprisin, a defensin from the dung beetle

    PubMed Central

    Lee, Daeun; Choi, Hyemin; Kim, Ha Hyung; Kim, Ho; Hwang, Jae Sam; Lee, Dong Gun; Kim, Jae Il

    2014-01-01

    Defensins, which are small cationic molecules produced by organisms as part of their innate immune response, share a common structural scaffold that is stabilized by three disulfide bridges. Coprisin is a 43-amino acid defensin-like peptide from Copris tripartitus. Here, we report the intramolecular disulfide connectivity of cysteine-rich coprisin, and show that it is the same as in other insect defensins. The disulfide bond pairings of coprisin were determined by combining the enzymatic cleavage and mass analysis. We found that the loss of any single disulfide bond in coprisin eliminated all antibacterial, but not antifungal, activity. Circular dichroism (CD) analysis showed that two disulfide bonds, Cys20-Cys39 and Cys24-Cys41, stabilize coprisin’s α-helical region. Moreover, a BLAST search against UniProtKB database revealed that coprisin’s α-helical region is highly homologous to those of other insect defensins. [BMB Reports 2014; 47(11): 625-630] PMID:24393527

  14. Human Islet Amyloid Polypeptide N-Terminus Fragment Self-Assembly: Effect of Conserved Disulfide Bond on Aggregation Propensity

    NASA Astrophysics Data System (ADS)

    Ilitchev, Alexandre I.; Giammona, Maxwell J.; Do, Thanh D.; Wong, Amy G.; Buratto, Steven K.; Shea, Joan-Emma; Raleigh, Daniel P.; Bowers, Michael T.

    2016-06-01

    Amyloid formation by human islet amyloid polypeptide (hIAPP) has long been implicated in the pathogeny of type 2 diabetes mellitus (T2DM) and failure of islet transplants, but the mechanism of IAPP self-assembly is still unclear. Numerous fragments of hIAPP are capable of self-association into oligomeric aggregates, both amyloid and non-amyloid in structure. The N-terminal region of IAPP contains a conserved disulfide bond between cysteines at position 2 and 7, which is important to hIAPP's in vivo function and may play a role in in vitro aggregation. The importance of the disulfide bond in this region was probed using a combination of ion mobility-based mass spectrometry experiments, molecular dynamics simulations, and high-resolution atomic force microscopy imaging on the wildtype 1-8 hIAPP fragment, a reduced fragment with no disulfide bond, and a fragment with both cysteines at positions 2 and 7 mutated to serine. The results indicate the wildtype fragment aggregates by a different pathway than either comparison peptide and that the intact disulfide bond may be protective against aggregation due to a reduction of inter-peptide hydrogen bonding.

  15. Reversible protection of disulfide bonds followed by oxidative folding render recombinant hCGbeta highly immunogenic.

    PubMed

    Mukhopadhyay, A

    2000-03-06

    Active immunization of women against human chorionic gonadotropin (hCG) has been considered as a promising option for contraception. However, prototype hCG vaccines based on natural sources of antigen are expected to be costlier for use by common people. In the present report, a functionally active, cost-effective antigen of bacterial origin has been described. Sulfonation of thiol groups of the protein, anion-exchange purification, refolding with concomitant formation of disulfide bonds in the presence of cysteamine-cystamine redox buffer, and slow removal of denaturant resulted in 95% homogeneous, monomeric form of the antigen. The recombinant processed antigen [CGbeta(p)] obtained this way was highly immunopotent. Cellular DNA and endotoxin contaminants were appreciably low in the final product. The immunogenic response was drastically reduced with the unprocessed antigen. This finding envisages better prospect of a cost-effective hCG vaccine for birth control.

  16. A Disulfide Bond Is Required for the Transmission of Forces through SUN-KASH Complexes

    PubMed Central

    Jahed, Zeinab; Shams, Hengameh; Mofrad, Mohammad R.K.

    2015-01-01

    Numerous biological functions of a cell, including polarization, differentiation, division, and migration, rely on its ability to endure mechanical forces generated by the cytoskeleton on the nucleus. Coupling of the cytoskeleton and nucleoskeleton is ultimately mediated by LINC complexes that are formed via a strong interaction between SUN- and KASH-domain-containing proteins in the nuclear envelope. These complexes are mechanosensitive and essential for the transmission of forces between the cytoskeleton and nucleoskeleton, and the progression of cellular mechanotransduction. Herein, using molecular dynamics, we examine the effect of tension on the human SUN2-KASH2 complex and show that it is remarkably stable under physiologically relevant tensile forces and large strains. However, a covalent disulfide bond between two highly conserved cysteine residues of SUN2 and KASH2 is crucial for the stability of this interaction and the transmission of forces through the complex. PMID:26244732

  17. Transmembrane domain interactions and residue proline 378 are essential for proper structure, especially disulfide bond formation, in the human vitamin K-dependent gamma-glutamyl carboxylase.

    PubMed

    Tie, Jian-Ke; Zheng, Mei-Yan; Hsiao, Kuang-Ling N; Perera, Lalith; Stafford, Darrel W; Straight, David L

    2008-06-17

    We used recombinant techniques to create a two-chain form (residues 1-345 and residues 346-758) of the vitamin K-dependent gamma-glutamyl carboxylase, a glycoprotein located in the endoplasmic reticulum containing five transmembrane domains. The two-chain carboxylase had carboxylase and epoxidase activities similar to those of one-chain carboxylase. In addition, it had normal affinity for the propeptide of factor IX. We employed this molecule to investigate formation of the one disulfide bond in carboxylase, the transmembrane structure of carboxylase, and the potential interactions among the carboxylase's transmembrane domains. Our results indicate that the two peptides of the two-chain carboxylase are joined by a disulfide bond. Proline 378 is important for the structure necessary for disulfide formation. Results with the P378L carboxylase indicate that noncovalent bonds maintain the two-chain structure even when the disulfide bond is disrupted. As we had previously proposed, the fifth transmembrane domain of carboxylase is the last and only transmembrane domain in the C-terminal peptide of the two-chain carboxylase. We show that the noncovalent association between the two chains of carboxylase involves an interaction between the fifth transmembrane domain and the second transmembrane domain. Results of a homology model of transmembrane domains 2 and 5 suggest that not only do these two domains associate but that transmembrane domain 2 may interact with another transmembrane domain. This latter interaction may be mediated at least in part by a motif of glycine residues in the second transmembrane domain.

  18. Disruption of disulfide bond formation alters the trafficking of prothyrotropin releasing hormone (proTRH)-derived peptides.

    PubMed

    Mulcahy, Lawrence R; Barker, Alison J; Nillni, Eduardo A

    2006-01-15

    Rat prothyrotropin releasing hormone (proTRH) is processed in the regulated secretory pathway (RSP) of neuroendocrine cells yielding five TRH peptides and several non-TRH peptides. It is not understood how these peptides are targeted to the RSP. We show here that a disulfide bond in the carboxy-terminus of proTRH plays an important role in the trafficking of this prohormone. Recombinant proTRH was observed to migrate faster on a native gel when treated with dithiothreitol (DTT) suggesting the presence of a disulfide bond. In vitro disulfide bond formation was prevented either by DTT treatment or by mutating cysteines 213 and 219 to glycines. In both cases the peptides derived from these mutants exhibited increased constitutive release and processing defects when expressed in AtT20 cells, a neuroendocrine cell line used in our prior studies on proTRH processing. Immunocytochemistry revealed that wild-type proTRH and mutant proTRH localized in a punctate pattern typical of proteins sorted to the regulated secretory pathway. These data suggest that the proposed disulfide bond of proTRH is involved in sorting of proTRH-derived peptides and in their retention within maturing secretory granules. This is the first evidence of structural motifs being important for the sorting of proTRH.

  19. A dielectric barrier discharge terminally inactivates RNase A by oxidizing sulfur-containing amino acids and breaking structural disulfide bonds

    NASA Astrophysics Data System (ADS)

    Lackmann, J.-W.; Baldus, S.; Steinborn, E.; Edengeiser, E.; Kogelheide, F.; Langklotz, S.; Schneider, S.; Leichert, L. I. O.; Benedikt, J.; Awakowicz, P.; Bandow, J. E.

    2015-12-01

    RNases are among the most stable proteins in nature. They even refold spontaneously after heat inactivation, regaining full activity. Due to their stability and universal presence, they often pose a problem when experimenting with RNA. We investigated the capabilities of nonthermal atmospheric-pressure plasmas to inactivate RNase A and studied the inactivation mechanism on a molecular level. While prolonged heating above 90 °C is required for heat inactivating RNase A, direct plasma treatment with a dielectric barrier discharge (DBD) source caused permanent inactivation within minutes. Circular dichroism spectroscopy showed that DBD-treated RNase A unfolds rapidly. Raman spectroscopy indicated methionine modifications and formation of sulfonic acid. A mass spectrometry-based analysis of the protein modifications that occur during plasma treatment over time revealed that methionine sulfoxide formation coincides with protein inactivation. Chemical reduction of methionine sulfoxides partially restored RNase A activity confirming that sulfoxidation is causal and sufficient for RNase A inactivation. Continued plasma exposure led to over-oxidation of structural disulfide bonds. Using antibodies, disulfide bond over-oxidation was shown to be a general protein inactivation mechanism of the DBD. The antibody’s heavy and light chains linked by disulfide bonds dissociated after plasma exposure. Based on their ability to inactivate proteins by oxidation of sulfur-containing amino acids and over-oxidation of disulfide bonds, DBD devices present a viable option for inactivating undesired or hazardous proteins on heat or solvent-sensitive surfaces.

  20. Effects of designed sulfhydryl groups and disulfide bonds into soybean proglycinin on its structural stability and heat-induced gelation.

    PubMed

    Adachi, Motoyasu; Chunying, Ho; Utsumi, Shigeru

    2004-09-08

    The gel-forming ability of glycinin is one of soybean's most important functional properties. The proglycinin A1aB1b homotrimer was engineered to introduce sulfhydryl groups and disulfide bonds, and their effects on the structural stability and the heat-induced gelation were evaluated. On the basis of the crystal structure, five mutants were designed and prepared: R161C and F163C forming an interprotomer disulfide bond with the inherent free cysteine residue of Cys377, N116C/P248C forming a new intraprotomer disulfide bond, and N116C and P248C introducing a new sulfhydryl group. Mutants of R161C, F163C, and N116C/P248C formed a new disulfide bond as expected. N116C/P248C was significantly more stable than the wild type against chemical and thermal denaturation and more resistant to alpha-chymotrypsin digestion, whereas F163C showed significantly increased thermal stability. All mutants exhibited greater hardness of heat-induced gels than wild type, and in particular, N116C/P248C gave the hardest gel. This result indicates that it is possible to increase hardness of glycinin gel by introduction of cysteine residues using protein engineering.

  1. Lysine Decarboxylase with an Enhanced Affinity for Pyridoxal 5-Phosphate by Disulfide Bond-Mediated Spatial Reconstitution

    PubMed Central

    Sagong, Hye-Young; Kim, Kyung-Jin

    2017-01-01

    Lysine decarboxylase (LDC) catalyzes the decarboxylation of l-lysine to produce cadaverine, an important industrial platform chemical for bio-based polyamides. However, due to high flexibility at the pyridoxal 5-phosphate (PLP) binding site, use of the enzyme for cadaverine production requires continuous supplement of large amounts of PLP. In order to develop an LDC enzyme from Selenomonas ruminantium (SrLDC) with an enhanced affinity for PLP, we introduced an internal disulfide bond between Ala225 and Thr302 residues with a desire to retain the PLP binding site in a closed conformation. The SrLDCA225C/T302C mutant showed a yellow color and the characteristic UV/Vis absorption peaks for enzymes with bound PLP, and exhibited three-fold enhanced PLP affinity compared with the wild-type SrLDC. The mutant also exhibited a dramatically enhanced LDC activity and cadaverine conversion particularly under no or low PLP concentrations. Moreover, introduction of the disulfide bond rendered SrLDC more resistant to high pH and temperature. The formation of the introduced disulfide bond and the maintenance of the PLP binding site in the closed conformation were confirmed by determination of the crystal structure of the mutant. This study shows that disulfide bond-mediated spatial reconstitution can be a platform technology for development of enzymes with enhanced PLP affinity. PMID:28095457

  2. Topological plasticity of enzymes involved in disulfide bond formation allows catalysis in either the periplasm or the cytoplasm.

    PubMed

    Hatahet, Feras; Ruddock, Lloyd W

    2013-09-23

    The transmembrane enzymes disulfide bond forming enzyme B (DsbB) and vitamin K epoxide reductase (VKOR) are central to oxidative protein folding in the periplasm of prokaryotes. Catalyzed formation of structural disulfide bonds in proteins also occurs in the cytoplasm of some hyperthermophilic prokaryotes through currently, poorly defined mechanisms. We aimed to determine whether DsbB and VKOR can be inverted in the membrane with retention of activity. By rational design of inversion of membrane topology, we engineered DsbB mutants that catalyze disulfide bond formation in the cytoplasm of Escherichia coli. This represents the first engineered inversion of a transmembrane protein with demonstrated conservation of activity and substrate specificity. This successful designed engineering led us to identify two naturally occurring and oppositely oriented VKOR homologues from the hyperthermophile Aeropyrum pernix that promote oxidative protein folding in the periplasm or cytoplasm, respectively, and hence defines the probable route for disulfide bond formation in the cytoplasm of hyperthermophiles. Our findings demonstrate how knowledge on the determinants of membrane protein topology can be used to de novo engineer a metabolic pathway and to unravel an intriguingly simple evolutionary scenario where a new "adaptive" cellular process is constructed by means of membrane protein topology inversion.

  3. Stabilizing the integrin alpha M inserted domain in alternative conformations with a range of engineered disulfide bonds.

    PubMed

    Shimaoka, Motomu; Lu, Chafen; Salas, Azucena; Xiao, Tsan; Takagi, Junichi; Springer, Timothy A

    2002-12-24

    Conformational movement of the C-terminal alpha7 helix in the integrin inserted (I) domain, a major ligand-binding domain that adopts an alpha/beta Rossmann fold, has been proposed to allosterically regulate ligand-binding activity. Disulfide bonds were engineered here to reversibly lock the position of the alpha7 helix in one of two alternative conformations seen in crystal structures, termed open and closed. Our results show that pairs of residues with Cbeta atoms farther apart than optimal for disulfide bond stereochemistry can be successfully replaced by cysteine, suggesting that backbone movement accommodates disulfide formation. We also find more success with substituting partially exposed than buried residues. Disulfides stabilizing the open conformation resulted in constitutively active alphaMbeta2 heterodimers and isolated alphaM inserted domains, which were reverted to an inactive form by dithiothreitol reduction. By contrast, a disulfide stabilizing the closed conformation resulted in inactive alphaMbeta2 that was resistant to activation but became activatable after dithiothreitol treatment.

  4. Genetic Selection for Enhanced Folding In Vivo Targets the Cys14-Cys38 Disulfide Bond in Bovine Pancreatic Trypsin Inhibitor

    PubMed Central

    Foit, Linda; Mueller-Schickert, Antje; Mamathambika, Bharath S.; Gleiter, Stefan; Klaska, Caitlyn L.; Ren, Guoping

    2011-01-01

    Abstract The periplasm provides a strongly oxidizing environment; however, periplasmic expression of proteins with disulfide bonds is often inefficient. Here, we used two different tripartite fusion systems to perform in vivo selections for mutants of the model protein bovine pancreatic trypsin inhibitor (BPTI) with the aim of enhancing its expression in Escherichia coli. This trypsin inhibitor contains three disulfides that contribute to its extreme stability and protease resistance. The mutants we isolated for increased expression appear to act by eliminating or destabilizing the Cys14-Cys38 disulfide in BPTI. In doing so, they are expected to reduce or eliminate kinetic traps that exist within the well characterized in vitro folding pathway of BPTI. These results suggest that elimination or destabilization of a disulfide bond whose formation is problematic in vitro can enhance in vivo protein folding. The use of these in vivo selections may prove a valuable way to identify and eliminate disulfides and other rate-limiting steps in the folding of proteins, including those proteins whose in vitro folding pathways are unknown. Antioxid. Redox Signal. 14, 973–984. PMID:21110786

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

    PubMed

    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.

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

    SciTech Connect

    Matak, Mehdi Youssefi; Moghaddam, Majid Erfani

    2009-12-11

    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.

  7. 30 CFR 256.53 - Additional bonds.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 2 2011-07-01 2011-07-01 false Additional bonds. 256.53 Section 256.53 Mineral Resources BUREAU OF OCEAN ENERGY MANAGEMENT, REGULATION, AND ENFORCEMENT, DEPARTMENT OF THE INTERIOR... the Government and the estimated costs of lease abandonment and cleanup are less than the...

  8. Bond additivity corrections for quantum chemistry methods

    SciTech Connect

    C. F. Melius; M. D. Allendorf

    1999-04-01

    In the 1980's, the authors developed a bond-additivity correction procedure for quantum chemical calculations called BAC-MP4, which has proven reliable in calculating the thermochemical properties of molecular species, including radicals as well as stable closed-shell species. New Bond Additivity Correction (BAC) methods have been developed for the G2 method, BAC-G2, as well as for a hybrid DFT/MP2 method, BAC-Hybrid. These BAC methods use a new form of BAC corrections, involving atomic, molecular, and bond-wise additive terms. These terms enable one to treat positive and negative ions as well as neutrals. The BAC-G2 method reduces errors in the G2 method due to nearest-neighbor bonds. The parameters within the BAC-G2 method only depend on atom types. Thus the BAC-G2 method can be used to determine the parameters needed by BAC methods involving lower levels of theory, such as BAC-Hybrid and BAC-MP4. The BAC-Hybrid method should scale well for large molecules. The BAC-Hybrid method uses the differences between the DFT and MP2 as an indicator of the method's accuracy, while the BAC-G2 method uses its internal methods (G1 and G2MP2) to provide an indicator of its accuracy. Indications of the average error as well as worst cases are provided for each of the BAC methods.

  9. Peptide-drug conjugate linked via a disulfide bond for kidney targeted drug delivery.

    PubMed

    Geng, Qian; Sun, Xun; Gong, Tao; Zhang, Zhi-Rong

    2012-06-20

    Chronic kidney disease (CKD) is a worldwide public health problem, and unfortunately, the therapeutic index of clinically available drugs is limited. Thus, there is a great need to exploit effective treatment strategies, and the carrier-drug approach is an attractive method to improve the kidney specificity of the therapeutic agents. The aim of this present study is to develop a peptide-drug conjugate for the kidney targeted delivery of angiotensin-converting enzyme (ACE) inhibitor captopril (CAP), since G3-C12 peptide (ANTPCGPYTHDCPVKR) could specifically accumulate in the kidney after intravenous injection. Therefore, FITC labeled G3-C12 peptide (G3-C12-FITC) and peptide-drug conjugate (G3-C12-CAP) with a disulfide bond which can be cleaved by reduced glutathione in the kidney were prepared by solid-phase peptide synthesis. The fluorescence imaging of G3-C12-FITC revealed that the labeled peptide specifically accumulated in the kidney soon after i.v. injection to mice, and the accumulation is due largely to the reabsorption of the peptide by the proximal renal tubule cells. Furthermore, in comparison with the corresponding nonconjugated form, a 2.7-fold increase in renal area under concentration-time curve produced by the conjugate was observed in mice. Interestingly, the CAP entirely released in the kidney even at 0.05 h postinjection through disulfide reduction. As a consequence, the in vivo renal ACE inhibition was significantly increased. In conclusion, these findings suggest the potential of G3-C12 peptide serving as a suitable candidate carrier for kidney-targeted drug delivery.

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

    PubMed Central

    2012-01-01

    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. PMID:22380681

  11. Molecular dynamics simulations on pars intercerebralis major peptide-C (PMP-C) reveal the role of glycosylation and disulfide bonds in its enhanced structural stability and function.

    PubMed

    Kaushik, Sandeep; Mohanty, Debasisa; Surolia, Avadhesha

    2012-01-01

    Fucosylation of Thr 9 in pars intercerebralis major peptide-C (PMP-C) enhances its structural stability and functional ability as a serine protease inhibitor. In order to understand the role of disulfide bonds and glycosylation on the structure and function of PMP-C, we have carried out multiple explicit solvent molecular dynamics (MD) simulations on fucosylated and non-fucosylated forms of PMP-C, both in the presence and absence of the disulfide bonds. Our simulations revealed that there were no significant structural changes in the native disulfide bonded forms of PMP-C due to fucosylation. On the other hand, the non-fucosylated form of PMP-C without disulfide bonds showed larger deviations from the starting structure than the fucosylated form. However, the structural deviations were restricted to the terminal regions while core β-sheet retained its hydrogen bonded structure even in absence of disulfide bonds as well as fucosylation. Interestingly, fucosylation of disulfide bonded native PMP-C led to a decreased thermal flexibility in the residue stretch 29-32 which is known to interact with the active site of the target proteases. Our analysis revealed that disulfide bonds covalently connect the residue stretch 29-32 to the central β-sheet of PMP-C and using a novel network of side chain interactions and disulfide bonds fucosylation at Thr 9 is altering the flexibility of the stretch 29-32 located at a distal site. Thus, our simulations explain for the first time, how presence of disulfide bonds between conserved cysteines and fucosylation enhance the function of PMP-C as a protease inhibitor.

  12. Application of MALDI TOF/TOF mass spectrometry and collision-induced dissociation for the identification of disulfide-bonded peptides.

    PubMed

    Janecki, Dariusz J; Nemeth, Jennifer F

    2011-07-01

    This paper describes a method for the fast identification and composition of disulfide-bonded peptides. A unique fragmentation signature of inter-disulfide-bonded peptides is detected using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF mass spectrometry and high-energy collision-induced dissociation (CID). This fragmentation pattern identifies peptides with an interconnected disulfide bond and provides information regarding the composition of the peptides involved in the pairing. The distinctive signature produced using CID is a triplet of ions resulting from the cleavage of the disulfide bond to produce dehydroalanine, cysteine or thiocysteine product ions. This method is not applicable to intra-peptide disulfide bonds, as the cleavage mechanism is not the same and a triplet pattern is not observed. This method has been successfully applied to identifying disulfide-bonded peptides in a number of control digestions, as well as study samples where disulfide bond networks were postulated and/or unknown.

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

    PubMed

    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.

  14. Chemical synthesis of La1 isolated from the venom of the scorpion Liocheles australasiae and determination of its disulfide bonding pattern.

    PubMed

    Nagao, Junya; Miyashita, Masahiro; Nakagawa, Yoshiaki; Miyagawa, Hisashi

    2015-08-01

    La1 is a 73-residue cysteine-rich peptide isolated from the scorpion Liocheles australasiae venom. Although La1 is the most abundant peptide in the venom, its biological function remains unknown. Here, we describe a method for efficient chemical synthesis of La1 using the native chemical ligation (NCL) strategy, in which three peptide components of less than 40 residues were sequentially ligated. The peptide thioester necessary for NCL was synthesized using an aromatic N-acylurea approach with Fmoc-SPPS. After completion of sequential NCL, disulfide bond formation was carried out using a dialysis method, in which the linear peptide dissolved in an acidic solution was dialyzed against a slightly alkaline buffer to obtain correctly folded La1. Next, we determined the disulfide bonding pattern of La1. Enzymatic and chemical digests of La1 without reduction of disulfide bonds were analyzed by liquid chromatography/mass spectrometry (LC/MS), which revealed two of four disulfide bond linkages. The remaining two linkages were assigned based on MS/MS analysis of a peptide fragment containing two disulfide bonds. Consequently, the disulfide bonding pattern of La1 was found to be similar to that of a von Willebrand factor type C (VWC) domain. To our knowledge, this is the first report of the experimental determination of the disulfide bonding pattern of peptides having a single VWC domain as well as their chemical synthesis. La1 synthesized in this study will be useful for investigation of its biological role in the venom.

  15. Inhibition of botulinum neurotoxins interchain disulfide bond reduction prevents the peripheral neuroparalysis of botulism.

    PubMed

    Zanetti, Giulia; Azarnia Tehran, Domenico; Pirazzini, Marcon; Binz, Thomas; Shone, Clifford C; Fillo, Silvia; Lista, Florigio; Rossetto, Ornella; Montecucco, Cesare

    2015-12-01

    Botulinum neurotoxins (BoNTs) form a growing family of metalloproteases with a unique specificity either for VAMP, SNAP25 or syntaxin. The BoNTs are grouped in seven different serotypes indicated by letters from A to G. These neurotoxins enter the cytosol of nerve terminals via a 100 kDa chain which binds to the presynaptic membrane and assists the translocation of a 50 kDa metalloprotease chain. These two chains are linked by a single disulfide bridge which plays an essential role during the entry of the metalloprotease chain in the cytosol, but thereafter it has to be reduced to free the proteolytic activity. Its reduction is mediated by thioredoxin which is continuously regenerated by its reductase. Here we show that inhibitors of thioredoxin reductase or of thioredoxin prevent the specific proteolysis of VAMP by the four VAMP-specific BoNTs: type B, D, F and G. These compounds are effective not only in primary cultures of neurons, but also in preventing the in vivo mouse limb neuroparalysis. In addition, one of these inhibitors, Ebselen, largely protects mice from the death caused by a systemic injection. Together with recent results obtained with BoNTs specific for SNAP25 and syntaxin, the present data demonstrate the essential role of the thioredoxin-thioredoxin reductase system in reducing the interchain disulfide during the nerve intoxication mechanism of all serotypes. Therefore its inhibitors should be considered for a possible use to prevent botulism and for treating infant botulism.

  16. Bond additivity corrections for quantum chemistry methods

    SciTech Connect

    Melius, C.F.; Allendorf, M.D.

    2000-03-23

    New bond additivity correction (BAC) methods have been developed for the G2 method, BAC-G2, as well as for a hybrid density functional theory (DFT) Moller-Plesset (MP)2 method, BAC-hybrid. These BAC methods use a new form of BAC corrections, involving atomic, molecular, and bond-wise additive terms. These terms enable one to treat positive and negative ions as well as neutrals. The BAC-G2 method reduces errors in the G2 method due to nearest-neighbor bonds. The parameters within the BAC-G2 method only depend on atom types. Thus the BAC-G2 method can be used to determine the parameters needed by BAC methods involving lower levels of theory, such as BAC-hybrid and BAC-MP4. The BAC-hybrid method is expected to scale well for large molecules. The BAC-hybrid method uses the differences between the DFT and MP2 predictions as an indication of the method's accuracy, whereas the BAC-G2 method uses its internal methods (G1 and G2MP2) to accomplish this. A statistical analysis of the error in each of the methods is presented on the basis of calculations performed for large sets (more than 120) of molecules.

  17. Artifactual formation of disulfide bonds during SDS-PAGE analysis of type I copper proteins

    SciTech Connect

    Kumar, M.A.; Davidson, V.L. )

    1991-03-11

    Amicyanin is a monomeric Type I blue' copper protein, which possesses a single cysteine that serves as one of the ligands to copper. Amicyanin denatured by heating in SDS in the presence of {beta}-mercaptoethanol ({beta}ME) migrated during SDS-PAGE with an M{sub r} = 15,000. When heated in SDS in the absence of {beta}ME it exhibited an M{sub r} = 30,000. If treated with {beta}ME, but not heated it exhibited an M{sub r} = 15,000. Similar data were obtained with the small blue copper protein, azurin. Ascorbate oxidase is a multicopper enzyme which is composed of two identical non-covalently bound subunits, each of which possesses a Type I copper center. When this blue' oxidase was denatured by heating in SDS with {beta}ME, or incubated with {beta}ME without heating, a single band of M{sub r} = 70,000 was observed. When heated in SDS without {beta}ME, a species twice that size was observed. Thus, exposure during denaturation of the free sulfhydryl of the Type I copper binding site can cause formation of disulfide bonds between otherwise unlinked polypeptides.

  18. Ion Channels of Alamethicin Dimer N-Terminally Linked by Disulfide Bond

    PubMed Central

    Okazaki, Takashi; Sakoh, Machiko; Nagaoka, Yasuo; Asami, Koji

    2003-01-01

    A covalent dimer of alamethicin Rf30 was synthesized by linking the N-termini by a disulfide bond. When the dimer peptides were added to the cis-side of a diphytanoyl PC membrane, macroscopic channel current was induced only at cis positive voltages. The single-channel recordings showed several conductance levels that were alternately stabilized. These results indicate that the dimer peptides form stable channels by N-terminal insertion like alamethicin and that most of the pores are assembled from even numbers of helices. Taking advantages of the long open duration of the dimer peptide channels, the current-voltage (I-V) relations of the single-channels were obtained by applying fast voltage ramps during the open states. The I-V relations showed rectification, such that current from the cis-side toward the trans-side is larger than that in the opposite direction. The intrinsic rectification is mainly attributed to the macro dipoles of parallel peptide helices surrounding a central pore. PMID:12829482

  19. Ion channels of alamethicin dimer N-terminally linked by disulfide bond.

    PubMed

    Okazaki, Takashi; Sakoh, Machiko; Nagaoka, Yasuo; Asami, Koji

    2003-07-01

    A covalent dimer of alamethicin Rf30 was synthesized by linking the N-termini by a disulfide bond. When the dimer peptides were added to the cis-side of a diphytanoyl PC membrane, macroscopic channel current was induced only at cis positive voltages. The single-channel recordings showed several conductance levels that were alternately stabilized. These results indicate that the dimer peptides form stable channels by N-terminal insertion like alamethicin and that most of the pores are assembled from even numbers of helices. Taking advantages of the long open duration of the dimer peptide channels, the current-voltage (I-V) relations of the single-channels were obtained by applying fast voltage ramps during the open states. The I-V relations showed rectification, such that current from the cis-side toward the trans-side is larger than that in the opposite direction. The intrinsic rectification is mainly attributed to the macro dipoles of parallel peptide helices surrounding a central pore.

  20. Calcium Binding and Disulfide Bonds Regulate the Stability of Secretagogin towards Thermal and Urea Denaturation

    PubMed Central

    Weiffert, Tanja; Ní Mhurchú, Niamh; O’Connell, David; Linse, Sara

    2016-01-01

    Secretagogin is a calcium-sensor protein with six EF-hands. It is widely expressed in neurons and neuro-endocrine cells of a broad range of vertebrates including mammals, fishes and amphibia. The protein plays a role in secretion and interacts with several vesicle-associated proteins. In this work, we have studied the contribution of calcium binding and disulfide-bond formation to the stability of the secretagogin structure towards thermal and urea denaturation. SDS-PAGE analysis of secretagogin in reducing and non-reducing conditions identified a tendency of the protein to form dimers in a redox-dependent manner. The denaturation of apo and Calcium-loaded secretagogin was studied by circular dichroism and fluorescence spectroscopy under conditions favoring monomer or dimer or a 1:1 monomer: dimer ratio. This analysis reveals significantly higher stability towards urea denaturation of Calcium-loaded secretagogin compared to the apo protein. The secondary and tertiary structure of the Calcium-loaded form is not completely denatured in the presence of 10 M urea. Reduced and Calcium-loaded secretagogin is found to refold reversibly after heating to 95°C, while both oxidized and reduced apo secretagogin is irreversibly denatured at this temperature. Thus, calcium binding greatly stabilizes the structure of secretagogin towards chemical and heat denaturation. PMID:27812162

  1. Synthesis strategies for disulfide bond-containing polymer-based drug delivery system for reduction-responsive controlled release

    NASA Astrophysics Data System (ADS)

    Liu, Lei; Liu, Peng

    2015-09-01

    Tumor micro-environment responsive drug delivery systems (DDSs) have been developed as a potential approach to reduce the side effects of cancer chemotherapy. Glutathione (GSH) has been supposed to the most significant signal of the difference between the normal tissue and the tumor cells, besides the media pH and temperature. In recent years, the reduction-responsive DDSs have attracted more and more attention for delivery of anti-cancer drugs, based on such physiological signal. Among them, disulfide bond-containing polymers have been designed as the main tool for the purpose. The recent progress in the synthesis strategies for the disulfide bond-containing polymer-based DDS is focused in the present review.

  2. The Effect of Tensile Stress on the Conformational Free Energy Landscape of Disulfide Bonds

    PubMed Central

    Anjukandi, Padmesh; Dopieralski, Przemyslaw; Ribas–Arino, Jordi; Marx, Dominik

    2014-01-01

    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, and . Moreover, the interplay of chemical reactivity and mechanical stress of disulfide switches has been recently elucidated using force–clamp spectroscopy and computer simulation. The and 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 –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 reaction step. All these findings constitute a step forward towards achieving a full understanding of functional disulfides. PMID:25286308

  3. Structural and biochemical characterization of the essential DsbA-like disulfide bond forming protein from Mycobacterium tuberculosis

    PubMed Central

    2013-01-01

    Background Bacterial Disulfide bond forming (Dsb) proteins facilitate proper folding and disulfide bond formation of periplasmic and secreted proteins. Previously, we have shown that Mycobacterium tuberculosis Mt-DsbE and Mt-DsbF aid in vitro oxidative folding of proteins. The M. tuberculosis proteome contains another predicted membrane-tethered Dsb protein, Mt-DsbA, which is encoded by an essential gene. Results Herein, we present structural and biochemical analyses of Mt-DsbA. The X-ray crystal structure of Mt-DsbA reveals a two-domain structure, comprising a canonical thioredoxin domain with the conserved CXXC active site cysteines in their reduced form, and an inserted α-helical domain containing a structural disulfide bond. The overall fold of Mt-DsbA resembles that of other DsbA-like proteins and not Mt-DsbE or Mt-DsbF. Biochemical characterization demonstrates that, unlike Mt-DsbE and Mt-DsbF, Mt-DsbA is unable to oxidatively fold reduced, denatured hirudin. Moreover, on the substrates tested in this study, Mt-DsbA has disulfide bond isomerase activity contrary to Mt-DsbE and Mt-DsbF. Conclusion These results suggest that Mt-DsbA acts upon a distinct subset of substrates as compared to Mt-DsbE and Mt-DsbF. One could speculate that Mt-DsbE and Mt-DsbF are functionally redundant whereas Mt-DsbA is not, offering an explanation for the essentiality of Mt-DsbA in M. tuberculosis. PMID:24134223

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

    PubMed

    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.

  5. Production of disulfide bond-rich peptides by fusion expression using small transmembrane proteins of Escherichia coli.

    PubMed

    Chang, Ziwei; Lu, Ming; Ma, Yunqi; Kwag, Dong-Geon; Kim, Seo-Hyun; Park, Ji-Min; Nam, Bo-Hye; Kim, Young-Ok; An, Cheul-Min; Li, Huayue; Jung, Jee H; Park, Jang-Su

    2015-03-01

    Recombinant expression in Escherichia coli allows the simple, economical, and effective production of bioactive peptides. On the other hand, the production of native peptides, particularly those rich in disulfide bonds, is a major problem. Previous studies have reported that the use of carrier proteins for fusion expression can result in good peptide yields, but few are folded correctly. In this study, two transmembrane small proteins in E. coli, YoaJ and YkgR, which both orientate with their N-termini in cytoplasm and their C-termini in periplasm, were used for fusion expression. The recombinant production of two peptides, asteropsin A (ASPA) and β-defensin (BD), was induced in the periplasm of E. coli using a selected carrier protein. Both peptides were expressed at high levels, at yields of approximately 5-10 mg/L of culture. Mass spectrometry showed that the resulting peptide had the same molecular weight as their natural forms. After purification, single peaks were observed by reversed phase high-performance liquid chromatography (RP-HPLC), demonstrating the absence of isoforms. Furthermore, cytoplasmically expressed fusion proteins with a carrier at their C-termini did not contain disulfide bonds. This study provides new carrier proteins for fusion expression of disulfide bond-rich peptides in E. coli.

  6. Intramolecular Disulfide Bond of Tim22 Protein Maintains Integrity of the TIM22 Complex in the Mitochondrial Inner Membrane*

    PubMed Central

    Okamoto, Hiroaki; Miyagawa, Akiko; Shiota, Takuya; Tamura, Yasushi; Endo, Toshiya

    2014-01-01

    Mitochondrial proteins require protein machineries called translocators in the outer and inner membranes for import into and sorting to their destination submitochondrial compartments. Among them, the TIM22 complex mediates insertion of polytopic membrane proteins into the inner membrane, and Tim22 constitutes its central insertion channel. Here we report that the conserved Cys residues of Tim22 form an intramolecular disulfide bond. By comparison of Tim22 Cys → Ser mutants with wild-type Tim22, we show that the disulfide bond of Tim22 stabilizes Tim22 especially at elevated temperature through interactions with Tim18, which are also important for the stability of the TIM22 complex. We also show that lack of the disulfide bond in Tim22 impairs the assembly of TIM22 pathway substrate proteins into the inner membrane especially when the TIM22 complex handles excess amounts of substrate proteins. Our findings provide a new insight into the mechanism of the maintenance of the structural and functional integrity of the TIM22 complex. PMID:24385427

  7. Air oxidation method employed for the disulfide bond formation of natural and synthetic peptides.

    PubMed

    Calce, Enrica; Vitale, Rosa Maria; Scaloni, Andrea; Amodeo, Pietro; De Luca, Stefania

    2015-08-01

    Among the available protocols, chemically driven approaches to oxidize cysteine may not be required for molecules that, under the native-like conditions, naturally fold in conformations ensuring an effective pairing of the right disulfide bridge pattern. In this contest, we successfully prepared the distinctin, a natural heterodimeric peptide, and some synthetic cyclic peptides that are inhibitors of the CXCR4 receptor. In the first case, the air oxidation reaction allowed to connect two peptide chains via disulfide bridge, while in the second case allowed the cyclization of rationally designed peptides by an intramolecular disulfide bridge. Computational approaches helped to either drive de-novo design or suggest structural modifications and optimal oxidization protocols for disulfide-containing molecules. They are able to both predict and to rationalize the propensity of molecules to spontaneously fold in suitable conformations to achieve the right disulfide bridges.

  8. Assignment of the disulfide bonds of Ole e 1, a major allergen of olive tree pollen involved in fertilization.

    PubMed

    González, E; Monsalve, R I; Puente, X S; Villalba, M; Rodríguez, R

    2000-01-01

    The most prevalent allergen from olive tree pollen, Ole e 1, consists of a single polymorphic polypeptide chain of 145 amino acids which includes six cysteine residues at positions 19, 22, 43, 78, 90 and 131. By using an homogeneous form of the allergen expressed in Pichia pastoris, the array of the disulfide bridges has been elucidated. Specific proteolysis with thermolysin and reverse-phase HPLC separation of the peptides allowed the determination of the disulfide bond between Cys43 and Cys78. Another thermolytic product, which contained three peptides linked by the remaining four cysteines, was digested with Glu-specific staphylococcal V8 protease and the products isolated by reverse-phase HPLC. Amino acid compositions and Edman degradation of the peptide products indicated the presence of the disulfide bonds at Cys19-Cys90 and Cys22-Cys131. These data can help in the analysis of the three-dimensional structure of the protein as well as in studies of its allergenic determinants.

  9. Palladium-catalyzed addition of disulfides and diselenides to alkynes under solvent free conditions.

    PubMed

    Ananikov, Valentine P; Beletskaya, Irina P

    2004-02-07

    An efficient methodology was developed for performing palladium-catalyzed E-E (E = S, Se) bond addition to alkynes under solvent free conditions. Compared to reaction in solvent significant enhancement of reaction rate, improved efficiency and remarkable catalyst stability were observed under solvent free conditions. The addition reactions were carried out with high stereoselectivity and yields in a short reaction time.

  10. Fragmentation of intra-peptide and inter-peptide disulfide bonds of proteolytic peptides by nanoESI collision-induced dissociation.

    PubMed

    Mormann, Michael; Eble, Johannes; Schwöppe, Christian; Mesters, Rolf M; Berdel, Wolfgang E; Peter-Katalinić, Jasna; Pohlentz, Gottfried

    2008-11-01

    Characterisation and identification of disulfide bridges is an important aspect of structural elucidation of proteins. Covalent cysteine-cysteine contacts within the protein give rise to stabilisation of the native tertiary structure of the molecules. Bottom-up identification and sequencing of proteins by mass spectrometry most frequently involves reductive cleavage and alkylation of disulfide links followed by enzymatic digestion. However, when using this approach, information on cysteine-cysteine contacts within the protein is lost. Mass spectrometric characterisation of peptides containing intra-chain disulfides is a challenging analytical task, because peptide bonds within the disulfide loop are believed to be resistant to fragmentation. In this contribution we show recent results on the fragmentation of intra and inter-peptide disulfide bonds of proteolytic peptides by nano electrospray ionisation collision-induced dissociation (nanoESI CID). Disulfide bridge-containing peptides obtained from proteolytic digests were submitted to low-energy nanoESI CID using a quadrupole time-of-flight (Q-TOF) instrument as a mass analyser. Fragmentation of the gaseous peptide ions gave rise to a set of b and y-type fragment ions which enabled derivation of the sequence of the amino acids located outside the disulfide loop. Surprisingly, careful examination of the fragment-ion spectra of peptide ions comprising an intramolecular disulfide bridge revealed the presence of low-abundance fragment ions formed by the cleavage of peptide bonds within the disulfide loop. These fragmentations are preceded by proton-induced asymmetric cleavage of the disulfide bridge giving rise to a modified cysteine containing a disulfohydryl substituent and a dehydroalanine residue on the C-S cleavage site.

  11. Macromolecule functionalization of disulfide-bonded polymer hydrogel capsules and cancer cell targeting.

    PubMed

    Shimoni, Olga; Postma, Almar; Yan, Yan; Scott, Andrew M; Heath, Joan K; Nice, Edouard C; Zelikin, Alexander N; Caruso, Frank

    2012-02-28

    We present a generic and versatile method for functionalization of disulfide-stabilized PMA hydrogel capsules (HCs) with macromolecules, including a number of specific antibodies to cancer cells. Functionalization was achieved by reversible addition-fragmentation chain transfer (RAFT) polymerization of poly(N-vinyl pyrrolidone) (PVPON), which introduced biorelevant heterotelechelic end groups (thiol and amine) to the polymer chain. The PVPON with heterotelechelic end groups was conjugated to the outermost layer of PMA HCs through the thiol groups and reacted with biotin via the amine groups to generate PMA/PVPON(biotin) HCs. On the basis of the high specific interaction and high affinity between biotin and avidin, and its derivates, such as NeutrAvidin (NAv), we functionalized the PMA HCs with biotinylated antibodies. We demonstrate significantly enhanced cellular binding and internalization of the antibody (Ab)-functionalized capsules compared with control human immunoglobulin (IgG)-functionalized capsules, suggesting these capsules can specifically interact with cells through antibody/antigen recognition. We anticipate that the versatility of the functionalization approach reported in this study will assist in targeted therapeutic delivery applications.

  12. 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

    PubMed Central

    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

  13. 30 CFR 556.53 - Additional bonds.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Resources BUREAU OF OCEAN ENERGY MANAGEMENT, DEPARTMENT OF THE INTERIOR OFFSHORE LEASING OF SULPHUR OR OIL... but before he/she approves drilling activities under the EP. (iii) You may satisfy the bond... drilling activities under the DPP or DOCD. (iii) You may satisfy the bond requirement of this paragraph...

  14. 30 CFR 556.53 - Additional bonds.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Resources BUREAU OF OCEAN ENERGY MANAGEMENT, DEPARTMENT OF THE INTERIOR OFFSHORE LEASING OF SULPHUR OR OIL... but before he/she approves drilling activities under the EP. (iii) You may satisfy the bond... drilling activities under the DPP or DOCD. (iii) You may satisfy the bond requirement of this paragraph...

  15. 30 CFR 556.53 - Additional bonds.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... Resources BUREAU OF OCEAN ENERGY MANAGEMENT, DEPARTMENT OF THE INTERIOR OFFSHORE LEASING OF SULPHUR OR OIL... but before he/she approves drilling activities under the EP. (iii) You may satisfy the bond... drilling activities under the DPP or DOCD. (iii) You may satisfy the bond requirement of this paragraph...

  16. Stabilizing Exposure of Conserved Epitopes by Structure Guided Insertion of Disulfide Bond in HIV-1 Envelope Glycoprotein

    PubMed Central

    Sarkar, Pampi; Labranche, Celia; Go, Eden P.; Clark, Daniel F.; Sun, Yide; Nandi, Avishek; Hartog, Karin; Desaire, Heather; Montefiori, David; Carfi, Andrea; Srivastava, Indresh K.; Barnett, Susan W.

    2013-01-01

    Entry of HIV-1 into target cells requires binding of the viral envelope glycoprotein (Env) to cellular receptors and subsequent conformational changes that culminates in fusion of viral and target cell membranes. Recent structural information has revealed that these conformational transitions are regulated by three conserved but potentially flexible layers stacked between the receptor-binding domain (gp120) and the fusion arm (gp41) of Env. We hypothesized that artificial insertion of a covalent bond will ‘snap’ Env into a conformation that is less mobile and stably expose conserved sites. Therefore, we analyzed the interface between these gp120 layers (layers 1, 2 and 3) and identified residues that may form disulfide bonds when substituted with cysteines. We subsequently probed the structures of the resultant mutant gp120 proteins by assaying their binding to a variety of ligands using Surface Plasmon Resonance (SPR) assay. We found that a single disulfide bond strategically inserted between the highly conserved layers 1 and 2 (C65-C115) is able to ‘lock’ gp120 in a CD4 receptor bound conformation (in the absence of CD4), as indicated by the lower dissociation constant (Kd) for the CD4-induced (CD4i) epitope binding 17b antibody. When disulfide-stabilized monomeric (gp120) and trimeric (gp140) Envs were used to immunize rabbits, they were found to elicit a higher proportion of antibodies directed against both CD4i and CD4 binding site epitopes than the wild-type proteins. These results demonstrate that structure-guided stabilization of inter-layer interactions within HIV-1 Env can be used to expose conserved epitopes and potentially overcome the sequence diversity of these molecules. PMID:24146829

  17. Stability of wild-type and mutant RTEM-1 beta-lactamases: effect of the disulfide bond.

    PubMed

    Schultz, S C; Dalbadie-McFarland, G; Neitzel, J J; Richards, J H

    1987-01-01

    Uniquely among class A beta-lactamases, the RTEM-1 and RTEM-2 enzymes contain a single disulfide bond between Cys 77 and Cys 123. To study the possible role of this naturally occurring disulfide in stabilizing RTEM-1 beta-lactamase and its mutants at residue 71, this bond was removed by introducing a Cys 77----Ser mutation. Both the wild-type enzyme and the single mutant Cys 77----Ser confer the same high levels of resistance to ampicillin in vivo to Escherichia coli; at 30 degrees C the specific activity of purified Cys 77----Ser mutant is also the same as that of the wild-type enzyme. Also, neither wild-type enzyme nor the Cys 77----Ser mutant is inactivated by brief exposure to p-hydroxymercuribenzoate. However, above 40 degrees C the mutant enzyme is less stable than wild-type enzyme. After introduction of the Cys 77----Ser mutation, none of the double mutants (containing the second mutations at residue 71) confer resistance to ampicillin in vivo at 37 degrees C; proteins with Ala, Val, Leu, Ile, Met, Pro, His, Cys, and Ser at residue 71 confer low levels of resistance to ampicillin in vivo at 30 degrees C. The use of electrophoretic blots stained with antibodies against beta-lactamase to analyze the relative quantities of mutant proteins in whole-cell extracts of E. coli suggests that all 19 of the doubly mutant enzymes are proteolyzed much more readily than their singly mutant analogues (at Thr 71) that contain a disulfide bond.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. From structure to redox: the diverse functional roles of disulfides and implications in disease

    PubMed Central

    Bechtel, Tyler J.; Weerapana, Eranthie

    2017-01-01

    This review provides a comprehensive overview of the functional roles of disulfide bonds and their relevance to human disease. The critical roles of disulfide bonds in protein structure stabilization and redox regulation of protein activity are addressed. Disulfide bonds are essential to the structural stability of many proteins within the secretory pathway and can exist as intramolecular or inter-domain disulfides. The proper formation of these bonds often relies on folding chaperones and oxidases such as members of the protein disulfide isomerase (PDI) family. Many of the PDI family members catalyze disulfide-bond formation, reduction and isomerization through redox-active disulfides and perturbed PDI activity is characteristic of carcinomas and neurodegenerative diseases. In addition to catalytic function in oxidoreductases, redox-active disulfides are also found on a diverse array of cellular proteins and act to regulate protein activity and localization in response to oxidative changes in the local environment. These redox-active disulfides are either dynamic intramolecular protein disulfides or mixed disulfides with small-molecule thiols generating glutathionylation and cysteinylation adducts. The oxidation and reduction of redox-active disulfides are mediated by cellular reactive oxygen species and activity of reductases, such as glutaredoxin and thioredoxin. Dysregulation of cellular redox conditions and resulting changes in mixed disulfide formation are directly linked to diseases such as cardiovascular disease and Parkinson’s disease. PMID:28044432

  19. Disulfide Bond Formation and N-Glycosylation Modulate Protein-Protein Interactions in GPI-Transamidase (GPIT)

    PubMed Central

    Yi, Lina; Bozkurt, Gunes; Li, Qiubai; Lo, Stanley; Menon, Anant K.; Wu, Hao

    2017-01-01

    Glycosylphosphatidylinositol (GPI) transamidase (GPIT), the enzyme that attaches GPI anchors to proteins as they enter the lumen of the endoplasmic reticulum, is a membrane-bound hetero-pentameric complex consisting of Gpi8, Gpi16, Gaa1, Gpi17 and Gab1. Here, we expressed and purified the luminal domain of Saccharomyces cerevisiae (S. cerevisiae) Gpi8 using different expression systems, and examined its interaction with insect cell expressed luminal domain of S. cerevisiae Gpi16. We found that the N-terminal caspase-like domain of Gpi8 forms a disulfide-linked dimer, which is strengthened by N-glycosylation. The non-core domain of Gpi8 following the caspase-like domain inhibits this dimerization. In contrast to the previously reported disulfide linkage between Gpi8 and Gpi16 in human and trypanosome GPIT, our data show that the luminal domains of S. cerevisiae Gpi8 and S. cerevisiae Gpi16 do not interact directly, nor do they form a disulfide bond in the intact S. cerevisiae GPIT. Our data suggest that subunit interactions within the GPIT complex from different species may vary, a feature that should be taken into account in future structural and functional studies. PMID:28374821

  20. Efficient export of prefolded, disulfide-bonded recombinant proteins to the periplasm by the Tat pathway in Escherichia coli CyDisCo strains.

    PubMed

    Matos, Cristina F R O; Robinson, Colin; Alanen, Heli I; Prus, Piotr; Uchida, Yuko; Ruddock, Lloyd W; Freedman, Robert B; Keshavarz-Moore, Eli

    2014-01-01

    Numerous high-value therapeutic proteins are produced in Escherichia coli and exported to the periplasm, as this approach simplifies downstream processing and enables disulfide bond formation. Most recombinant proteins are exported by the Sec pathway, which transports substrates across the plasma membrane in an unfolded state. The Tat system also exports proteins to the periplasm, but transports them in a folded state. This system has attracted interest because of its tendency to transport correctly folded proteins, but this trait renders it unable to export proteins containing disulfide bonds since these are normally acquired only in the periplasm; reduced substrates tend to be recognized as incorrectly folded and rejected. In this study we have used a series of novel strains (termed CyDisCo) which oxidise disulfide bonds in the cytoplasm, and we show that these cells efficiently export a range of disulfide-containing proteins when a Tat signal peptide is attached. These test proteins include alkaline phosphatase (PhoA), a phytase containing four disulfide bonds (AppA), an antiinterleukin 1β scFv and human growth hormone. No export of PhoA or AppA is observed in wild-type cells lacking the CyDisCo factors. The PhoA, AppA and scFv proteins were exported in an active form by Tat in the CyDisCo strain, and mass spectrometry showed that the vast majority of the scFv protein was disulfide-bonded and correctly processed. The evidence indicates that this combination of Tat + CyDisCo offers a novel means of exporting active, correctly folded disulfide bonded proteins to the periplasm.

  1. Selective disulfide bond cleavage in gold(I) cationized polypeptide ions formed via gas-phase ion/ion cation switching.

    PubMed

    Gunawardena, Harsha P; O'Hair, Richard A J; McLuckey, Scott A

    2006-09-01

    Gaseous multiply protonated disulfide-linked peptides have been subjected to reactions with AuCl2(-) ions to explore the possibility of effecting cation switching of Au+ for two protons and to determine whether cationization by Au+ ions affords selective dissociation of disulfide linkages. The incorporation of Au+ into several model disulfide-linked peptides proved to be straightforward. The primary ion/ion reaction channels were proton transfer, which does not lead to Au+ incorporation, and attachment of AuCl2(-) ions to the polypeptide cation, which does incorporate Au+. Fragmentation of the attachment product, the extent of which varied with peptide and charge state, led to losses of one or more molecules of HCl and, to some extent, cleavage of polypeptides at the disulfide linkage into its two constituent chains. Collisional activation of the intact metal-ion-incorporated peptides showed cleavage of the disulfide linkage to be a major, and in some cases exclusive, process. Cations with protons as the only cationizing agents showed only small contributions from cleavage of the disulfide linkage. These results indicate that Au+ incorporation into a disulfide-linked polypeptide ion is a promising way to effect selective dissociation of disulfide bonds. Cation switching via ion/ion reactions is a convenient means for incorporating gold and is attractive because it avoids the requirement of adding metal salts to the analyte solution.

  2. Differential roles for disulfide bonds in the structural integrity and biological activity of kappa-Bungarotoxin, a neuronal nicotinic acetylcholine receptor antagonist.

    PubMed

    Grant, G A; Luetje, C W; Summers, R; Xu, X L

    1998-09-01

    kappa-Bungarotoxin, a kappa-neurotoxin derived from the venom of the banded Krait, Bungarus multicinctus, is a homodimeric protein composed of subunits of 66 amino acid residues containing five disulfide bonds. kappa-Bungarotoxin is a potent, selective, and slowly reversible antagonist of alpha3 beta2 neuronal nicotinic acetylcholine receptors. kappa-Bungarotoxin is structurally related to the alpha-neurotoxins, such as alpha-bungarotoxin derived from the same snake, which are monomeric in solution and which effectively antagonize muscle type receptors (alpha1 beta1 gamma delta) and the homopentameric neuronal type receptors (alpha7, alpha8, and alpha9). Like the kappa-neurotoxins, the long alpha-neurotoxins contain the same five conserved disulfide bonds, while the short alpha-neurotoxins only contain four of the five. Systematic removal of single disulfide bonds in kappa-bungarotoxin by site-specific mutagenesis reveals a differential role for each of the disulfide bonds. Removal of either of the two disulfides connecting elements of the carboxy terminal loop of this toxin (Cys 46-Cys 58 and Cys 59-Cys 64) interferes with the ability of the toxin to fold. In contrast, removal of each of the other three disulfides does not interfere with the general folding of the toxin and yields molecules with biological activity. In fact, when either C3-C21 or C14-C42 are removed individually, no loss in biological activity is seen. However, removing both produces a polypeptide chain which fails to fold properly. Removal of the C27-C31 disulfide only reduces the activity of the toxin 46.6-fold. This disulfide may play a role in specific interaction of the toxin with specific neuronal receptors.

  3. Granulomorphometry: a suitable tool for identifying hydrophobic and disulfide bonds in β-lactoglobulin aggregates. Application to the study of β-lactoglobulin aggregation mechanism between 70 and 95°C.

    PubMed

    Petit, J; Herbig, A-L; Moreau, A; Le Page, J-F; Six, T; Delaplace, G

    2012-08-01

    This work deals with the investigation of β-lactoglobulin (β-LG) aggregation by granulomorphometry. In the first part of this study, we showed that the binding interactions involved in aggregate structure could be identified by their appearance in granulomorphometric pictures. The reliability of this analytical approach was demonstrated by comparing the appearance of β-LG aggregates in the presence and absence of a thiol-blocking agent (N-ethylmaleimide). The translucency of the aggregates was associated with hydrophobic interactions and their opacity was associated with disulfide bonds. We state, based on the morphology of the aggregates, along with the color of protein aggregates and insoluble materials, that hydrophobic interactions had a better water-holding capacity than disulfide bonds. Additionally, our results suggest that disulfide and hydrophobic bonds compete for β-LG aggregate shaping. In the second part of this work, interesting features of granulomorphometry useful for identifying aggregate binding interactions were highlighted to clarify the effect of temperature on the aggregation mechanisms occurring in a β-LG concentrate with a moderate calcium content (6.6mmol·L(-1)). Heat treatment experiments were performed between 70 and 95°C, and granulomorphometric measurements (aggregate size, aggregate number, and gray level of the picture) were conducted at different sampling times up to 4h. Results, which were interpreted in light of calculated β-LG denaturation levels, revealed that the aggregation mechanism could be split into 2 steps. Initially, β-LG denatured quickly, leading to fast β-LG aggregation by disulfide bonds. The denaturation rate then declined, which drastically slowed the disulfide aggregation mechanism. From that point on, a second aggregation path became preponderant. It consisted of the agglomeration of small aggregates by hydrophobic interactions and resulted in the formation of large aggregates containing both interaction

  4. Protein disulfide engineering.

    PubMed

    Dombkowski, Alan A; Sultana, Kazi Zakia; Craig, Douglas B

    2014-01-21

    Improving the stability of proteins is an important goal in many biomedical and industrial applications. A logical approach is to emulate stabilizing molecular interactions found in nature. Disulfide bonds are covalent interactions that provide substantial stability to many proteins and conform to well-defined geometric conformations, thus making them appealing candidates in protein engineering efforts. Disulfide engineering is the directed design of novel disulfide bonds into target proteins. This important biotechnological tool has achieved considerable success in a wide range of applications, yet the rules that govern the stabilizing effects of disulfide bonds are not fully characterized. Contrary to expectations, many designed disulfide bonds have resulted in decreased stability of the modified protein. We review progress in disulfide engineering, with an emphasis on the issue of stability and computational methods that facilitate engineering efforts.

  5. Role of the intra-A-chain disulfide bond of insulin-like peptide 3 in binding and activation of its receptor, RXFP2.

    PubMed

    Zhang, Suode; Hughes, Richard A; Bathgate, Ross A D; Shabanpoor, Fazel; Hossain, M Akhter; Lin, Feng; van Lierop, Bianca; Robinson, Andrea J; Wade, John D

    2010-09-01

    INSL3 is a member of the insulin-IGF-relaxin superfamily and plays a key role in male fetal development and in adult germ cell maturation. It is the cognate ligand for RXFP2, a leucine-rich repeat containing G-protein coupled receptor. To date, and in contrast to our current knowledge of the key structural features that are required for the binding of INSL3 to RXFP2, comparatively little is known about the key residues that are required to elicit receptor activation and downstream cell signaling. Early evidence suggests that these are contained principally within the A-chain. To further explore this hypothesis, we have undertaken an examination of the functional role of the intra-A-chain disulfide bond. Using solid-phase peptide synthesis together with regioselective disulfide bond formation, two analogs of human INSL3 were prepared in which the intra-chain disulfide bond was replaced, one in which the corresponding Cys residues were substituted with the isosteric Ser and the other in which the Cys were removed altogether. Both of these peptides retained nearly full RXFP2 receptor binding but were devoid of cAMP activity (receptor activation), indicating that the intra-A-chain disulfide bond makes a significant contribution to the ability of INSL3 to act as an RXFP2 agonist. Replacement of the disulfide bond with a metabolically stable dicarba bond yielded two isomers of INSL3 that each exhibited bioactivity similar to native INSL3. This study highlights the critical structural role played by the intra-A-chain disulfide bond of INSL3 in mediating agonist actions through the RXFP2 receptor.

  6. Reduced glutathione and procaine hydrochloride protect the nucleoprotein structure of boar spermatozoa during freeze-thawing by stabilising disulfide bonds.

    PubMed

    Yeste, Marc; Flores, Eva; Estrada, Efrén; Bonet, Sergi; Rigau, Teresa; Rodríguez-Gil, Joan E

    2013-01-01

    One important change the head of boar spermatozoa during freeze-thawing is the destabilisation of its nucleoprotein structure due to a disruption of disulfide bonds. With the aim of better understanding these changes in frozen-thawed spermatozoa, two agents, namely reduced glutathione (GSH) and procaine hydrochloride (ProHCl), were added at different concentrations to the freezing media at different concentrations and combinations over the range 1-2mM. Then, 30 and 240 min after thawing, cysteine-free residue levels of boar sperm nucleoproteins, DNA fragmentation and other sperm functional parameters were evaluated. Both GSH and ProHCl, at final concentrations of 2mM, induced a significant (P<0.05) increase in the number of non-disrupted sperm head disulfide bonds 30 and 240 min after thawing compared with the frozen-thawed control. This effect was accompanied by a significant (P<0.05) decrease in DNA fragmentation 240 min after thawing. Concomitantly, 1 and 2mM GSH, but not ProHCl at any of the concentrations tested, partially counteracted the detrimental effects caused by freeze-thawing on sperm peroxide levels, motility patterns and plasma membrane integrity. In conclusion, the results show that both GSH and ProHCl have a stabilising effect on the nucleoprotein structure of frozen-thawed spermatozoa, although only GSH exerts an appreciable effect on sperm viability.

  7. The Structure of Eukaryotic Translation Initiation Factor-4E from Wheat Reveals a Novel Disulfide Bond

    SciTech Connect

    Monzingo,A.; Dhaliwal, S.; Dutt-Chaudhuri, A.; Lyon, A.; Sadow, J.; Hoffman, D.; Robertus, J.; Browning, K.

    2007-01-01

    Eukaryotic translation initiation factor-4E (eIF4E) recognizes and binds the m{sup 7} guanosine nucleotide at the 5' end of eukaryotic messenger RNAs; this protein-RNA interaction is an essential step in the initiation of protein synthesis. The structure of eIF4E from wheat (Triticum aestivum) was investigated using a combination of x-ray crystallography and nuclear magnetic resonance (NMR) methods. The overall fold of the crystallized protein was similar to eIF4E from other species, with eight {beta}-strands, three {alpha}-helices, and three extended loops. Surprisingly, the wild-type protein did not crystallize with m{sup 7}GTP in its binding site, despite the ligand being present in solution; conformational changes in the cap-binding loops created a large cavity at the usual cap-binding site. The eIF4E crystallized in a dimeric form with one of the cap-binding loops of one monomer inserted into the cavity of the other. The protein also contained an intramolecular disulfide bridge between two cysteines (Cys) that are conserved only in plants. A Cys-to-serine mutant of wheat eIF4E, which lacked the ability to form the disulfide, crystallized with m{sup 7}GDP in its binding pocket, with a structure similar to that of the eIF4E-cap complex of other species. NMR spectroscopy was used to show that the Cys that form the disulfide in the crystal are reduced in solution but can be induced to form the disulfide under oxidizing conditions. The observation that the disulfide-forming Cys are conserved in plants raises the possibility that their oxidation state may have a role in regulating protein function. NMR provided evidence that in oxidized eIF4E, the loop that is open in the ligand-free crystal dimer is relatively flexible in solution. An NMR-based binding assay showed that the reduced wheat eIF4E, the oxidized form with the disulfide, and the Cys-to-serine mutant protein each bind m{sup 7}GTP in a similar and labile manner, with dissociation rates in the range of 20

  8. Structures of cGMP-Dependent Protein Kinase (PKG) Iα Leucine Zippers Reveal an Interchain Disulfide Bond Important for Dimer Stability.

    PubMed

    Qin, Liying; Reger, Albert S; Guo, Elaine; Yang, Matthew P; Zwart, Peter; Casteel, Darren E; Kim, Choel

    2015-07-28

    cGMP-dependent protein kinase (PKG) Iα is a central regulator of smooth muscle tone and vasorelaxation. The N-terminal leucine zipper (LZ) domain dimerizes and targets PKG Iα by interacting with G-kinase-anchoring proteins. The PKG Iα LZ contains C42 that is known to form a disulfide bond upon oxidation and to activate PKG Iα. To understand the molecular details of the PKG Iα LZ and C42-C42' disulfide bond, we determined crystal structures of the PKG Iα wild-type (WT) LZ and C42L LZ. Our data demonstrate that the C42-C42' disulfide bond dramatically stabilizes PKG Iα and that the C42L mutant mimics the oxidized WT LZ structurally.

  9. Thiol, disulfide, and trisulfide complexes of Ru porphyrins: potential models for iron-sulfur bonds in heme proteins.

    PubMed

    Rebouças, Júlio S; Patrick, Brian O; James, Brian R

    2012-02-22

    Thirty-two Ru(porp)L(2) complexes have been synthesized, where porp = the dianion of meso-tetramesitylporphyrin (TMP) or meso-tetrakis(4-methylphenyl)porphyrin (H(2)T-pMe-PP), and L = a thiol, a sulfide, a disulfide, or a trisulfide. Species studied were with RSH [R = Me, Et, (n)Pr, (i)Pr, (t)Bu, Bn (benzyl), and Ph], RSR (R = Me, Bn), RSSR (R = Me, Et, (n)Pr, Bn) and MeSS(t)Bu, and RSSSR (R = Me, Bn). All the species except two, which were the isolated Ru(T-pMe-PP)((t)BuSH)(2) and Ru(TMP)(MeSSMe)(2), were characterized in situ. The disulfide complex was characterized by X-ray analysis. (1)H NMR data for the coordinated thiols are the first reported within metalloporphyrin systems, and are especially informative because of the upfield shifts of the axial sulfur-containing ligands due to the porphyrin π-ring current effect, which is also present in the di- and trisulfide species. The disulfide in the solid state structure of Ru(TMP)(MeSSMe)(2) is η(1)(end-on) coordinated, the first example of such bonding in a nontethered, acyclic dialkyl disulfide; (1)H-(1)H EXSY NMR data in solution show that the species undergoes 1,2-S-metallotropic shifts. Stepwise formation of the bis(disulfide) complex from Ru(TMP)(MeCN)(2) in solution occurs with a cooperativity effect, resembling behavior of Fe(II)-porphyrin systems where crystal field effects dominate, but ligand trans-effects are more likely in the Ru system. The η(1)(end-on) coordination mode is also favored for the trisulfide ligand. Discussed also are the remarkable linear correlations that exist between the ring-current shielding shifts for the axial ligand C(1) protons of Ru(porp)(RS(x)R)(2) and x (the number of S atoms). The Introduction briefly reviews literature on Ru- and Fe porphyrins (including heme proteins) with sulfur-containing ligands or substrates, and relationships between our findings and this literature are discussed throughout the paper.

  10. Purely heterometallic lanthanide(III) macrocycles through controlled assembly of disulfide bonds for dual color emission.

    PubMed

    Lewis, David J; Glover, Peter B; Solomons, Melissa C; Pikramenou, Zoe

    2011-02-02

    Lanthanide complexes based on bis(amides) of diethylenetriaminepentaacetic acid with thiol functionalities are modified with 2,2'-dipyridyl disulfide to give activated complexes that can selectively react with thiol-functionalized complexes to form heterometallic lanthanide macrocycles. The preparation and full characterization of the polyaminocarboxylate ligands N,N''-bis[p-thiophenyl(aminocarbonyl)]diethylenetriamine-N,N',N''-triacetic acid (H(3)L(x)) and the activated N,N''-bis[p-(pyridyldithio)[phenyl(aminocarbonyl)

  11. Proteins in vacuo: A molecular dynamics study of the unfolding behavior of highly charged disulfide-bond-intact lysozyme subjected to a temperature pulse

    NASA Astrophysics Data System (ADS)

    Reimann, C. T.; Velázquez, I.; Bittner, M.; Tapia, O.

    1999-12-01

    Molecular dynamics simulations were used to interpret a variety of experimental data on highly charged disulfide-bond-intact lysozyme in vacuo. The simulation approach involved submitting a model of the protein [Reimann, Velázquez, and Tapia, J. Phys. Chem. B 102, 9344 (1998)] in a given charge state to a 3-ns-long heat pulse (usually at 500 K) followed by cooling or relaxation for 1 ns back to room temperature (293 K). This treatment yielded a charge threshold around Q0=8+ for obtaining significant unfolding, as indicated by an enhancement in collision cross section and conformer length. The collision cross sections and lengths theoretically obtained, along with the threshold charge state for initiating unfolding, were compatible with experimental results on lysozyme in vacuo. The unfolded, highly elongated conformations obtained for Q>=9+ displayed a significant level of non-native β-sheet content which appeared to be additionally stabilized by charge self-solvation.

  12. Molecular dynamics simulation to investigate the impact of disulfide bond formation on conformational stability of chicken cystatin I66Q mutant.

    PubMed

    He, Jianwei; Xu, Linan; Zou, Zhiyuan; Ueyama, Nobuhiro; Li, Hui; Kato, Akio; Jones, Gary W; Song, Youtao

    2013-10-01

    Chicken cystatin (cC) mutant I66Q is located in the hydrophobic core of the protein and increases the propensity for amyloid formation. Here, we demonstrate that under physiological conditions, the replacement of Ile with the Gln in the I66Q mutant increases the susceptibility for the disulfide bond Cys71-Cys81 to be reduced when compared to the wild type (WT) cC. Molecular dynamics (MD) simulations under conditions favoring cC amyloid fibril formation are in agreement with the experimental results. MD simulations were also performed to investigate the impact of disrupting the Cys71-Cys81 disulfide bond on the conformational stability of cC at the atomic level, and highlighted major disruption to the cC appendant structure. Domain swapping and extensive unfolding has been proposed as one of the possible mechanisms initiating amyloid fibril formation by cystatin. Our in silico studies suggest that disulfide bond formation between residues Cys95 and Cys115 is necessary to maintain conformational stability of the I66Q mutant following breakage of the Cys71-Cys81 disulfide bridge. Subsequent breakage of disulfide bond Cys95-Cys115 resulted in large structural destabilization of the I66Q mutant, which increased the α-β interface distance and expanded the hydrophobic core. These experimental and computational studies provide molecular-level insight into the relationship between disulfide bond formation and progressive unfolding of amyloidogenic cC mutant I66Q. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:23.

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

    PubMed

    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.

  14. Formation and reshuffling of disulfide bonds in bovine serum albumin demonstrated using tandem mass spectrometry with collision-induced and electron-transfer dissociation.

    PubMed

    Rombouts, Ine; Lagrain, Bert; Scherf, Katharina A; Lambrecht, Marlies A; Koehler, Peter; Delcour, Jan A

    2015-07-20

    Thermolysin hydrolyzates of freshly isolated, extensively stored (6 years, 6 °C, dry) and heated (60 min, 90 °C, in excess water) bovine serum albumin (BSA) samples were analyzed with liquid chromatography (LC) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using alternating electron-transfer dissociation (ETD) and collision-induced dissociation (CID). The positions of disulfide bonds and free thiol groups in the different samples were compared to those deduced from the crystal structure of native BSA. Results revealed non-enzymatic posttranslational modifications of cysteine during isolation, extensive dry storage, and heating. Heat-induced extractability loss of BSA was linked to the impact of protein unfolding on the involvement of specific cysteine residues in intermolecular and intramolecular thiol-disulfide interchange and thiol oxidation reactions. The here developed approach holds promise for exploring disulfide bond formation and reshuffling in various proteins under conditions relevant for chemical, biochemical, pharmaceutical and food processing.

  15. 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.

    PubMed

    Jacquez, Pedro; Avila, Gustavo; Boone, Kyle; Altiyev, Agamyrat; Puschhof, Jens; Sauter, Roland; Arigi, Emma; Ruiz, Blanca; Peng, Xiuli; Almeida, Igor; Sherman, Michael; Xiao, Chuan; Sun, Jianjun

    2015-01-01

    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.

  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

    PubMed Central

    Boone, Kyle; Altiyev, Agamyrat; Puschhof, Jens; Sauter, Roland; Arigi, Emma; Ruiz, Blanca; Peng, Xiuli; Almeida, Igor; Sherman, Michael; Xiao, Chuan; Sun, Jianjun

    2015-01-01

    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. PMID:26107617

  17. The Role of Individual Disulfide Bonds of μ-Conotoxin GIIIA in the Inhibition of NaV1.4

    PubMed Central

    Han, Penggang; Wang, Kang; Dai, Xiandong; Cao, Ying; Liu, Shangyi; Jiang, Hui; Fan, Chongxu; Wu, Wenjian; Chen, Jisheng

    2016-01-01

    μ-Conotoxin GIIIA, a peptide toxin isolated from Conus geographus, preferentially blocks the skeletal muscle sodium channel NaV1.4. GIIIA folds compactly to a pyramidal structure stabilized by three disulfide bonds. To assess the contributions of individual disulfide bonds of GIIIA to the blockade of NaV1.4, seven disulfide-deficient analogues were prepared and characterized, each with one, two, or three pairs of disulfide-bonded Cys residues replaced with Ala. The inhibitory potency of the analogues against NaV1.4 was assayed by whole cell patch-clamp on rNaV1.4, heterologously expressed in HEK293 cells. The corresponding IC50 values were 0.069 ± 0.005 μM for GIIIA, 2.1 ± 0.3 μM for GIIIA-1, 3.3 ± 0.2 μM for GIIIA-2, and 15.8 ± 0.8 μM for GIIIA-3 (-1, -2 and -3 represent the removal of disulfide bridges Cys3–Cys15, Cys4–Cys20 and Cys10–Cys21, respectively). Other analogues were not active enough for IC50 measurement. Our results indicate that all three disulfide bonds of GIIIA are required to produce effective inhibition of NaV1.4, and the removal of any one significantly lowers its sodium channel binding affinity. Cys10–Cys21 is the most important for the NaV1.4 potency. PMID:27869701

  18. Contrasting the individual reactive pathways in protein unfolding and disulfide bond reduction observed within a single protein.

    PubMed

    Garcia-Manyes, Sergi; Kuo, Tzu-Ling; Fernández, Julio M

    2011-03-09

    Identifying the dynamics of individual molecules along their reactive pathways remains a major goal of modern chemistry. For simple chemical reactions, the transition state position is thought to be highly localized. Conversely, in the case of more complex reactions involving proteins, the potential energy surfaces become rougher, resulting in heterogeneous reaction pathways with multiple transition state structures. Force-clamp spectroscopy experimentally probes the individual reaction pathways sampled by a single protein under the effect of a constant stretching force. Herein, we examine the distribution of conformations that populate the transition state of two different reactions; the unfolding of a single protein and the reduction of a single disulfide bond, both occurring within the same single protein. By applying the recently developed static disorder theory, we quantify the variance of the barrier heights, σ(2), governing each distinct reaction. We demonstrate that the unfolding of the I27 protein follows a nonexponential kinetics, consistent with a high value of σ(2) ∼ 18 (pN nm)(2). Interestingly, shortening of the protein upon introduction of a rigid disulfide bond significantly modulates the disorder degree, spanning from σ(2) ∼ 8 to ∼21 (pN nm)(2). These results are in sharp contrast with the exponential distribution of times measured for an S(N)2 chemical reaction, implying the absence of static disorder σ(2) ∼ 0 (pN nm)(2). Our results demonstrate the high sensitivity of the force-clamp technique to capture the signatures of disorder in the individual pathways that define two distinct force-induced reactions, occurring within the core of a single protein.

  19. Redox‐dependent disulfide bond formation in SAP30L corepressor protein: Implications for structure and function

    PubMed Central

    Laitaoja, Mikko; Tossavainen, Helena; Pihlajamaa, Tero; Valjakka, Jarkko; Viiri, Keijo; Lohi, Olli; Permi, Perttu

    2015-01-01

    Abstract Sin3A‐associated protein 30‐like (SAP30L) is one of the key proteins in a multi‐subunit protein complex involved in transcriptional regulation via histone deacetylation. SAP30L, together with a highly homologous SAP30 as well as other SAP proteins (i.e., SAP25, SAP45, SAP130, and SAP180), is an essential component of the Sin3A corepressor complex, although its actual role has remained elusive. SAP30L is thought to function as an important stabilizing and bridging molecule in the complex and to mediate its interactions with other corepressors. SAP30L has been previously shown to contain an N‐terminal Cys3His type zinc finger (ZnF) motif, which is responsible for the key protein–protein, protein–DNA, and protein–lipid interactions. By using high‐resolution mass spectrometry, we studied a redox‐dependent disulfide bond formation in SAP30L ZnF as a regulatory mechanism for its structure and function. We showed that upon oxidative stress SAP30L undergoes the formation of two specific disulfide bonds, a vicinal Cys29‐Cys30 and Cys38‐Cys74, with a concomitant release of the coordinated zinc ion. The oxidized protein was shown to remain folded in solution and to bind signaling phospholipids. We also determined a solution NMR structure for SAP30L ZnF that showed an overall fold similar to that of SAP30, determined earlier. The NMR titration experiments with lipids and DNA showed that the binding is mediated by the C‐terminal tail as well as both α‐helices of SAP30L ZnF. The implications of these results for the structure and function of SAP30L are discussed. PMID:26609676

  20. Cytotoxicities of two disulfide-bond-linked conjugates of methotrexate with monoclonal anti-MM46 antibody.

    PubMed

    Umemoto, N; Kato, Y; Hara, T

    1989-01-01

    In studies on (antitumor antibody)-drug conjugates as potential antitumor agents, the amide derivatives of methotrexate (MTX) with cysteine and with 2-mercaptoethylamine (cysteamine) (MTX-Cys and MTX-MEA, respectively) were linked via a disulfide bond with a monoclonal antibody (alpha MM46) to a mouse mammary tumor MM46 with attached 3-(2-pyridyldithio) propionyl groups to give conjugates of MTX with alpha MM46 (MTX-Cys-SS-alpha MM46 and MTX-MEA-SS-alpha MM46, respectively). These two conjugates are both linked by a disulfide bond and are very similar in structure, but MTX-MEA-SS-alpha MM46 showed only weak in vitro cytotoxicity against MM46 cells, whereas MTX-Cys-SS-alpha MM46 had strong cytotoxicity. The cytotoxicity of the latter was comparable to that of the conventional direct MTX-alpha MM46 conjugate prepared with an MTX-active ester. However, this conjugate had a greater selectivity than that of the direct conjugate, calculated as the IC50 (concentration of a conjugate by MTX equivalence required for suppression of the number of viable MM46 cells to 50% of that of the untreated control) for the corresponding nonspecific conjugate divided by the IC50 for the alpha MM46 conjugate. The inhibitory activities of MTX-Cys and MTX-MEA on dihydrofolate reductase were similar. The cytotoxicity of MTX-Cys-SS-alpha MM46 was not affected by thiamine pyrophosphate, an inhibitor of the active transport of MTX across the cell membrane, but was decreased significantly by ammonium chloride, a lysosomotropic amine. However, the cytotoxicity was decreased only to a small extent by leupeptin, an inhibitor of lysosomal cysteine proteases cathepsins B, H, and L. These results suggest that the cytotoxicity is mediated by lysosomes, and may involve lysosomal enzymes other than cathepsins B, H, and L.

  1. Effect of immunoglobulin G (IgG) interchain disulfide bond cleavage on efficacy of intravenous immunoglobulin for immune thrombocytopenic purpura (ITP).

    PubMed

    Machino, Y; Ohta, H; Suzuki, E; Higurashi, S; Tezuka, T; Nagashima, H; Kohroki, J; Masuho, Y

    2010-12-01

    Intravenous immunoglobulin (IVIG) has been used widely to treat immune thrombocytopenic purpura (ITP), but the mechanisms of its action remain unclear. We investigated the affinity for Fcγ receptors (FcγRs) and the thrombocytopenia-ameliorating effect of S-sulfonated gammaglobulin (SGG) and S-alkylated gammaglobulin (AGG), in comparison with unmodified gammaglobulin (GG), in a mouse ITP model. Cleavage of immunoglobulin (Ig)G interchain disulfide bonds by either S-sulfonation or S-alkylation did not decrease the affinity for FcγRIIA (CD32A) and FcγRIIB (CD32B), but did decrease the affinity for FcγRIA (CD64A) and FcγRIIIA (CD16A), presumably because of changes in H-chain configuration. The interchain disulfide bond cleavage decreased the affinity much more for mouse FcγRIV than for mouse FcγRIIB. The ability of AGG to ameliorate ITP was greatly diminished, while SGG, whose disulfide bonds are reconstituted in vivo, was as effective as GG. These results suggest that the interchain disulfide bonds are important for therapeutic effect. It is also suggested that the interaction of IVIG with the inhibitory receptor FcγRIIB is insufficient for effective amelioration of ITP and that, at least in this model, direct binding of IVIG to FcγRIIIA is also required.

  2. Inverse-Micelle-Encapsulated Water-Enabled Bond Breaking of Dialkyl Diselenide/Disulfide: A Critical Step for Synthesizing High- Quality Gold Nanoparticles

    SciTech Connect

    Zaluzhna, Oksana; Li, Ying; Allison, Thomas C.; Tong, Yu ye J.

    2012-10-09

    Inverse-micelle-encapsulated water formed in the two-phase Brust-Schiffrin method (BSM) synthesis of Au nanoparticles (NPs) is identified as essential for dialkyl diselenide/disulfide to react with the Au(III) complex in which the Se-Se/S-S bond is broken, leading to formation of higher-quality Au NPs.

  3. Role of disulfide bonds in maintaining the structural integrity of the sheath of Leptothrix discophora SP-6.

    PubMed Central

    Emerson, D; Ghiorse, W C

    1993-01-01

    Isolated sheaths of Leptothrix discophora SP-6 (ATCC 51168) were tested for susceptibility to degradation by a variety of chemical denaturants and lytic enzymes and found to be resistant to many reagents and enzyme treatments. However, disulfide bond-reducing agents such as dithiothreitol (DTT), beta-mercaptoethanol, sodium cyanide, and sodium sulfite degraded the sheath, especially at elevated pH (pH 9) and temperature (50 degrees C). DTT and beta-mercaptoethanol caused more rapid degradation of the sheath than cyanide or sulfite. Treatment of the sheath with 1 N NaOH resulted in rapid breakdown, while treatment with 1 N HCl resulted in slow but significant hydrolysis. Transmission electron microscopy showed that the 6.5-nm fibrils previously shown to be an integral structural element of the sheath fabric (D. Emerson and W. C. Ghiorse, J. Bacteriol. 175:7808-7818, 1993) were progressively dissociated into random masses during DTT-induced degradation. Quantitation of disulfide bonds with DTT showed that the sheaths contained approximately 2.2 mumol of disulfides per mg of sheath protein. Reaction with 5,5'-dithio-bis-(2-nitrobenzoic acid) showed that sheaths also contained approximately 0.8 mumol of free sulfhydryls per mg of protein. A sulfhydryl-specific fluorescent probe (fluorescein 5-maleimide) showed that the free sulfhydryls in sheathed cell filaments were evenly distributed throughout the sheath. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography of [14C]iodoacetamide-labeled sheaths and DTT-dissociated sheath fibril suspensions showed that the majority of 14C-labeled sulfhydryls in the sheaths did not enter the gel. However, low-molecular-mass silver-staining bands (14 to 45 kDa) did appear in the gels after iodoacetic acid or iodoacetamide alkylation of the dissociated fibrils. These bands did not stain with Coomassie blue. Their migration in gels was slightly affected by digestion with pronase. The fibrils contained 20 to 25

  4. Elucidating the Role of Disulfide Bond on Amyloid Formation and Fibril Reversibility of Somatostatin-14

    PubMed Central

    Anoop, Arunagiri; Ranganathan, Srivastav; Dhaked, Bhagwan Das; Jha, Narendra Nath; Pratihar, Supriya; Ghosh, Saikat; Sahay, Shruti; Kumar, Santosh; Das, Subhadeep; Kombrabail, Mamata; Agarwal, Kumud; Jacob, Reeba S.; Singru, Praful; Bhaumik, Prasenjit; Padinhateeri, Ranjith; Kumar, Ashutosh; Maji, Samir K.

    2014-01-01

    The storage of protein/peptide hormones within subcellular compartments and subsequent release are crucial for their native function, and hence these processes are intricately regulated in mammalian systems. Several peptide hormones were recently suggested to be stored as amyloids within endocrine secretory granules. This leads to an apparent paradox where storage requires formation of aggregates, and their function requires a supply of non-aggregated peptides on demand. The precise mechanism behind amyloid formation by these hormones and their subsequent release remain an open question. To address this, we examined aggregation and fibril reversibility of a cyclic peptide hormone somatostatin (SST)-14 using various techniques. After proving that SST gets stored as amyloid in vivo, we investigated the role of native structure in modulating its conformational dynamics and self-association by disrupting the disulfide bridge (Cys3–Cys14) in SST. Using two-dimensional NMR, we resolved the initial structure of somatostatin-14 leading to aggregation and further probed its conformational dynamics in silico. The perturbation in native structure (S-S cleavage) led to a significant increase in conformational flexibility and resulted in rapid amyloid formation. The fibrils formed by disulfide-reduced noncyclic SST possess greater resistance to denaturing conditions with decreased monomer releasing potency. MD simulations reveal marked differences in the intermolecular interactions in SST and noncyclic SST providing plausible explanation for differential aggregation and fibril reversibility observed experimentally in these structural variants. Our findings thus emphasize that subtle changes in the native structure of peptide hormone(s) could alter its conformational dynamics and amyloid formation, which might have significant implications on their reversible storage and secretion. PMID:24782311

  5. The Kringle-like Domain Facilitates Post-endoplasmic Reticulum Changes to Premelanosome Protein (PMEL) Oligomerization and Disulfide Bond Configuration and Promotes Amyloid Formation*

    PubMed Central

    Ho, Tina; Watt, Brenda; Spruce, Lynn A.; Seeholzer, Steven H.; Marks, Michael S.

    2016-01-01

    The formation of functional amyloid must be carefully regulated to prevent the accumulation of potentially toxic products. Premelanosome protein (PMEL) forms non-toxic functional amyloid fibrils that assemble into sheets upon which melanins ultimately are deposited within the melanosomes of pigment cells. PMEL is synthesized in the endoplasmic reticulum but forms amyloid only within post-Golgi melanosome precursors; thus, PMEL must traverse the secretory pathway in a non-amyloid form. Here, we identified two pre-amyloid PMEL intermediates that likely regulate the timing of fibril formation. Analyses by non-reducing SDS-PAGE, size exclusion chromatography, and sedimentation velocity revealed two native high Mr disulfide-bonded species that contain Golgi-modified forms of PMEL. These species correspond to disulfide bond-containing dimeric and monomeric PMEL isoforms that contain no other proteins as judged by two-dimensional PAGE of metabolically labeled/immunoprecipitated PMEL and by mass spectrometry of affinity-purified complexes. Metabolic pulse-chase analyses, small molecule inhibitor treatments, and evaluation of site-directed mutants suggest that the PMEL dimer forms around the time of endoplasmic reticulum exit and is resolved by disulfide bond rearrangement into a monomeric form within the late Golgi or a post-Golgi compartment. Mutagenesis of individual cysteine residues within the non-amyloid cysteine-rich Kringle-like domain stabilizes the disulfide-bonded dimer and impairs fibril formation as determined by electron microscopy. Our data show that the Kringle-like domain facilitates the resolution of disulfide-bonded PMEL dimers and promotes PMEL functional amyloid formation, thereby suggesting that PMEL dimers must be resolved to monomers to generate functional amyloid fibrils. PMID:26694611

  6. Stereochemistry of enzymatic water addition to C=C bonds.

    PubMed

    Chen, Bi-Shuang; Otten, Linda G; Hanefeld, Ulf

    2015-01-01

    Water addition to carbon-carbon double bonds using hydratases is attracting great interest in biochemistry. Most of the known hydratases are involved in primary metabolism and to a lesser extent in secondary metabolism. New hydratases have recently been added to the toolbox, both from natural sources or artificial metalloenzymes. In order to comprehensively understand how the hydratases are able to catalyse the water addition to carbon-carbon double bonds, this review will highlight the mechanistic and stereochemical studies of the enzymatic water addition to carbon-carbon double bonds, focusing on the syn/anti-addition and stereochemistry of the reaction.

  7. 29 CFR 2580.412-20 - Use of existing bonds, separate bonds and additional bonding.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... bonding. 2580.412-20 Section 2580.412-20 Labor Regulations Relating to Labor (Continued) EMPLOYEE BENEFITS SECURITY ADMINISTRATION, DEPARTMENT OF LABOR TEMPORARY BONDING RULES UNDER THE EMPLOYEE RETIREMENT INCOME SECURITY ACT OF 1974 TEMPORARY BONDING RULES General Bond Rules § 2580.412-20 Use of existing...

  8. 29 CFR 2580.412-20 - Use of existing bonds, separate bonds and additional bonding.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... bonding. 2580.412-20 Section 2580.412-20 Labor Regulations Relating to Labor (Continued) EMPLOYEE BENEFITS SECURITY ADMINISTRATION, DEPARTMENT OF LABOR TEMPORARY BONDING RULES UNDER THE EMPLOYEE RETIREMENT INCOME SECURITY ACT OF 1974 TEMPORARY BONDING RULES General Bond Rules § 2580.412-20 Use of existing...

  9. Accumulation of β-Conglycinin in Soybean Cotyledon through the Formation of Disulfide Bonds between α′- and α-Subunits1[W][OA

    PubMed Central

    Wadahama, Hiroyuki; Iwasaki, Kensuke; Matsusaki, Motonori; Nishizawa, Keito; Ishimoto, Masao; Arisaka, Fumio; Takagi, Kyoko; Urade, Reiko

    2012-01-01

    β-Conglycinin, one of the major soybean (Glycine max) seed storage proteins, is folded and assembled into trimers in the endoplasmic reticulum and accumulated into protein storage vacuoles. Prior experiments have used soybean β-conglycinin extracted using a reducing buffer containing a sulfhydryl reductant such as 2-mercaptoethanol, which reduces both intermolecular and intramolecular disulfide bonds within the proteins. In this study, soybean proteins were extracted from the cotyledons of immature seeds or dry beans under nonreducing conditions to prevent the oxidation of thiol groups and the reduction or exchange of disulfide bonds. We found that approximately half of the α′- and α-subunits of β-conglycinin were disulfide linked, together or with P34, prior to amino-terminal propeptide processing. Sedimentation velocity experiments, size-exclusion chromatography, and two-dimensional polyacrylamide gel electrophoresis (PAGE) analysis, with blue native PAGE followed by sodium dodecyl sulfate-PAGE, indicated that the β-conglycinin complexes containing the disulfide-linked α′/α-subunits were complexes of more than 720 kD. The α′- and α-subunits, when disulfide linked with P34, were mostly present in approximately 480-kD complexes (hexamers) at low ionic strength. Our results suggest that disulfide bonds are formed between α′/α-subunits residing in different β-conglycinin hexamers, but the binding of P34 to α′- and α-subunits reduces the linkage between β-conglycinin hexamers. Finally, a subset of glycinin was shown to exist as noncovalently associated complexes larger than hexamers when β-conglycinin was expressed under nonreducing conditions. PMID:22218927

  10. Efficient export of human growth hormone, interferon α2b and antibody fragments to the periplasm by the Escherichia coli Tat pathway in the absence of prior disulfide bond formation.

    PubMed

    Alanen, Heli I; Walker, Kelly L; Lourdes Velez Suberbie, M; Matos, Cristina F R O; Bönisch, Sarah; Freedman, Robert B; Keshavarz-Moore, Eli; Ruddock, Lloyd W; Robinson, Colin

    2015-03-01

    Numerous therapeutic proteins are expressed in Escherichia coli and targeted to the periplasm in order to facilitate purification and enable disulfide bond formation. Export is normally achieved by the Sec pathway, which transports proteins through the plasma membrane in a reduced, unfolded state. The Tat pathway is a promising alternative means of export, because it preferentially exports correctly folded proteins; however, the reducing cytoplasm of standard strains has been predicted to preclude export by Tat of proteins that contain disulfide bonds in the native state because, in the reduced state, they are sensed as misfolded and rejected. Here, we have tested a series of disulfide-bond containing biopharmaceuticals for export by the Tat pathway in CyDisCo strains that do enable disulfide bond formation in the cytoplasm. We show that interferon α2b, human growth hormone (hGH) and two antibody fragments are exported with high efficiency; surprisingly, however, they are efficiently exported even in the absence of cytoplasmic disulfide formation. The exported proteins acquire disulfide bonds in the periplasm, indicating that the normal disulfide oxidation machinery is able to act on the proteins. Tat-dependent export of hGH proceeds even when the disulfide bonds are removed by substitution of the Cys residues involved, suggesting that these substrates adopt tertiary structures that are accepted as fully-folded by the Tat machinery.

  11. Insights into the mechanism of X-ray-induced disulfide-bond cleavage in lysozyme crystals based on EPR, optical absorption and X-ray diffraction studies

    SciTech Connect

    Sutton, Kristin A.; Black, Paul J.; Mercer, Kermit R.; Garman, Elspeth F.; Owen, Robin L.; Snell, Edward H.; Bernhard, William A.

    2013-12-01

    Electron paramagnetic resonance (EPR) and online UV–visible absorption microspectrophotometry with X-ray crystallography have been used in a complementary manner to follow X-ray-induced disulfide-bond cleavage, to confirm a multi-track radiation-damage process and to develop a model of that process. Electron paramagnetic resonance (EPR) and online UV–visible absorption microspectrophotometry with X-ray crystallography have been used in a complementary manner to follow X-ray-induced disulfide-bond cleavage. Online UV–visible spectroscopy showed that upon X-irradiation, disulfide radicalization appeared to saturate at an absorbed dose of approximately 0.5–0.8 MGy, in contrast to the saturating dose of ∼0.2 MGy observed using EPR at much lower dose rates. The observations suggest that a multi-track model involving product formation owing to the interaction of two separate tracks is a valid model for radiation damage in protein crystals. The saturation levels are remarkably consistent given the widely different experimental parameters and the range of total absorbed doses studied. The results indicate that even at the lowest doses used for structural investigations disulfide bonds are already radicalized. Multi-track considerations offer the first step in a comprehensive model of radiation damage that could potentially lead to a combined computational and experimental approach to identifying when damage is likely to be present, to quantitate it and to provide the ability to recover the native unperturbed structure.

  12. Roles of tryptophan residue and disulfide bond in the variable lid region of oxidized polyvinyl alcohol hydrolase.

    PubMed

    Yang, Yu; Ko, Tzu-Ping; Liu, Long; Li, Jianghua; Huang, Chun-Hsiang; Chen, Jian; Guo, Rey-Ting; Du, Guocheng

    2014-09-26

    Oxidized polyvinyl alcohol hydrolase (OPH) catalyzes the cleavage of C-C bond in β-diketone. It belongs to the α/β-hydrolase family and contains a unique lid region that covers the active site. The lid is the most variable region when pOPH from Pseudomonas sp. VM15C and sOPH from Sphingopyxis sp. 113P3 are compared. The wild-type enzymes and the pOPH mutants W255A, W255Y and W255F were analyzed for lipase activity by using p-nitrophenyl (pNP) esters as the substrates. The wild-type enzymes showed increased Km and decreased kcat/Km with the acyl chain length, and the mutants showed reduced kcat/Km for pNP acetate, indicating the importance of Trp255 in sequestering the active site from solvent. The significantly lower activity for pNP butyrate can be a result of product inhibition, as suggested by the complex crystal structures, in which butyric acid, DMSO or PEG occupied the same substrate-binding cleft. The mutant activity was retained with pNP caprylate and pNP laurate as the substrates, reflecting the amphipathic nature of the cleft. Moreover, the disulfide bond formation of Cys257/267 is important for the activity of pOPH, but it is not essential for sOPH, which has a shorter lid structure.

  13. Evidence for disulfide bonds in SR Protein Kinase 1 (SRPK1) that are required for activity and nuclear localization

    PubMed Central

    Koutroumani, Maria; Papadopoulos, Georgios E.; Vlassi, Metaxia; Nikolakaki, Eleni; Giannakouros, Thomas

    2017-01-01

    Serine/arginine protein kinases (SRPKs) phosphorylate Arg/Ser dipeptide-containing proteins that play crucial roles in a broad spectrum of basic cellular processes. The existence of a large internal spacer sequence that separates the bipartite kinase catalytic core is a unique structural feature of SRPKs. Previous structural studies on a catalytically active fragment of SRPK1, which lacks the main part of the spacer domain, revealed that SRPK1 remains in an active state without any post-translational modifications or specific intra-protein interactions, while the spacer domain is depicted as a loop structure, outside the kinase core. Using systematic mutagenesis we now provide evidence that replacement of any individual cysteine residue in the spacer, apart from Cys414, or in its proximal flaking ends of the two kinase catalytic domains has an impact on kinase activity. Furthermore, the cysteine residues are critical for nuclear translocation of SRPK1 in response to genotoxic stress and SRPK1-dependent splicing of a reporter gene. While replacement of Cys207, Cys502 and Cys539 of the catalytic domains is predicted to distort the kinase active structure, our findings suggest that Cys356, Cys386, Cys427 and Cys455 of the spacer domain and Cys188 of the first catalytic domain are engaged in disulfide bridging. We propose that such a network of intramolecular disulfide bonds mediates the bending of the spacer region thus allowing the proximal positioning of the two catalytic subunits which is a prerequisite for SRPK1 activity. PMID:28166275

  14. Hypertriglyceridemia associated with the c.553G>T APOA5 SNP results from aberrant hetero-disulfide bond formation

    PubMed Central

    Sharma, Vineeta; Witkowski, Andrzej; Witkowska, H. Ewa; Dykstra, Andrew; Simonsen, Jens B.; Nelbach, Lisa; Beckstead, Jennifer A.; Pullinger, Clive R.; Kane, John P.; Malloy, Mary J.; Watson, Gordon; Forte, Trudy M.; Ryan, Robert O.

    2014-01-01

    Objective Apolipoprotein (apo) A-V is a low abundance plasma protein that modulates triacylglycerol (TG) homeostasis. Gene transfer studies were undertaken in apoa5 (−/−) mice to define the mechanism underlying the correlation between the single nucleotide polymorphism (SNP) c.553G>T in APOA5 and hypertriglyceridemia (HTG). Approach and Results Adeno-associated virus (AAV) 2/8 mediated gene transfer of wild type (WT) apoA-V induced a dramatic lowering of plasma TG in apoa5 (−/−) mice while AAV2/8-Gly162Cys apoA-V (corresponding to the c.553G>T SNP: rs2075291) had a modest effect. Characterization studies revealed that plasma levels of WT- and G162C apoA-V in transduced mice were similar and within the physiological range. Fractionation of plasma from mice transduced with AAV2/8-G162C apoA-V indicated that, unlike WT apoA-V, >50% of G162C apoA-V was recovered in the lipoprotein-free fraction. Non-reducing SDS-PAGE immunoblot analysis provided evidence that G162C apoA-V present in the lipoprotein-free fraction, but not that portion associated with lipoproteins, displayed altered electrophoretic mobility consistent with disulfide-linked hetero-dimer formation. Immunoprecipitation followed by liquid chromatography/mass spectrometry of human plasma from subjects homozygous for WT APOA5 and c.553G>T APOA5 revealed that G162C apoA-V forms adducts with extraneous plasma proteins including fibronectin, kininogen-1 and others. Conclusion Substitution of Cys for Gly at position 162 of mature apoA-V introduces a free cysteine that forms disulfide bonds with plasma proteins such that its lipoprotein binding and TG modulation functions are compromised. PMID:25127531

  15. Structure-activity relation of human beta-defensin 3: influence of disulfide bonds and cysteine substitution on antimicrobial activity and cytotoxicity.

    PubMed

    Klüver, Enno; Schulz-Maronde, Sandra; Scheid, Svenja; Meyer, Bernd; Forssmann, Wolf-Georg; Adermann, Knut

    2005-07-19

    Human beta-defensins form a group of cysteine-rich antimicrobial peptides which have been found in epithelial tissue and, more recently, in the male genital tract. They play a role in the defense against microbial pathogens in innate immunity and display additional chemotactic functions in the adaptive immune system. An important characteristic of antimicrobial peptides is that they also exhibit toxic potential on eukaryotic cells. Very little is known about the structure dependence of antimicrobial and cytotoxic effects. We investigated human beta-defensin 3 (hBD-3), a potent broad-spectrum antimicrobial effector peptide, regarding the influence of structural parameters on the antimicrobial and cytotoxic activity. We have established a structure-activity relation of the hBD-3 using synthetic derivatives differing in length, charge, disulfide connectivity, and overall hydrophobicity. The antimicrobial activity of the peptides was compared to the cyctotoxic effects on monocytic THP-1 cells and the hemolytic activity on human erythrocytes. We found that it is not important for antimicrobial and cytotoxic activity whether and how cysteine residues are arranged to form disulfide bonds. Substitution of half-cystinyl residues by tryptophan resulted in increased activities, while other substitutions did not change activity. Correlation of activities with the structural changes demonstrates that the activity on eukaryotic cells appears to depend strongly on the overall hydrophobicity. In contrast, the antimicrobial potency of hBD-3 peptides is determined by the distribution of positively charged amino acid residues and hydrophobic side chains. The results facilitate the understanding of beta-defensin interaction with different cell types and guide the design of antimicrobially active peptides.

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

    PubMed Central

    Xu, Xin; Li, Qi

    2016-01-01

    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. PMID:27100881

  17. Prediction of the disulfide-bonding state of cysteines in proteins at 88% accuracy

    PubMed Central

    Martelli, Pier Luigi; Fariselli, Piero; Malaguti, Luca; Casadio, Rita

    2002-01-01

    The task of predicting the cysteine-bonding state in proteins starting from the residue chain is addressed by implementing a new hybrid system that combines a neural network and a hidden Markov model (hidden neural network). Training is performed using 4136 cysteine-containing segments extracted from 969 nonhomologous proteins of well-resolved three-dimensional structure. After a 20-fold cross-validation procedure, the efficiency of the prediction scores as high as 88% and 84%, when measured on cysteine and protein basis, respectively. These results outperform previously described methods for the same task. PMID:12381855

  18. OX133, a monoclonal antibody recognizing protein-bound N-ethylmaleimide for the identification of reduced disulfide bonds in proteins

    PubMed Central

    Holbrook, Lisa-Marie; Kwong, Lai-Shan; Metcalfe, Clive L.; Fenouillet, Emmanuel; Jones, Ian M.; Barclay, A. Neil

    2016-01-01

    ABSTRACT In vivo, enzymatic reduction of some protein disulfide bonds, allosteric disulfide bonds, provides an important level of structural and functional regulation. The free cysteine residues generated can be labeled by maleimide reagents, including biotin derivatives, allowing the reduced protein to be detected or purified. During the screening of monoclonal antibodies for those specific for the reduced forms of proteins, we isolated OX133, a unique antibody that recognizes polypeptide resident, N-ethylmaleimide (NEM)-modified cysteine residues in a sequence-independent manner. OX133 offers an alternative to biotin-maleimide reagents for labeling reduced/alkylated antigens and capturing reduced/alkylated proteins with the advantage that NEM-modified proteins are more easily detected in mass spectrometry, and may be more easily recovered than is the case following capture with biotin based reagents. PMID:26986548

  19. OX133, a monoclonal antibody recognizing protein-bound N-ethylmaleimide for the identification of reduced disulfide bonds in proteins.

    PubMed

    Holbrook, Lisa-Marie; Kwong, Lai-Shan; Metcalfe, Clive L; Fenouillet, Emmanuel; Jones, Ian M; Barclay, A Neil

    2016-01-01

    In vivo, enzymatic reduction of some protein disulfide bonds, allosteric disulfide bonds, provides an important level of structural and functional regulation. The free cysteine residues generated can be labeled by maleimide reagents, including biotin derivatives, allowing the reduced protein to be detected or purified. During the screening of monoclonal antibodies for those specific for the reduced forms of proteins, we isolated OX133, a unique antibody that recognizes polypeptide resident, N-ethylmaleimide (NEM)-modified cysteine residues in a sequence-independent manner. OX133 offers an alternative to biotin-maleimide reagents for labeling reduced/alkylated antigens and capturing reduced/alkylated proteins with the advantage that NEM-modified proteins are more easily detected in mass spectrometry, and may be more easily recovered than is the case following capture with biotin based reagents.

  20. A disulfide-bonded dimer of the core protein of hepatitis C virus is important for virus-like particle production.

    PubMed

    Kushima, Yukihiro; Wakita, Takaji; Hijikata, Makoto

    2010-09-01

    Hepatitis C virus (HCV) core protein forms the nucleocapsid of the HCV particle. Although many functions of core protein have been reported, how the HCV particle is assembled is not well understood. Here we show that the nucleocapsid-like particle of HCV is composed of a disulfide-bonded core protein complex (dbc-complex). We also found that the disulfide-bonded dimer of the core protein (dbd-core) is formed at the endoplasmic reticulum (ER), where the core protein is initially produced and processed. Mutational analysis revealed that the cysteine residue at amino acid position 128 (Cys128) of the core protein, a highly conserved residue among almost all reported isolates, is responsible for dbd-core formation and virus-like particle production but has no effect on the replication of the HCV RNA genome or the several known functions of the core protein, including RNA binding ability and localization to the lipid droplet. The Cys128 mutant core protein showed a dominant negative effect in terms of HCV-like particle production. These results suggest that this disulfide bond is critical for the HCV virion. We also obtained the results that the dbc-complex in the nucleocapsid-like structure was sensitive to proteinase K but not trypsin digestion, suggesting that the capsid is built up of a tightly packed structure of the core protein, with its amino (N)-terminal arginine-rich region being concealed inside.

  1. The Disulfide Bond of the Peptide Thanatin Is Dispensible for Its Antimicrobial Activity In Vivo and In Vitro.

    PubMed

    Ma, Bo; Niu, Chao; Zhou, Ying; Xue, Xiaoyan; Meng, Jingru; Luo, Xiaoxing; Hou, Zheng

    2016-07-01

    Thanatin (THA) displays potent antibiotic activity, especially against extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli both in vitro and in vivo, with minimal hemolytic toxicity and satisfactory stability in plasma. However, the high cost of thanatin significantly limits its development and clinical application. To reduce the cost of peptide synthesis, a formulation of cyclic thanatin (C-thanatin) called linear thanatin (L-thanatin) was synthesized and its activity was evaluated in vivo and in vitro Results showed that C-thanatin and L-thanatin MICs did not differ against eight Gram-negative and two Gram-positive bacterial strains. Furthermore, the survival rates of ESBL-producing-E. coli-infected mice were consistent after C-thanatin or L-thanatin treatment at 5 or 10 mg/kg of body weight. Neither C-thanatin nor L-thanatin showed toxicity for human red blood cells (hRBCs) and human umbilical vein endothelial cells (HUVECs) at a concentration as high as 256 μg/ml. Results of circular dichroism spectroscopy indicated that the secondary structure of L-thanatin is extremely similar to that of C-thanatin. Membrane permeabilization and depolarization assays showed that C-thanatin and L-thanatin have similar abilities to permeabilize the outer and inner membranes and to induce membrane depolarization in ESBL-producing E. coli However, neither of them caused significant HUVEC membrane permeability. These findings indicate that the two peptides have similar effects on bacterial cell membranes and that the disulfide bond in thanatin is not essential for its antimicrobial activities in vivo and in vitro L-thanatin is thus a promising low-cost peptide candidate for treating ESBL-producing E. coli infections.

  2. The Disulfide Bond of the Peptide Thanatin Is Dispensible for Its Antimicrobial Activity In Vivo and In Vitro

    PubMed Central

    Ma, Bo; Niu, Chao; Zhou, Ying; Xue, Xiaoyan; Meng, Jingru

    2016-01-01

    Thanatin (THA) displays potent antibiotic activity, especially against extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli both in vitro and in vivo, with minimal hemolytic toxicity and satisfactory stability in plasma. However, the high cost of thanatin significantly limits its development and clinical application. To reduce the cost of peptide synthesis, a formulation of cyclic thanatin (C-thanatin) called linear thanatin (L-thanatin) was synthesized and its activity was evaluated in vivo and in vitro. Results showed that C-thanatin and L-thanatin MICs did not differ against eight Gram-negative and two Gram-positive bacterial strains. Furthermore, the survival rates of ESBL-producing-E. coli-infected mice were consistent after C-thanatin or L-thanatin treatment at 5 or 10 mg/kg of body weight. Neither C-thanatin nor L-thanatin showed toxicity for human red blood cells (hRBCs) and human umbilical vein endothelial cells (HUVECs) at a concentration as high as 256 μg/ml. Results of circular dichroism spectroscopy indicated that the secondary structure of L-thanatin is extremely similar to that of C-thanatin. Membrane permeabilization and depolarization assays showed that C-thanatin and L-thanatin have similar abilities to permeabilize the outer and inner membranes and to induce membrane depolarization in ESBL-producing E. coli. However, neither of them caused significant HUVEC membrane permeability. These findings indicate that the two peptides have similar effects on bacterial cell membranes and that the disulfide bond in thanatin is not essential for its antimicrobial activities in vivo and in vitro. L-thanatin is thus a promising low-cost peptide candidate for treating ESBL-producing E. coli infections. PMID:27161645

  3. Association of poly(ADP-ribose) polymerase with the nuclear matrix: the role of intermolecular disulfide bond formation, RNA retention, and cell type.

    PubMed

    Kaufmann, S H; Brunet, G; Talbot, B; Lamarr, D; Dumas, C; Shaper, J H; Poirier, G

    1991-02-01

    The recovery of the enzyme poly(ADP-ribose) polymerase (pADPRp) in the nuclease- and 1.6 M NaCl-resistant nuclear subfraction prepared from a number of different sources was assessed by Western blotting. When rat liver nuclei were treated with DNase I and RNase A followed by 1.6 M NaCl, approximately 10% of the nuclear pADPRp was recovered in the sedimentable fraction. The proportion of pADPRp recovered with the residual fraction decreased to less than 5% of the total nuclear polymerase when nuclei were prepared in the presence of the sulfhydryl blocking reagent iodoacetamide and increased to approximately 50% of the total nuclear pADPRp when nuclei were treated with the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) prior to fractionation. To determine whether this effect of disulfide bond formation was unique to rat liver nuclei, nuclear matrix/cytoskeleton structures were prepared in situ by sequentially treating monolayers of tissue culture cells with Nonidet-P40, DNase I and RNase A, and 1.6 M NaCl (S.H. Kaufmann and J.H. Shaper (1991) Exp. Cell Res. 192, 511-523). When nuclear monolayers were prepared from HTC rat hepatoma cells, CaLu-1 human lung carcinoma cells, and CHO hamster ovary cells in the absence of NaTT, pADPRp was undetectable in the nuclease- and 1.6 M NaCl-resistant fraction. In contrast, when nuclear monolayers were isolated in the presence of NaTT, from 5% (CaLu-1) to 26% (HTC cells) of the total nuclear pADPRp was recovered with the nuclease- and salt-resistant fraction. Examination of these residual structures by SDS-polyacrylamide gel electrophoresis under nonreducing conditions suggested that pADPRp was present as a component of disulfide cross-linked complexes. Further analysis by immunofluorescence revealed that the pADPRp was diffusely distributed throughout the CaLu-1 or CHO nuclear matrix. In addition, when matrices were prepared in the absence of RNase A, pADPRp was also observed in the residual nucleoli. These

  4. Insights into the mechanism of X-ray-induced disulfide-bond cleavage in lysozyme crystals based on EPR, optical absorption and X-ray diffraction studies.

    PubMed

    Sutton, Kristin A; Black, Paul J; Mercer, Kermit R; Garman, Elspeth F; Owen, Robin L; Snell, Edward H; Bernhard, William A

    2013-12-01

    Electron paramagnetic resonance (EPR) and online UV-visible absorption microspectrophotometry with X-ray crystallography have been used in a complementary manner to follow X-ray-induced disulfide-bond cleavage. Online UV-visible spectroscopy showed that upon X-irradiation, disulfide radicalization appeared to saturate at an absorbed dose of approximately 0.5-0.8 MGy, in contrast to the saturating dose of ∼0.2 MGy observed using EPR at much lower dose rates. The observations suggest that a multi-track model involving product formation owing to the interaction of two separate tracks is a valid model for radiation damage in protein crystals. The saturation levels are remarkably consistent given the widely different experimental parameters and the range of total absorbed doses studied. The results indicate that even at the lowest doses used for structural investigations disulfide bonds are already radicalized. Multi-track considerations offer the first step in a comprehensive model of radiation damage that could potentially lead to a combined computational and experimental approach to identifying when damage is likely to be present, to quantitate it and to provide the ability to recover the native unperturbed structure.

  5. Formation and reshuffling of disulfide bonds in bovine serum albumin demonstrated using tandem mass spectrometry with collision-induced and electron-transfer dissociation

    PubMed Central

    Rombouts, Ine; Lagrain, Bert; Scherf, Katharina A.; Koehler, Peter; Delcour, Jan A.

    2015-01-01

    Thermolysin hydrolyzates of freshly isolated, extensively stored (6 years, 6 °C, dry) and heated (60 min, 90 °C, in excess water) bovine serum albumin (BSA) samples were analyzed with liquid chromatography (LC) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using alternating electron-transfer dissociation (ETD) and collision-induced dissociation (CID). The positions of disulfide bonds and free thiol groups in the different samples were compared to those deduced from the crystal structure of native BSA. Results revealed non-enzymatic posttranslational modifications of cysteine during isolation, extensive dry storage, and heating. Heat-induced extractability loss of BSA was linked to the impact of protein unfolding on the involvement of specific cysteine residues in intermolecular and intramolecular thiol-disulfide interchange and thiol oxidation reactions. The here developed approach holds promise for exploring disulfide bond formation and reshuffling in various proteins under conditions relevant for chemical, biochemical, pharmaceutical and food processing. PMID:26193081

  6. The Dynamic Disulfide Relay of Quiescin Sulfhydryl Oxidase

    PubMed Central

    Alon, Assaf; Grossman, Iris; Gat, Yair; Kodali, Vamsi K.; DiMaio, Frank; Mehlman, Tevie; Haran, Gilad; Baker, David; Thorpe, Colin; Fass, Deborah

    2012-01-01

    Protein stability, assembly, localization, and regulation often depend on formation of disulfide cross-links between cysteine side chains. Enzymes known as sulfhydryl oxidases catalyze de novo disulfide formation and initiate intra- and intermolecular dithiol/disulfide relays to deliver the disulfides to substrate proteins1,2. Quiescin sulfhydryl oxidase (QSOX) is a unique, multi-domain disulfide catalyst that is localized primarily to the Golgi apparatus and secreted fluids3 and has attracted attention due to its over-production in tumors4,5. In addition to its physiological importance, QSOX is a mechanistically intriguing enzyme, encompassing functions typically carried out by a series of proteins in other disulfide formation pathways. How disulfides are relayed through the multiple redox-active sites of QSOX and whether there is a functional benefit to concatenating these sites on a single polypeptide are open questions. We determined the first crystal structure of an intact QSOX enzyme, derived from a trypanosome parasite. Notably, sequential sites in the disulfide relay were found more than 40 Å apart in this structure, too far for direct disulfide transfer. To resolve this puzzle, we trapped and crystallized an intermediate in the disulfide hand-off, which showed a 165° domain rotation relative to the original structure, bringing the two active sites within disulfide bonding distance. The comparable structure of a mammalian QSOX enzyme, also presented herein, reveals additional biochemical features that facilitate disulfide transfer in metazoan orthologs. Finally, we quantified the contribution of concatenation to QSOX activity, providing general lessons for the understanding of multi-domain enzymes and the design of novel catalytic relays. PMID:22801504

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

    PubMed Central

    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

  8. Presence of interchain disulfide bonds between two gene products that compose the secreted form of an antigen-specific suppressor factor

    PubMed Central

    1981-01-01

    The secreted form of the suppressor T cell factor specific for keyhole limpet hemocyanin derived from the hybridoma 34S-704 was found to consist of the two distinct polypeptide chains, i.e., the antigen- binding and the I-J-encoded chains. They were linked in covalent association with disulfide bonds. The two chains were cleaved by the reduction with dithiothreitol and were easy to reconstitute the active form of TsF. The association of the two distinct chains was suggested to be essential for the expression of the TsF activity. PMID:6166720

  9. Chemical shift and coupling constant analysis of dibenzyloxy disulfides

    NASA Astrophysics Data System (ADS)

    Stoutenburg, Eric G.; Gryn'ova, Ganna; Coote, Michelle L.; Priefer, Ronny

    2015-02-01

    Dialkoxy disulfides have found applications in the realm of organic synthesis as an S2 or alkoxy donor, under thermal and photolytic decompositions conditions, respectively. Spectrally, dibenzyloxy disulfides possess an ABq in the 1H NMR, which can shift by over 1.1 ppm depending on the substituents present on the aromatic ring, as well as the solvent employed. The effect of the said substituents and solvent were analyzed and compared to the center of the ABq, geminal coupling, and the differences in chemical shifts of the individual doublets. Additionally, quantum-chemical calculations demonstrated the intramolecular H-bonding arrangement, found within the dibenzyloxy disulfides.

  10. Enthalpic and entropic contributions mediate the role of disulfide bonds on the conformational stability of Interleukin-4

    PubMed Central

    Vaz, Daniela C.; Rodrigues, J. Rui; Sebald, Walter; Dobson, Christopher M.; Brito, Rui M.M.

    2006-01-01

    The role of disulfide bridges in the structure, stability, and folding pathways of proteins has been the subject of wide interest in the fields of protein design and engineering. However, the relative importance of entropic and enthalpic contributions for the stabilization of proteins provided by disulfides is not always clear. Here, we perform a detailed analysis of the role of disulfides in the conformational stability of human Interleukin-4 (IL4), a four-helix bundle protein. In order to evaluate the contribution of two out of the three disulfides to the structure and stability of IL4, two IL4 mutants, C3T-IL4 and C24T-IL4, were used. NMR and ANS binding experiments were compatible with altered dynamics and an increase of the nonpolar solvent-accessible surface area of the folded state of the mutant proteins. Chemical and thermal unfolding experiments followed by fluorescence and circular dichroism revealed that both mutant proteins have lower conformational stability than the wild-type protein. Transition temperatures of unfolding decreased 14°C for C3T-IL4 and 10°C for C24T-IL4, when compared to WT-IL4, and the conformational stability, at 25°C, decreased 4.9 kcal/mol for C3T-IL4 and 3.2 kcal/mol for C24T-IL4. Interestingly, both the enthalpy and the entropy of unfolding, at the transition temperature, decreased in the mutant proteins. Moreover, a smaller change in heat capacity of unfolding was also observed for the mutants. Thus, disulfide bridges in IL4 play a critical role in maintaining the thermodynamic stability and core packing of the helix bundle. PMID:16373475

  11. Quantification of Thiols and Disulfides

    PubMed Central

    Winther, Jakob R.; Thorpe, Colin

    2013-01-01

    Background 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 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. Scope of the review In the present account, we briefly survey the toolbox available to the experimentalist for the chemical determination of thiols and disulfides. We have chosen to focus on the key chemical aspects of current methodology, together with identifying potential difficulties inherent in their experimental implementation. Major conclusions While many reagents have been described for the measurement and manipulation of the redox status of thiols and disulfides, a number of these methods remain underutilized. The ability to effectively quantify changes in redox conditions in living cells presents a continuing challenge. General Significance Many unresolved questions in the metabolic interconversion of thiols and disulfides remain. For example, while pool sizes of redox pairs and their intracellular distribution are being uncovered, very little is known about the flux in thiol-disulfide exchange pathways. New tools are needed to address this important aspect of cellular metabolism. PMID:23567800

  12. Characterization of Helicobacter pylori HP0231 (DsbK): role in disulfide bond formation, redox homeostasis and production of Helicobacter cystein-rich protein HcpE.

    PubMed

    Lester, Jeffrey; Kichler, Sari; Oickle, Brandon; Fairweather, Spencer; Oberc, Alexander; Chahal, Jaspreet; Ratnayake, Dinath; Creuzenet, Carole

    2015-04-01

    Helicobacter pylori is a human gastric pathogen that colonizes ∼ 50% of the world's population. It can cause gastritis, gastric or duodenal ulcers and also gastric cancer. The numerous side effects of available treatments and the emergence of antibiotic resistant strains are severe concerns that justify further research into H. pylori's pathogenic mechanisms. H. pylori produces secreted proteins that may play a role in virulence, including the Helicobacter cysteine-rich protein HcpE (aka HP0235). We demonstrate herein that HcpE is secreted in the culture supernatant both as a soluble protein and in association with outer membrane vesicles. We show that the structure of HcpE comprises an organized array of disulfide bonds. We identify DsbK (aka HP0231) as a folding factor necessary for HcpE production and secretion in H. pylori and show that recombinant DsbK can interact with and refold unprocessed, reduced HcpE in vitro. These experiments highlight the first biologically relevant substrate for DsbK. Furthermore, we show that DsbK has disulfide bond (Dsb) forming activity on reduced lysozyme and demonstrate a DsbA-type of activity for DsbK upon expression in E. coli, despite its similarity with DsbG. Finally, we show a role of DsbK in maintaining redox homeostasis in H. pylori.

  13. Disulfides as cyanide antidotes: evidence for a new in vivo oxidative pathway for cyanide detoxification.

    PubMed

    Zottola, Mark A; Beigel, Keith; Soni, Sunil-Datta; Lawrence, Richard

    2009-12-01

    It is known that cyanide is converted to thiocyanate in the presence of the enzyme rhodanese. The enzyme is activated by sulfur transfer from an appropriate sulfur donor. The activated enzyme then binds cyanide and transfers the sulfur atom to cyanide to form thiocyanate. This project began as an exploration of the ability of disulfides to act as sulfur donors in the rhodanese-mediated detoxification of cyanide. To our surprise, and contrary to expectations based on efficacy studies in vivo, our in vitro results showed that disulfides are rather poor sulfur donors. The transfer of a sulfur atom from a disulfide to the enzyme must occur via cleavage of a carbon-sulfur bond either of the original disulfide or in a mixed disulfide arising from the reaction of rhodanese with the original disulfide. Extending the reaction time and addition of chloride anion (a nucleophile) did not significantly change the results of the experiment. Using ultrasound as a means of accelerating bond cleavage also had a minimal effect. Those results ruled out cleavage of the carbon-sulfur bond in the original disulfide but did not preclude formation of a mixed disulfide. S-Methyl methylthiosulfonate (MTSO) was used to determine whether a mixed disulfide, if formed, would result in transfer of a sulfur atom to rhodanese. While no thiocyanate was formed in the reaction between cyanide and rhodanese exposed to MTSO, NMR analysis revealed that MTSO reacted directly with cyanide anion to form methyl thiocyanate. This result reveals the body's possible use of oxidized disulfides as a first line of defense against cyanide intoxication. The oxidation of disulfides to the corresponding thiosulfinate or thiosulfonate will result in facilitating their reaction with other nucleophiles. The reaction of an oxidized disulfide with a sulfur nucleophile from glutathione could be a plausible origin for the cyanide metabolite 2-aminothiazoline-4-carboxylic acid.

  14. Early events in the disulfide-coupled folding of BPTI.

    PubMed Central

    Bulaj, G.; Goldenberg, D. P.

    1999-01-01

    Recent studies of the refolding of reduced bovine pancreatic trypsin inhibitor (BPTI) have shown that a previously unidentified intermediate with a single disulfide is formed much more rapidly than any other one-disulfide species. This intermediate contains a disulfide that is present in the native protein (between Cys14 and 38), but it is thermodynamically less stable than the other two intermediates with single native disulfides. To characterize the role of the [14-38] intermediate and the factors that favor its formation, detailed kinetic and mutational analyses of the early disulfide-formation steps were carried out. The results of these studies indicate that the formation of [14-38] from the fully reduced protein is favored by both local electrostatic effects, which enhance the reactivities of the Cys14 and 38 thiols, and conformational tendencies that are diminished by the addition of urea and are enhanced at lower temperatures. At 25 degrees C and pH 7.3, approximately 35% of the reduced molecules were found to initially form the 14-38 disulfide, but the majority of these molecules then undergo intramolecular rearrangements to generate non-native disulfides, and subsequently the more stable intermediates with native disulfides. Amino acid replacements, other than those involving Cys residues, were generally found to have only small effects on either the rate of forming [14-38] or its thermodynamic stability, even though many of the same substitutions greatly destabilized the native protein and other disulfide-bonded intermediates. In addition, those replacements that did decrease the steady-state concentration of [14-38] did not adversely affect further folding and disulfide formation. These results suggest that the weak and transient interactions that are often detected in unfolded proteins and early folding intermediates may, in some cases, not persist or promote subsequent folding steps. PMID:10493584

  15. Disulfide bond involvement in the maintenance of the cryptic nature of the cross-reacting determinant of metacyclic forms of Trypanosoma congolense

    SciTech Connect

    Fish, W.R.; Muriuki, C.W.; Muthiani, A.M.; Grab, D.J.; Lonsdale-Eccles, J.D. )

    1989-06-27

    The variable surface glycoprotein (VSG) of African trypanosomes possesses a 1,2-dimyristoylglycosylphosphatidylinositol at the carboxy terminus. Cleavage of the 1,2-dimyristoylglycerol (1,2-DMG) moiety from the VSG reportedly results in a higher apparent molecular mass and an increased binding of antibodies against the cross-reacting determinant (CRD), a cryptic epitope present on most VSGs. Using metacyclic forms of Trypanosoma congolense, the authors show that the process involved are more complex than heretofore presumed and that the removal of the 1,2-DMG moiety may not be necessary for binding of anti-CRD antibodies (RxCRD). Among other findings, they observe the following: (1) in sonicated samples of trypanosomes metabolically labeled with ({sup 3}H)myristate, the binding of RxCRD on Western blots is coincident with bands containing labeled (membrane form) VSGs; (2) disulfide reduction of trypanosome sonicates suffices to promote RxCRD binding in the presence or absence of inhibitors of a glycosylphosphatidylinositol-specific phospholipase C; (3) trypanosomes directly solubilized in detergents show quantitative and qualitative differences in RxCRD binding which depend upon the detergent used and the order of addition of disulfide reducing agents. The authors conclude that the binding of RxCRD to T. congolense metacyclic VSGs depends upon the degree of unfolding of the molecule and is clearly a complex, multistep process in which structural changes and disulfide reduction play pivotal roles.

  16. Elimination of a Free Cysteine by Creation of a Disulfide Bond Increases the Activity and Stability of Candida boidinii Formate Dehydrogenase

    PubMed Central

    Zheng, Junxian; Yang, Taowei; Zhou, Junping; Xu, Meijuan; Zhang, Xian

    2016-01-01

    ABSTRACT NAD+-dependent formate dehydrogenase (FDH; EC 1.2.1.2) is an industrial enzyme widely used for NADH regeneration. However, enzyme inactivation caused by the oxidation of cysteine residues is a flaw of native FDH. In this study, we relieved the oxidation of the free cysteine of FDH from Candida boidinii (CboFDH) through the construction of disulfide bonds between A10 and C23 as well as I239 and C262. Variants A10C, I239C, and A10C/I239C were obtained by the site-directed mutagenesis and their properties were studied. Results showed that there were no significant changes in the optimum temperature and pH between variants and wild-type CboFDH. However, the stabilities of all variant enzymes were improved. Specifically, the CboFDH variant A10C (A10Cfdh) showed a significant increase in copper ion resistance and acid resistance, a 6.7-fold increase in half-life at 60°C, and a 1.4-fold increase in catalytic efficiency compared with the wild type. Asymmetric synthesis of l-tert-leucine indicated that the process time was reduced by 40% with variant A10Cfdh, which benefited from the increase in catalytic efficiency. Circular dichroism analysis and molecular dynamics simulation indicated that variants that contained disulfide bonds lowered the overall root mean square deviation (RMSD) and consequently increased the protein rigidity without affecting the secondary structure of enzyme. This work is expected to provide a viable strategy to avoid the microbial enzyme inactivation caused by the oxidation of the free cysteine residues and improving their performances. IMPORTANCE FDH is widely used for NADH regeneration in dehydrogenase-based synthesis of optically active compounds to decrease the cost of production. This study highlighted a viable strategy that was used to eliminate the oxidation of free cysteine residues of FDH from Candida boidinii by the introduction of disulfide bonds. Using this strategy, we obtained a variant FDH with improved activity and

  17. Synthesis of bicyclic organo-peptide hybrids via oxime/intein-mediated macrocyclization followed by disulfide bond formation.

    PubMed

    Smith, Jessica M; Hill, Nicholas C; Krasniak, Peter J; Fasan, Rudi

    2014-02-21

    A new strategy is described to generate bicyclic peptides that incorporate non-peptidic backbone elements starting from recombinant polypeptide precursors. These compounds are produced via a one-pot, two-step sequence, in which peptide macrocyclization by means of a bifunctional oxyamine/1,3-amino-thiol synthetic precursor is followed by intramolecular disulfide formation between the synthetic precursor-borne thiol and a cysteine embedded in the peptide sequence. This approach was found to be compatible with the cysteine residue occupying different positions within 8mer and 10mer target peptide sequences and across different synthetic precursor scaffolds, thereby enabling the formation of a variety of diverse bicyclic scaffolds.

  18. Association of topoisomerase II with the hepatoma cell nuclear matrix: the role of intermolecular disulfide bond formation.

    PubMed

    Kaufmann, S H; Shaper, J H

    1991-02-01

    Previous studies have resulted in conflicting data regarding the recovery of the nuclear enzymes topoisomerase (topo) II and topo I in the nuclear matrix fraction. In the present study we have assessed the effect of systematically altering a single extraction procedure on the distribution of these enzymes during the subfractionation of nuclei from HTC hepatoma tissue culture cells. When nuclear monolayers (prepared by treating attached cells in situ with the neutral detergent Nonidet-P40 at 4 degrees C) were isolated in the presence of the irreversible sulfhydryl blocking reagent iodoacetamide, subsequent treatment with DNase I and RNase A followed by 1.6 M NaCl resulted in structures which were extensively depleted of intranuclear components as assessed by phase contrast microscopy and conventional transmission electron microscopy. These structures contained 12 +/- 4% of the total protein present in the original nuclear monolayers. The lamins and polypeptides with molecular weights comparable to those of actin and vimentin were the predominant polypeptides present on SDS-polyacrylamide gels. Western blotting revealed that less than 5% of the total nuclear topo II molecules were present in these structures. In contrast, when the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) was substituted for iodoacetamide, the same extraction procedure yielded structures containing components of the nucleolus and an extensive intranuclear network. These structures contained a wide variety of nonlamin, nonhistone nuclear polypeptides including 23 +/- 4% of the total nuclear topo II. SDS-polyacrylamide gel electrophoresis performed under nonreducing conditions revealed that topo II in these nuclear matrices was present as part of a large disulfide cross-linked complex. Treatment of these structures with reducing agents in 1.6 M NaCl released the topo II. In contrast, topo I did not form disulfide cross-linked oligomers and was not detectable in any of these nuclease

  19. Formation of reversible disulfide bonds with the protein matrix of the endoplasmic reticulum correlates with the retention of unassembled Ig light chains.

    PubMed Central

    Reddy, P; Sparvoli, A; Fagioli, C; Fassina, G; Sitia, R

    1996-01-01

    Exposed thiols act as intracellular retention elements for unassembled secretory molecules. Yet, some free Ig lambda light chains are secreted despite the presence of an unpaired cysteine (Cys214). This is due largely to the presence of a flanking acidic residue: substitution of Asp213 for Gly or Lys increases pre-Golgi retention and degradation of free lambda. Secretion is restored by exogenous reducing agents or by assembly with heavy chains. In the endoplasmic reticulum (ER), lambda chains form covalent complexes with many proteins through Cys214. These complexes are absent from the Golgi. They are more abundant in transfectants expressing the lambdaGly2I3 and lambdaLys213 mutants that are poorly secreted. Radioactive N-ethylmaleimide labels some monomeric lambda chains isolated from the ER, but not from the Golgi or from the medium, indicating that the Cys214 thiol is masked during ER-Golgi transport. Mass spectrometry reveals the presence of a free cysteine residue disulfide-linked to Cys214. We suggest that thiol-mediated retention involves the formation of reversible disulfide bonds with the protein matrix of the ER. The presence of an acidic residue next to the critical cysteine may allow the masking of the thiol and transport to the Golgi. Images PMID:8641273

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

    PubMed

    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.

  1. Cell-free synthesis system suitable for disulfide-containing proteins

    SciTech Connect

    Matsuda, Takayoshi; Watanabe, Satoru; Kigawa, Takanori

    2013-02-08

    Highlights: ► Cell-free synthesis system suitable for disulfide-containing proteins is proposed. ► Disulfide bond formation was facilitated by the use of glutathione buffer. ► DsbC catalyzed the efficient shuffling of incorrectly formed disulfide bonds. ► Milligram quantities of functional {sup 15}N-labeled BPTI and lysozyme C were obtained. ► Synthesized proteins were both catalytically functional and properly folded. -- Abstract: Many important therapeutic targets are secreted proteins with multiple disulfide bonds, such as antibodies, cytokines, hormones, and proteases. The preparation of these proteins for structural and functional analyses using cell-based expression systems still suffers from several issues, such as inefficiency, low yield, and difficulty in stable-isotope labeling. The cell-free (or in vitro) protein synthesis system has become a useful protein production method. The openness of the cell-free system allows direct control of the reaction environment to promote protein folding, making it well suited for the synthesis of disulfide-containing proteins. In this study, we developed the Escherichia coli (E. coli) cell lysate-based cell-free synthesis system for disulfide-containing proteins, which can produce sufficient amounts of functional proteins for NMR analyses. Disulfide bond formation was facilitated by the use of glutathione buffer. In addition, disulfide isomerase, DsbC, catalyzed the efficient shuffling of incorrectly formed disulfide bonds during the protein synthesis reaction. We successfully synthesized milligram quantities of functional {sup 15}N-labeled higher eukaryotic proteins, bovine pancreatic trypsin inhibitor (BPTI) and human lysozyme C (LYZ). The NMR spectra and functional analyses indicated that the synthesized proteins are both catalytically functional and properly folded. Thus, the cell-free system is useful for the synthesis of disulfide-containing proteins for structural and functional analyses.

  2. Centipede venom peptide SsmTX-I with two intramolecular disulfide bonds shows analgesic activities in animal models.

    PubMed

    Wang, Ying; Li, Xiaojie; Yang, Meifeng; Wu, Chunyun; Zou, Zhirong; Tang, Jing; Yang, Xinwang

    2017-03-01

    Pain is a major symptom of many diseases and results in enormous pressures on human body or society. Currently, clinically used analgesic drugs, including opioids and nonsteroidal anti-inflammatory drugs, have adverse reactions, and thus, the development of new types of analgesic drug candidates is urgently needed. Animal venom peptides have proven to have potential as new types of analgesic medicine. In this research, we describe the isolation and characterization of an analgesic peptide from the crude venom of centipede, Scolopendra subspinipes mutilans. The amino acid sequence of this peptide was identical with SsmTX-I that was previously reported as a specific Kv2.1 ion channel blocker. Our results revealed that SsmTX-I was produced by posttranslational processing of a 73-residue prepropeptide. The intramolecular disulfide bridge motifs of SsmTX-I was Cys1-Cys3 and Cys2-Cys4. Functional assay revealed that SsmTX-I showed potential analgesic activities in formalin-induced paw licking, thermal pain, and acetic acid-induced abdominal writhing mice models. Our research provides the first report of cDNA sequences, disulfide motif, successful synthesis, and analgesic potential of SsmTX-I for the development of pain-killing drugs. It indicates that centipede peptide toxins could be a treasure trove for the search of novel analgesic drug candidates. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

  3. Substitution of lysine for arginine at position 42 of human transforming growth factor-alpha eliminates biological activity without changing internal disulfide bonds.

    PubMed Central

    Defeo-Jones, D; Tai, J Y; Vuocolo, G A; Wegrzyn, R J; Schofield, T L; Riemen, M W; Oliff, A

    1989-01-01

    Transforming growth factor-alpha (TGF-alpha) is a growth-promoting protein that binds to the epidermal growth factor (EGF) receptor. To identify critical residues that govern TGF-alpha-EGF receptor binding, we prepared site-specific substitution mutants of TGF-alpha. Mutant proteins were tested in receptor-binding and mitogenesis assays. Semiconservative substitutions at positions 4, 12, 18, and 45 decreased biological activity 2.1- to 14-fold. The conservative substitution of lysine for arginine at position 42 completely eliminated biological activity. Amino acid composition analysis of proteolytic fragments from TGF-alpha and the Lys-42 mutant indicated that these proteins contained the same disulfide bonds. These studies suggest that arginine 42 may be a contact point for TGF-alpha-EGF receptor interaction. PMID:2506441

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

    SciTech Connect

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

    2010-04-16

    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{sub 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.

  5. Functional coupling of Cys-226 and Cys-296 in the glucagon-like peptide-1 (GLP-1) receptor indicates a disulfide bond that is close to the activation pocket.

    PubMed

    Mann, Rosalind J; Al-Sabah, Suleiman; de Maturana, Rakel López; Sinfield, John K; Donnelly, Dan

    2010-12-01

    G protein-coupled receptors (GPCRs) are seven transmembrane α-helical (7TM) integral membrane proteins that play a central role in both cell signaling and in the action of many pharmaceuticals. The crystal structures of several Family A GPCRs have shown the presence of a disulfide bond linking transmembrane helix 3 (TM3) to the second extracellular loop (ECL2), enabling ECL2 to stabilize and contribute to the ligand binding pocket. Family B GPCRs share no significant sequence identity with those in Family A but nevertheless share two conserved cysteines in topologically equivalent positions. Since there are no available crystal structures for the 7TM domain of any Family B GPCR, we used mutagenesis alongside pharmacological analysis to investigate the role of ECL2 and the conserved cysteine residues. We mutated Cys-226, at the extracellular end of TM3 of the glucagon-like peptide-1 (GLP-1) receptor, to alanine and observed a 38-fold reduction in GLP-1 potency. Interestingly, this potency loss was restored by the additional substitution of Cys-296 in ECL2 to alanine. Alongside the complete conservation of these cysteine residues in Family B GPCRs, this functional coupling suggested the presence of a disulfide bond. Further mutagenesis demonstrated that the low potency observed at the C226A mutant, compared with the C226A-C296A double mutant, was the result of the bulky nature of the released Cys-296 side chain. Since this suggested that ECL2 was in close proximity to the agonist activation pocket, an alanine scan of ECL2 was carried out which confirmed the important role of this loop in agonist-induced receptor activation.

  6. Function and stability of human transcobalamin II: role of intramolecular disulfide bonds C98-C291 and C147-C187.

    PubMed

    Kalra, Seema; Li, Ning; Seetharam, Shakuntla; Alpers, David H; Seetharam, Bellur

    2003-07-01

    The current studies have investigated the role of three disulfide bonds of human transcobalamin II (TC II), a plasma transporter of cobalamin (Cbl; vitamin B12), in its function and stability. When translated in vitro in the presence or absence of microsomal vesicles, TC II constructs with a single substitution, C3S or C249S, demonstrated synthesis of a stable functional protein. However, TC II synthesized in the presence of microsomal vesicles using constructs with a single (C98S, C147S, C187S, C291S), double (C3/147/S, C98/147/S) or triple (C3/98/147/S) substitution was unstable. In the absence of microsomal vesicles, the percentage of binding to Cbl-Sepharose matrix by TC II expressed by constructs C3S, C3/147/S, C98/147/S, or C3/98/147/S was 100, 49, 52, and 35%, respectively. Upon their reductive alkylation, the binding of TC II expressed by these constructs was reduced to approximately 25-30%. TC II constructs C3S or C249S, when expressed in TC II-deficient fibroblasts, produced a stable functional protein, but those expressed by constructs C147S, C187S, C291S, C3/147/S, C98/147/S, or C3/98/147/S were rapidly degraded. The intracellular degradation of TC II expressed by these constructs was inhibited by lactacystin or MG-132 but not by the lysosomal degradation inhibitors ammonium chloride or chloroquine. These studies suggest that optimal binding of Cbl by human TC II is supported by disulfide bonds C98-C291 and C147-C187 and that their disruption results in loss of Cbl binding and their rapid degradation by the proteasomal machinery.

  7. Transition-Metal-Catalyzed C-H Bond Addition to Carbonyls, Imines, and Related Polarized π Bonds.

    PubMed

    Hummel, Joshua R; Boerth, Jeffrey A; Ellman, Jonathan A

    2016-12-12

    The transition-metal-catalyzed addition of C-H bonds to carbonyls, imines, and related polarized π bonds has emerged as a particularly efficient and powerful approach for the construction of an incredibly diverse array of heteroatom-substituted products. Readily available and stable inputs are typically employed, and reactions often proceed with very high functional group compatibility and without the production of waste byproducts. Additionally, many transition-metal-catalyzed C-H bond additions to polarized π bonds occur within cascade reaction sequences to provide rapid access to a diverse array of different heterocyclic as well as carbocyclic products. This review highlights the diversity of transformations that have been achieved, catalysts that have been used, and types of products that have been prepared through the transition-metal-catalyzed addition of C-H bonds to carbonyls, imines, and related polarized π bonds.

  8. Isolation of Metarhizium anisopliae carboxypeptidase A with native disulfide bonds from the cytosol of Escherichia coli BL21(DE3)

    PubMed Central

    Austin, Brian P.; Waugh, David S.

    2011-01-01

    The carboxypeptidase A enzyme from Metarhizium anisopliae (MeCPA) has broader specificity than the mammalian A-type carboxypeptidases, making it a more useful reagent for the removal of short affinity tags and disordered residues from the C-termini of recombinant proteins. When secreted from baculovirus-infected insect cells, the yield of pure MeCPA was 0.25 mg per liter of conditioned medium. Here, we describe a procedure for the production of MeCPA in the cytosol of Escherichia coli that yields approximately 0.5 mg of pure enzyme per liter of cell culture. The bacterial system is much easier to scale up and far less expensive than the insect cell system. The expression strategy entails maintaining the proMeCPA zymogen in a soluble state by fusing it to the C-terminus of maltose-binding protein (MBP) while simultaneously overproducing the protein disulfide isomerase DsbC in the cytosol from a separate plasmid. Unexpectedly, we found that the yield of active and properly oxidized MeCPA was highest when coexpressed with DsbC in BL21(DE3) cells that do not also contain mutations in the trxB and gor genes. Moreover, the formation of active MeCPA was only partially dependent on the disulfide-isomerase activity of DsbC. Intriguingly, we observed that most of the active MeCPA was generated after cell lysis and amylose affinity purification of the MBP-proMeCPA fusion protein, during the time that the partially purified protein was held overnight at 4 °C prior to activation with thermolysin. Following removal of the MBP-propeptide by thermolysin digestion, active MeCPA (with a C-terminal polyhistidine tag) was purified to homogeneity by immobilized metal affinity chromatography (IMAC), ion exchange chromatography and gel filtration. PMID:22197595

  9. 31 CFR 315.91 - Additional requirements; bond of indemnity.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... (Continued) FISCAL SERVICE, DEPARTMENT OF THE TREASURY BUREAU OF THE PUBLIC DEBT REGULATIONS GOVERNING U.S. SAVINGS BONDS, SERIES A, B, C, D, E, F, G, H, J, AND K, AND U.S. SAVINGS NOTES Miscellaneous...

  10. Comparison of the Effects of British Anti-Lewisite (BAL) and Beta Mercapto Ethanol on the Reduction and Cleavage of Disulfide Bonds in IgG and Human Keratinocyte Proteins

    DTIC Science & Technology

    1988-02-01

    34. Din1a.Z’atic representation of anticipated :-su ts I<•:;di.,’¢,i two-dimensionai e cto h rsswhen- sŕ,’ý" l s are trertet with a disulfide redu cer...Development Laboraliory A TTN: SGRD-UBG;-M l ;ort Detrick, Bldg 568 F~rederick, MD 21701-5010 2/88 S~WkWWW ~1W,$~/ b)AlEmm ...Cleavage of Disulfide Bonds in IgG and Human Keratinocyte Proteins Michael A . Deaton, PhD, CPT, MS Charles A . Barba, BS, SP4 Carlos R. Flores, BS, SP4

  11. The remarkable efficiency of a Pin-II proteinase inhibitor sans two conserved disulfide bonds is due to enhanced flexibility and hydrogen bond density in the reactive site loop.

    PubMed

    Joshi, Rakesh S; Mishra, Manasi; Tamhane, Vaijayanti A; Ghosh, Anirban; Sonavane, Uddhavesh; Suresh, C G; Joshi, Rajendra; Gupta, Vidya S; Giri, Ashok P

    2014-01-01

    Capsicum annuum (L.) expresses diverse potato type II family proteinase inhibitors comprising of inhibitory repeat domain (IRD) as basic functional unit. Most IRDs contain eight conserved cysteines forming four disulfide bonds, which are indispensible for their stability and activity. We investigated the functional significance of evolutionary variations in IRDs and their role in mediating interaction between the inhibitor and cognate proteinase. Among the 18 IRDs encoded by C. annuum, IRD-7, -9, and -12 were selected for further characterization on the basis of variation in their reactive site loop, number of conserved cysteine residues, and higher theoretical ΔGbind for interaction with Helicoverpa armigera trypsin. Moreover, inhibition kinetics showed that IRD-9, despite loss of some of the disulfide bonds, was a more potent proteinase inhibitor among the three selected IRDs. Molecular dynamic simulations revealed that serine residues in the place of cysteines at seventh and eighth positions of IRD-9 resulted in an increase in the density of intramolecular hydrogen bonds and reactive site loop flexibility. Results of the serine residues chemical modification also supported this observation and provided a possible explanation for the remarkable inhibitory potential of IRD-9. Furthermore, this natural variant among IRDs showed special attributes like stability to proteolysis and synergistic inhibitory effect on other IRDs. It is likely that IRDs have coevolved selective specialization of their structure and function as a response towards specific insect proteases they encountered. Understanding the molecular mechanism of pest protease-plant proteinaceous inhibitor interaction will help in developing effective pest control strategies. An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:39.

  12. Uptake and intracellular fate of disulfide-bonded polymer hydrogel capsules for Doxorubicin delivery to colorectal cancer cells.

    PubMed

    Yan, Yan; Johnston, Angus P R; Dodds, Sarah J; Kamphuis, Marloes M J; Ferguson, Charles; Parton, Robert G; Nice, Edouard C; Heath, Joan K; Caruso, Frank

    2010-05-25

    Understanding the interactions between drug carriers and cells is of importance to enhance the delivery of therapeutics. The release of therapeutics into different intracellular environments, such as the lysosomes or the cell cytoplasm, will impact their pharmacological activity. Herein, we investigate the intracellular fate of layer-by-layer (LbL)-assembled, submicrometer-sized polymer hydrogel capsules in a human colon cancer derived cell line, LIM1899. The cellular uptake of the disulfide-stabilized poly(methacrylic acid) (PMA(SH)) capsules by colon cancer cells is a time-dependent process. Confocal laser scanning microscopy and transmission electron microscopy reveal that the internalized capsules are deformed in membrane-enclosed compartments, which further mature to late endosomes or lysosomes. We further demonstrate the utility of these redox-responsive PMA(SH) capsules for the delivery of doxorubicin (DOX) to colon cancer cells. The DOX-loaded PMA(SH) capsules demonstrate a 5000-fold enhanced cytotoxicity in cell viability studies compared to free DOX.

  13. Free radical addition of butanethiol to vegetable oil double bonds.

    PubMed

    Bantchev, Grigor B; Kenar, James A; Biresaw, Girma; Han, Moon Gyu

    2009-02-25

    Butanethiol was used in ultraviolet-initiated thiol-ene reaction with canola and corn oils to produce sulfide-modified vegetable oils (SMVO). The crude SMVO product was successfully purified by solvent extraction, vacuum evaporation, and silica gel chromatography. The SMVO products were characterized by nuclear magnetic resonance and Fourier transform infrared spectroscopy. Further product characterization and analysis was conducted using GC and GC-MS on the fatty acid methyl esters obtained by the transesterification of the SMVO products. Investigation of the effect of reaction conditions showed that high yield and high conversion of double bonds into thiol were favored at low reaction temperatures and high butanethiol/vegetable oil ratios. Canola and corn oils gave similar double-bond conversions and yields of the desired SMVO product even though they have big differences in the relative numbers of single and multiple double bonds in their structures. Under best reaction conditions, up to 97% of double-bond conversion and 61% isolated yields of the purified SMVO products were attained.

  14. Antileishmanial Activity of Disulfiram and Thiuram Disulfide Analogs in an Ex Vivo Model System Is Selectively Enhanced by the Addition of Divalent Metal Ions

    PubMed Central

    Peniche, Alex G.; Renslo, Adam R.; Melby, Peter C.

    2015-01-01

    Current treatments for cutaneous and visceral leishmaniasis are toxic, expensive, difficult to administer, and limited in efficacy and availability. Disulfiram has primarily been used to treat alcoholism. More recently, it has shown some efficacy as therapy against protozoan pathogens and certain cancers, suggesting a wide range of biological activities. We used an ex vivo system to screen several thiuram disulfide compounds for antileishmanial activity. We found five compounds (compound identifier [CID] 7188, 5455, 95876, 12892, and 3117 [disulfiram]) with anti-Leishmania activity at nanomolar concentrations. We further evaluated these compounds with the addition of divalent metal salts based on studies that indicated these salts could potentiate the action of disulfiram. In addition, clinical studies suggested that zinc has some efficacy in treating cutaneous leishmaniasis. Several divalent metal salts were evaluated at 1 μM, which is lower than the normal levels of copper and zinc in plasma of healthy individuals. The leishmanicidal activity of disulfiram and CID 7188 were enhanced by several divalent metal salts at 1 μM. The in vitro therapeutic index (IVTI) of disulfiram and CID 7188 increased 12- and 2.3-fold, respectively, against L. major when combined with ZnCl2. The combination of disulfiram with ZnSO4 resulted in a 1.8-fold increase in IVTI against L. donovani. This novel combination of thiuram disulfides and divalent metal ions salts could have application as topical and/or oral therapies for treatment of cutaneous and visceral leishmaniasis. PMID:26239994

  15. Oxidative addition of methane and benzene C--H bonds to rhodium center: A DFT study

    NASA Astrophysics Data System (ADS)

    Bi, Siwei; Zhang, Zhenwei; Zhu, Shufen

    2006-11-01

    A density functional theory study on mechanisms of the oxidative addition of methane and benzene C-H bonds to the rhodium center containing Cp and PMe 3 ligands has been performed. Our calculated results confirm that the C-H bond cleavage from a sigma complex to a hydride alkyl complex is the rate-determining step. Compared with the case of methane C-H bond, the oxidative addition of benzene C-H bond is more favorable kinetically and thermodynamically. Stronger backdonation from metal center to the σ ∗ antibonding orbital of benzene C-H bond is responsible for the observations.

  16. Use of a temporary "solubilizing" peptide tag for the Fmoc solid-phase synthesis of human insulin glargine via use of regioselective disulfide bond formation.

    PubMed

    Hossain, Mohammed Akhter; Belgi, Alessia; Lin, Feng; Zhang, Suode; Shabanpoor, Fazel; Chan, Linda; Belyea, Chris; Truong, Hue-Trung; Blair, Amy R; Andrikopoulos, Sof; Tregear, Geoffrey W; Wade, John D

    2009-07-01

    Solid-phase peptide synthesis has been refined to a stage where efficient preparation of long and complex peptides is now achievable. However, the postsynthesis handling of poorly soluble peptides often remains a significant hindrance to their purification and further use. Several synthetic schemes have been developed for the preparation of such peptides containing modifications to aid their solubility. However, these require the use of complex chemistry or yield non-native sequences. We describe a simple approach based on the use of penta-lysine "tags" that are linked to the C-terminus of the peptide of interest via a base-labile linker. After ready purification of the now freely solubilized peptide, the "tag" is removed by simple, brief base treatment giving the native sequence in much higher overall yield. The applicability of the method was demonstrated by the novel preparation of insulin glargine via solid-phase synthesis of each of the two chains--including the notoriously poorly soluble A-chain--followed by their combination in solution via regioselective disulfide bond formation. At the conclusion of the chain combination, the solubilizing peptide tag was removed from the A-chain to provide synthetic human glargine in nearly 10% overall yield. This approach should facilitate the development of new insulin analogues as well as be widely applicable to the improved purification and acquisition of otherwise poorly soluble synthetic peptides.

  17. Structural Basis of IgE Binding to α- and γ-Gliadins: Contribution of Disulfide Bonds and Repetitive and Nonrepetitive Domains.

    PubMed

    Mameri, Hamza; Brossard, Chantal; Gaudin, Jean-Charles; Gohon, Yann; Paty, Evelyne; Beaudouin, Etienne; Moneret-Vautrin, Denise-Anne; Drouet, Martine; Solé, Véronique; Wien, Frank; Lupi, Roberta; Larré, Colette; Snégaroff, Jacques; Denery-Papini, Sandra

    2015-07-29

    Wheat products cause IgE-mediated allergies. The present study aimed to decipher the molecular basis of α- and γ-gliadin allergenicity. Gliadins and their domains, the repetitive N-terminal and the nonrepetitive C-terminal domains, were cloned and expressed in Escherichia coli. Their secondary structures and their IgE binding capacity were compared with those of natural proteins before and after reduction/alkylation. Allergenicity was evaluated with sera from patients who had a wheat food allergy or baker's asthma. The secondary structures of natural and recombinant proteins were slightly different. Compared with natural gliadins, recombinant proteins retained IgE binding but with reduced reactivity. Reduction/alkylation decreased IgE binding for both natural and recombinant gliadins. Although more continuous epitopes were identified in the N-terminal domains of α- and γ-gliadins, both the N-terminal and C-terminal domains contributed to IgE binding. As for other members of the prolamin superfamily, disulfide bonds appear to be of high importance for IgE binding.

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

    PubMed Central

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

    2015-01-01

    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 α-helical membrane protein sequences and folds. In conclusion, we suggest a chronology for the evolution of the five extant VKOR clades. PMID:26230708

  19. Role of cysteine residues and disulfide bonds in the activity of a legume root nodule-specific, cysteine-rich peptide.

    PubMed

    Haag, Andreas F; Kerscher, Bernhard; Dall'Angelo, Sergio; Sani, Monica; Longhi, Renato; Baloban, Mikhail; Wilson, Heather M; Mergaert, Peter; Zanda, Matteo; Ferguson, Gail P

    2012-03-30

    The root nodules of certain legumes including Medicago truncatula produce >300 different nodule-specific cysteine-rich (NCR) peptides. Medicago NCR antimicrobial peptides (AMPs) mediate the differentiation of the bacterium, Sinorhizobium meliloti into a nitrogen-fixing bacteroid within the legume root nodules. In vitro, NCR AMPs such as NCR247 induced bacteroid features and exhibited antimicrobial activity against S. meliloti. The bacterial BacA protein is critical to prevent S. meliloti from being hypersensitive toward NCR AMPs. NCR AMPs are cationic and have conserved cysteine residues, which form disulfide (S-S) bridges. However, the natural configuration of NCR AMP S-S bridges and the role of these in the activity of the peptide are unknown. In this study, we found that either cysteine replacements or S-S bond modifications influenced the activity of NCR247 against S. meliloti. Specifically, either substitution of cysteines for serines, changing the S-S bridges from cysteines 1-2, 3-4 to 1-3, 2-4 or oxidation of NCR247 lowered its activity against S. meliloti. We also determined that BacA specifically protected S. meliloti against oxidized NCR247. Due to the large number of different NCRs synthesized by legume root nodules and the importance of bacterial BacA proteins for prolonged host infections, these findings have important implications for analyzing the function of these novel peptides and the protective role of BacA in the bacterial response toward these peptides.

  20. The attack of the phytopathogens and the trumpet solo: Identification of a novel plant antifungal peptide with distinct fold and disulfide bond pattern.

    PubMed

    Mandal, Santi M; Porto, William F; Dey, Prabuddha; Maiti, Mrinal K; Ghosh, Ananta K; Franco, Octavio L

    2013-10-01

    Phytopathogens cause economic losses in agribusiness. Plant-derived compounds have been proposed to overcome this problem, including the antimicrobial peptides (AMPs). This paper reports the identification of Ps-AFP1, a novel AMP isolated from the Pisum sativum radicle. Ps-AFP1 was purified and evaluated against phytopathogenic fungi, showing clear effectiveness. In silico analyses were performed, suggesting an unusual fold and disulfide bond pattern. A novel fold and a novel AMP class were here proposed, the αβ-trumpet fold and αβ-trumpet peptides, respectively. The name αβ-trumpet was created due to the peptide's fold, which resembles the musical instrument. The Ps-AFP1 mechanism of action was also proposed. Microscopic analyses revealed that Ps-AFP1 could affect the fungus during the hyphal elongation from spore germination. Furthermore, confocal microscopy performed with Ps-AFP1 labeled with FITC shows that the peptide was localized at high concentration along the fungal cell surface. Due to low cellular disruption rates, it seems that the main target is the fungal cell wall. The binding thermogram and isothermal titration, molecular dynamics and docking analyses were also performed, showing that Ps-AFP1 could bind to chitin producing a stable complex. Data here reported provided novel structural-functional insights into the αβ-trumpet peptide fold.

  1. Conjugation to octa-arginine via disulfide bonds confers solubility to denatured proteins in physiological solution and enables efficient cell internalization.

    PubMed

    Kuwada, Eri; Tadaki, Toshimasa; Kambara, Kaori; Egawa, Kohji; Noguchi, Katsuo

    2011-01-01

    Some protein transduction methods have already been developed for regenerative medicine application. These methods can be applied to soluble proteins but not to insoluble proteins, such as those that originate from inclusion bodies, for example, Escherichia coli. We have developed a method that allows the in vitro solubilization of denatured proteins without refolding and their efficient cellular internalization through conjugation to the peptide, octa-arginine (R8), via disulfide bonds with cysteine residues. Ovalbumin (OVA), denatured in urea solution containing dithiothreitol, was used as a model protein. The R8 peptide was conjugated with OVA in urea solution. Denatured OVA was recovered in the insoluble fraction after dialysis against phosphate-buffered saline. However, almost all the R8-conjugated OVA was recovered in the soluble fraction and used for translocation experiments in HeLa, Chinese hamster ovary-K1, Cos-7, and matured dendritic cells, where efficient internalization of the protein conjugate was observed. Furthermore, we formulated R8-conjugated β-galactosidase and R8-conjugated luciferase using a similar procedure, and investigated how the conjugated proteins are processed after cell internalization. We also observed that only a small fraction of these proteins refolded and almost all underwent intracellular degradation. These results suggest that this method is suitable for the transduction of antigen-presenting cells and will benefit research and innovation in vaccine design and discovery.

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

    PubMed

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

    2014-09-16

    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.

  3. Transition-metal-catalyzed additions of C-H bonds to C-X (X = N, O) multiple bonds via C-H bond activation.

    PubMed

    Yan, Guobing; Wu, Xiangmei; Yang, Minghua

    2013-09-14

    Chemical transformations via catalytic C-H bond activation have been established as one of the most powerful tools in organic synthetic chemistry. Transition-metal-catalyzed addition reactions of C-H bonds to polar C-X (X = N, O) multiple bonds, such as aldehydes, ketones, imines, isocyanates, nitriles, isocyanides, carbon monoxide and carbon dioxide, have undergone a rapid development in recent years. In this review, recent advances in this active area have been highlighted and their mechanisms have been discussed.

  4. Site-Selective Disulfide Modification of Proteins: Expanding Diversity beyond the Proteome.

    PubMed

    Kuan, Seah Ling; Wang, Tao; Weil, Tanja

    2016-11-21

    The synthetic transformation of polypeptides with molecular accuracy holds great promise for providing functional and structural diversity beyond the proteome. Consequently, the last decade has seen an exponential growth of site-directed chemistry to install additional features into peptides and proteins even inside living cells. The disulfide rebridging strategy has emerged as a powerful tool for site-selective modifications since most proteins contain disulfide bonds. In this Review, we present the chemical design, advantages and limitations of the disulfide rebridging reagents, while summarizing their relevance for synthetic customization of functional protein bioconjugates, as well as the resultant impact and advancement for biomedical applications.

  5. Carbon disulfide

    Integrated Risk Information System (IRIS)

    Carbon disulfide ; CASRN 75 - 15 - 0 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic E

  6. Thiol-copper(I) and disulfide-dicopper(I) complex O2-reactivity leading to sulfonate-copper(II) complex or the formation of a cross-linked thioether-phenol product with phenol addition.

    PubMed

    Lee, Yunho; Lee, Dong-Heon; Sarjeant, Amy A Narducci; Karlin, Kenneth D

    2007-11-01

    In order to better understand copper mediated oxidative chemistry via ligand-Cu(I)/O(2) reactivity employing S-donor ligands for copper, O(2)-reactivity studies of the copper(I) complexes (1 and 2, Chart 2) have been carried out with a tridentate N(2)S thiol ligand (1-(N-methyl-N-(2-(pyridin-2-yl)ethyl)amino)propane-2-thiol; L(SH)) or its oxidized disulfide form (L(SS)). Reactions of [L(SH)Cu(I)](+) (1) and [L(SS)(Cu(I))(2)(X)(2)](2+) (2) with O(2) give approximately 90% and approximately 70% yields of [L(SO3)Cu(II)(MeOH)(2)](+) (3), respectively, where L(SO3) is S-oxygenated sulfonate; 3 was characterized by electrospray ionization (ESI) mass spectrometry and X-ray crystallography. Mimicking TyrCys galactose oxidase cofactor biogenesis, a new C-S bond is formed (within new thioether moiety L(SPhOH)) from cuprous complex (both 1 and 2) dioxygen reactivity in the presence of 2,4-tBu(2)-phenolate. In addition, the disulfide ligand (L(SS)) reacts with 2equiv. cupric ion salts and the phenolate to efficiently give the cross-linked product L(SPhOH) in high yield (>90%) under anaerobic conditions. Separately, complex [L(SPhO)Cu(II)(ClO(4))] (4), possessing the cross-linked L(SPhOH), was characterized by ESI mass spectrometry and X-ray crystallography.

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

    PubMed

    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.

  8. Identification of Allosteric Disulfides from Prestress Analysis

    PubMed Central

    Zhou, Beifei; Baldus, Ilona B.; Li, Wenjin; Edwards, Scott A.; Gräter, Frauke

    2014-01-01

    Disulfide bonds serve to form physical cross-links between residues in protein structures, thereby stabilizing the protein fold. Apart from this purely structural role, they can also be chemically active, participating in redox reactions, and they may even potentially act as allosteric switches controlling protein functions. Specific types of disulfide bonds have been identified in static protein structures from their distinctive pattern of dihedral bond angles, and the allosteric function of such bonds is purported to be related to the torsional strain they store. Using all-atom molecular-dynamics simulations for ∼700 disulfide bonded proteins, we analyzed the intramolecular mechanical forces in 20 classes of disulfide bonds. We found that two particular classes, the −RHStaple and the −/+RHHook disulfides, are indeed more stressed than other disulfide bonds, but the stress is carried primarily by stretching of the S-S bond and bending of the neighboring bond angles, rather than by dihedral torsion. This stress corresponds to a tension force of magnitude ∼200 pN, which is balanced by repulsive van der Waals interactions between the cysteine Cα atoms. We confirm stretching of the S-S bond to be a general feature of the −RHStaples and the −/+RHHooks by analyzing ∼20,000 static protein structures. Given that forced stretching of S-S bonds is known to accelerate their cleavage, we propose that prestress of allosteric disulfide bonds has the potential to alter the reactivity of a disulfide, thereby allowing us to readily switch between functional states. PMID:25099806

  9. The Variable Transition State in Polar Additions to Pi Bonds

    ERIC Educational Resources Information Center

    Weiss, Hilton M.

    2010-01-01

    A vast majority of polar additions of Bronsted acids to alkynes involve a termolecular transition state. With strong acids, considerable positive charge is developed on carbon and Markovnikov addition predominates. In less acidic solutions, however, the reaction is much slower and the transition state more closely resembles the olefinic product.…

  10. Homolytic S-S bond dissociation of 11 bis(thiocarbonyl)disulfides R-C(=S)-S-S-C(=S)R and prediction of a novel rubber vulcanization accelerator.

    PubMed

    Mak, Adrian Matthew; Steudel, Ralf; Wong, Ming Wah

    2008-06-02

    The structures and energetics of eight substituted bis(thiocarbonyl)disulfides (RCS(2))(2), their associated radicals RCS(2)(*), and their coordination compounds with a lithium cation have been studied at the G3X(MP2) level of theory for R = H, Me, F, Cl, OMe, SMe, NMe(2), and PMe(2). The effects of substituents on the dissociation of (RCS(2))(2) to RCS(2)(*) were analyzed using isodesmic stabilization reactions. Electron-donating groups with an unshared pair of electrons have a pronounced stabilization effect on both (RCS(2))(2) and RCS(2)(*). The S-S bond dissociation enthalpy of tetramethylthiuram disulfide (TMTD, R = NMe(2)) is the lowest in the above series (155 kJ mol(-1)), attributed to the particular stability of the formed Me(2)NCS(2)(*) radical. Both (RCS(2))(2) and the fragmented radicals RCS(2)(*) form stable chelate complexes with a Li(+) cation. The S-S homolytic bond cleavage in (RCS(2))(2) is facilitated by the reaction [Li(RCS(2))(2)](+)+Li(+)-->2 [Li(RCS(2))](*+). Three other substituted bis(thiocarbonyl) disulfides with the unconventional substituents R = OSF(5), Gu(1), and Gu(2) have been explored to find suitable alternative rubber vulcanization accelerators. Bis(thiocarbonyl)disulfide with a guanidine-type substituent, (Gu(1)CS(2))(2), is predicted to be an effective accelerator in sulfur vulcanization of rubber. Compared to TMTD, (Gu(1)CS(2))(2) is calculated to have a lower bond dissociation enthalpy and smaller associated barrier for the S-S homolysis.

  11. 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.

    PubMed

    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 (VH+CH1) 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.

  12. Carbon–carbon bond activation of cyclobutenones enabled by the addition of chiral organocatalyst to ketone

    PubMed Central

    Li, Bao-Sheng; Wang, Yuhuang; Jin, Zhichao; Zheng, Pengcheng; Ganguly, Rakesh; Chi, Yonggui Robin

    2015-01-01

    The activation of carbon–carbon (C–C) bonds is an effective strategy in building functional molecules. The C–C bond activation is typically accomplished via metal catalysis, with which high levels of enantioselectivity are difficult to achieve due to high reactivity of metal catalysts and the metal-bound intermediates. It remains largely unexplored to use organocatalysis for C–C bond activation. Here we describe an organocatalytic activation of C–C bonds through the addition of an NHC to a ketone moiety that initiates a C–C single bond cleavage as a key step to generate an NHC-bound intermediate for chemo- and stereo-selective reactions. This reaction constitutes an asymmetric functionalization of cyclobutenones using organocatalysts via a C–C bond activation process. Structurally diverse and multicyclic compounds could be obtained with high optical purities via an atom and redox economic process. PMID:25652912

  13. Engineering out motion: a surface disulfide bond alters the mobility of tryptophan 22 in cytochrome b5 as probed by time-resolved fluorescence and 1H NMR experiments.

    PubMed

    Storch, E M; Grinstead, J S; Campbell, A P; Daggett, V; Atkins, W M

    1999-04-20

    the wild type, and the fractional contribution increased significantly upon raising the temperature. The fast rotational component of the S18C:R47C mutant was less sensitive to increasing temperature. A comparison of the 1H NMR monitored temperature titration of the delta-methyl protons of Ile 76 for wild type and oxidized disulfide mutant, S18C:R47C, showed a significantly smaller downfield shift for the mutant protein, suggesting that Trp 22 in the mutant protein experiences comparatively decreased cleft dynamics in core 2 at higher temperatures. Furthermore, comparison of the delta'-methyl protons of Leu 25 in the two proteins revealed a difference in the ratio of the equilibrium heme conformers of 1.2:1 for S18C:R47C versus 1.5:1 for wild type at 40 degrees C. The difference in equilibrium heme orientations between wild type and S18C:R47C suggests that the disulfide bond affects heme binding within core 1, possibly through damped cleft fluctuations. Taken together, the NMR and fluorescence studies support the proposal that an engineered disulfide bond inhibits the formation of a dynamic cleft on the surface of cyt b5.

  14. Effects of adding low levels of a disulfide reducing agent on the disulfide interactions of β-lactoglobulin and κ-casein in skim milk.

    PubMed

    Nguyen, Nguyen H A; Wong, Marie; Anema, Skelte G; Havea, Palatasa; Guyomarc'h, Fanny

    2012-03-07

    Low concentrations of a disulfide reducing agent were added to unheated and heated (80 °C for 30 min) skim milk, with and without added whey protein. The reduction of the β-lactoglobulin and κ-casein disulfide bonds was monitored over time using electrophoresis. The distribution of the proteins between the colloidal and serum phases was also investigated. κ-Casein disulfide bonds were reduced in preference to those of β-lactoglobulin in both unheated and heated skim milk (with or without added whey protein). In addition, in heated skim milk, while the serum κ-casein was reduced more readily than the colloidal κ-casein, the distribution of κ-casein between the two phases was not affected.

  15. Soft Computing Methods for Disulfide Connectivity Prediction

    PubMed Central

    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. PMID:26523116

  16. Transition metal-catalyzed process for addition of amines to carbon-carbon double bonds

    DOEpatents

    Hartwig, John F.; Kawatsura, Motoi; Loeber, Oliver

    2002-01-01

    The present invention is directed to a process for addition of amines to carbon-carbon double bonds in a substrate, comprising: reacting an amine with a compound containing at least one carbon-carbon double bond in the presence a transition metal catalyst under reaction conditions effective to form a product having a covalent bond between the amine and a carbon atom of the former carbon-carbon double bond. The transition metal catalyst comprises a Group 8 metal and a ligand containing one or more 2-electron donor atoms. The present invention is also directed to enantioselective reactions of amine compounds with compounds containing carbon-carbon double bonds, and a calorimetric assay to evaluate potential catalysts in these reactions.

  17. 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

    SciTech Connect

    Torgomyan, Heghine; Trchounian, Armen

    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.

  18. The effect of additional etching and curing mechanism of composite resin on the dentin bond strength

    PubMed Central

    Lee, In-Su; Son, Sung-Ae; Hur, Bock; Kwon, Yong-Hoon

    2013-01-01

    PURPOSE The aim of this study was to evaluate the effects of additional acid etching and curing mechanism (light-curing or self-curing) of a composite resin on the dentin bond strength and compatibility of one-step self-etching adhesives. MATERIALS AND METHODS Sixteen human permanent molars were randomly divided into eight groups according to the adhesives used (All-Bond Universal: ABU, Clearfil S3 Bond: CS3), additional acid etching (additional acid etching performed: EO, no additional acid etching performed: EX), and composite resins (Filtek Z-250: Z250, Clearfil FII New Bond: CFNB). Group 1: ABU-EO-Z250, Group 2: ABU-EO-CFNB, Group 3: ABU-EX-Z250, Group 4: ABU-EX-CFNB, Group 5: CS3-EO-Z250, Group 6: CS3-EO-CFNB, Group 7: CS3-EX-Z250, Group 8: CS3-EX-CFNB. After bonding procedures, composite resins were built up on dentin surfaces. After 24-hour water storage, the teeth were sectioned to make 10 specimens for each group. The microtensile bond strength test was performed using a microtensile testing machine. The failure mode of the fractured specimens was examined by means of an optical microscope at ×20 magnification. The data was analyzed using a one-way ANOVA and Scheffe's post-hoc test (α=.05). RESULTS Additional etching groups showed significantly higher values than the no additional etching group when using All-Bond Universal. The light-cured composite resin groups showed significantly higher values than the self-cured composite resin groups in the Clearfil S3 Bond. CONCLUSION The additional acid etching is beneficial for the dentin bond strength when using low acidic one-step self-etch adhesives, and low acidic one-step self-etch adhesives are compatible with self-cured composite resin. The acidity of the one-step self-etch adhesives is an influencing factor in terms of the dentin bonding strength and incompatibility with a self-cured composite resin. PMID:24353889

  19. Eight at one stroke - a synthetic tetra-disulfide peptide epitope.

    PubMed

    Schrimpf, Andreas; Linne, Uwe; Geyer, Armin

    2017-02-13

    We have designed a cysteine-rich β-hairpin peptide which dimerises spontaneously to the antiparallel double β-hairpin motif C1-C12', C1'-C12, C5-C8, C5'-C8'-tricyclo-(CHWECCitGCRLVC)2. The highly regioselective oxidation of eight cysteines yields an intermolecular bi-disulfide 24mer hinge peptide from two individual 12mer β-hairpins, each rigidified by an additional intramolecular disulfide bond - all in all a tetra-disulfide. The reaction kinetics of air-oxidation were followed by HPLC and the constitutional isomer was identified by mass spectrometry. The hairpin conformation was characterised in detail by NMR spectroscopy and the opening angle of the antiparallel hinge was estimated from drift times obtained by ion-mobility spectrometry. Based on a set of investigated disulfide motifs, we are able to rationalise how the unbalanced number of bonded and non-bonded hydrogen pairs in a 12 mer hairpin causes their dimerisation. The unique dimeric bi-/tetra-disulfides provide systematic insights into β-hairpin formation. They can serve as a standalone structural element for the oligomerisation of peptide epitopes where structural diversity is generated from a minimal number of amino acids.

  20. The disulfide relay system of mitochondria is required for the biogenesis of mitochondrial Ccs1 and Sod1.

    PubMed

    Reddehase, Silvia; Grumbt, Barbara; Neupert, Walter; Hell, Kai

    2009-01-16

    Cells protect themselves against oxygen stress and reactive oxygen species. An important enzyme in this process is superoxide dismutase, Sod1, which converts superoxide radicals into water and hydrogen peroxide. The biogenesis of functional Sod1 is dependent on its copper chaperone, Ccs1, which introduces a disulfide bond and a copper ion into Sod1. Ccs1 and Sod1 are present in the cytosol but are also found in the mitochondrial intermembrane space (IMS), the compartment between the outer and the inner membrane of mitochondria. Ccs1 mediates mitochondrial localization of Sod1. Here, we report on the biogenesis of the fractions of Ccs1 and Sod1 present in mitochondria of Saccharomyces cerevisiae. The IMS of mitochondria harbors a disulfide relay system consisting of the import receptor Mia40 and the thiol oxidase Erv1, which drives the import of substrates with conserved cysteine residues arranged in typical twin Cx(3)C and twin Cx(9)C motifs. We show that depletion of Mia40 results in decreased levels of Ccs1 and Sod1. On the other hand, overexpression of Mia40 increased the mitochondrial fraction of both proteins. In addition, the import rates of Ccs1 were enhanced by increased levels of Mia40 and reduced upon depletion of Mia40. Mia40 forms mixed disulfides with Ccs1, suggesting a role of Mia40 for the generation of disulfide bonds in Ccs1. We suggest that the disulfide relay system transfers disulfide bonds via Mia40 to Ccs1, which then shuttles disulfide bonds to Sod1. In conclusion, the disulfide relay system is crucial for the import of Ccs1, thereby affecting the transport of Sod1, and it can control the distribution of Ccs1 and Sod1 between the IMS of mitochondria and the cytosol.

  1. A mutation in either dsbA or dsbB, a gene encoding a component of a periplasmic disulfide bond-catalyzing system, is required for high-level expression of the Bacteroides fragilis metallo-beta-lactamase, CcrA, in Escherichia coli.

    PubMed Central

    Alksne, L E; Keeney, D; Rasmussen, B A

    1995-01-01

    The metallo-beta-lactamase gene, ccrA, from Bacteroides fragilis is functionally expressed in Escherichia coli only in the presence of a genomic mutation in iarA or iarB (increased ampicillin resistance), identified in this study as dsbA or dsbB, respectively. DsbA and DsbB are components of a periplasmic protein disulfide bond-catalyzing system. Data indicated that DsbA interacted with CcrA, creating aberrant disulfide bond linkages that render CcrA proteolytically unstable. Mutations in dsbA or dsbB permissive for CcrA expression eliminated or greatly reduced DsbA activity, allowing CcrA to assume a disulfide bond-free and proteolytically stable conformation. PMID:7814337

  2. Evidence for thiol/disulfide exchange reactions between tubulin and glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Landino, Lisa M; Hagedorn, Tara D; Kennett, Kelly L

    2014-12-01

    While thiol redox reactions are a common mechanism to regulate protein structure and function, protein disulfide bond formation is a marker of oxidative stress that has been linked to neurodegeneration. Both tubulin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) contain multiple cysteines that have been identified as targets for oxidation to disulfides, S-nitrosation and S-glutathionylation. We show that GAPDH is one of three prominent brain microtubule-associated proteins (MAPs), in addition to MAP-2 and tau, with reactive cysteines. We detected a threefold to fourfold increase in tubulin cysteine oxidation by hydrogen peroxide in the presence of rabbit muscle GAPDH by 5-iodoacetamidofluorescein labeling and by Western blot detection of higher molecular weight inter-chain tubulin disulfides. In thiol/disulfide exchange experiments, tubulin restored ∼50% of oxidized GAPDH cysteines and the equilibrium favored reduced GAPDH. Further, we report that oxidized GAPDH is repaired by the thioredoxin reductase system (TRS). Restoration of GAPDH activity after reduction by both tubulin and the TRS was time-dependent suggesting conformational changes near the active site cysteine149. The addition of brain MAPs to oxidized tubulin reduced tubulin disulfides and labeling of MAP-2 and of GAPDH decreased. Because the extent of tubulin repair of oxidized GAPDH was dependent on buffer strength, we conclude that electrostatics influence thiol/disulfide exchange between the two proteins. The novel interactions presented herein may protect GAPDH from inhibition under oxidative stress conditions.

  3. Protein stabilization by introduction of cross-strand disulfides.

    PubMed

    Chakraborty, Kausik; Thakurela, Sudhir; Prajapati, Ravindra Singh; Indu, S; Ali, P Shaik Syed; Ramakrishnan, C; Varadarajan, Raghavan

    2005-11-08

    Disulfides cross-link residues in a protein that are separated in primary sequence and stabilize the protein through entropic destabilization of the unfolded state. While the removal of naturally occurring disulfides leads to protein destabilization, introduction of engineered disulfides does not always lead to significant stabilization of a protein. We have analyzed naturally occurring disulfides that span adjacent antiparallel strands of beta sheets (cross-strand disulfides). Cross-strand disulfides have recently been implicated as redox-based conformational switches in proteins such as gp120 and CD4. The propensity of these disulfides to act as conformational switches was postulated on the basis of the hypothesis that this class of disulfide is conformationally strained. In the present analysis, there was no evidence to suggest that cross-strand disulfides are more strained compared to other disulfides as assessed by their torsional energy. It was also observed that these disulfides occur solely at non-hydrogen-bonded (NHB) registered pairs of adjacent antiparallel strands and not at hydrogen-bonded (HB) positions as suggested previously. One of the half-cystines involved in cross-strand disulfide formation often occurs at an edge strand. Experimental confirmation of the stabilizing effects of such disulfides was carried out in Escherichia coli thioredoxin. Four pairs of cross-strand cysteines were introduced, two at HB and two at NHB pairs. Disulfides were formed in all four cases. However, as predicted from our analysis, disulfides at NHB positions resulted in an increase in melting temperature of 7-10 degrees C, while at HB positions there was a corresponding decrease of -7 degrees C. The reduced state of all proteins had similar stability.

  4. Highly enantioselective Michael addition of nitroalkanes to chalcones using chiral squaramides as hydrogen bonding organocatalysts.

    PubMed

    Yang, Wen; Du, Da-Ming

    2010-12-03

    A series of squaramide-based organocatalysts were facilely synthesized and applied as hydrogen bonding organocatalysts in the enantioselective Michael addition of nitroalkanes to chalcones. These organocatalysts promoted the Michael addition with low catalyst loading under high temperature (80 °C), affording the desired R or S enantiomers of the products flexibly in high yields with excellent enantioselectivities (93-96% ee) by the appropriate choice of organocatalysts.

  5. Oxidative addition of C--H bonds in organic molecules to transition metal centers

    SciTech Connect

    Bergman, R.G.

    1989-04-01

    Alkanes are among the most chemically inert organic molecules. They are reactive toward a limited range of reagents, such as highly energetic free radicals and strongly electrophilic and oxidizing species. This low reactivity is a consequence of the C--H bond energies in most saturated hydrocarbons. These values range from 90 to 98 kcal/mole for primary and secondary C--H bonds; in methane, the main constituent of natural gas, the C--H bond energy is 104 kcal/mole. This makes methane one of the most common but least reactive organic molecules in nature. This report briefly discusses the search for metal complexes capable of undergoing the C--H oxidative addition process allowing alkane chemistry to be more selective than that available using free radical reagents. 14 refs.

  6. Synthesis of substituted β-diketiminate gallium hydrides via oxidative addition of H-O bonds.

    PubMed

    Herappe-Mejía, Eduardo; Trujillo-Hernández, Karla; Carlos Garduño-Jiménez, Juan; Cortés-Guzmán, Fernando; Martínez-Otero, Diego; Jancik, Vojtech

    2015-10-14

    Oxidative addition of LGa into the OH bonds from HCCCH2OH, Ph2Si(OH)2, (nBuO)2P(O)(OH) and 4-MeC6H4S(O)2(OH) results in the formation of four compounds of the general formula LGa(H)(O-X). The correlation of the Ga-O bond length and the strength of the Ga-H bond depending on the acidity of the OH group in the starting materials has been demonstrated. The molecular structures of all four compounds have been determined using single crystal X-ray diffraction experiments. DFT calculations were performed on the reacting complex of LGa with propargyl alcohol and show an OHGa hydrogen bond as the first interaction between the reagents. This reacting complex changes into a D-A complex where the oxygen atom of the propargyl alcohol coordinates to the gallium atom and in a concerted reaction the oxidative addition product is formed.

  7. Influence of additional adhesive application on the microtensile bond strength of adhesive systems.

    PubMed

    de Silva, André Luís Faria; Lima, Débora Alves Nunes Leite; de Souza, Grace Mendonça Dias; dos Santos, Carlos Tadeu Dias; Paulillo, Luís Alexandre Maffei Sartini

    2006-01-01

    This study evaluated microtensile bond strength (pTBS) when an additional adhesive layer was applied to the dentin surface. Thirty-five human third molars were flattened to expose the occlusal dentin surface. The teeth were randomly assigned to 7 experimental groups: G1-Single Bond (SB); G2-additional layer of SB; G3--a layer of Scotchbond Multi-purpose (SMP) adhesive applied over SB; G4-Clearfil SE Bond (CE); G5-additional layer of CE; G6-Adper Prompt (AP) and G7-additional layer of AP. For the G2, G3, G5 and G7 groups, the first adhesive layer was light-cured before application of the additional layer. After bonding procedures, 5-mm high composite crowns were incrementally built up. The samples were sectioned to obtain 0.9 x 0.9 beams, which were tested under tension at a crosshead speed of 0.5-mm/minute until failure. The failure mode and adhesive thickness were evaluated under SEM. The pTBS data were analyzed by 1-way ANOVA and post-hoc Ducan's Test (a=0.05). Mean adhesive thickness was analyzed by 1-way ANOVA and post-hoc Tukey's test (a=0.05). The results indicated that G3 presented the highest microTBS and the thickest adhesive layer. G6 and G7 presented the lowest microTBS values. When solvent-free adhesives systems were used, microTBS values were not affected by the thicker layer.

  8. Rh(III)-Catalyzed C-H Bond Addition/Amine-Mediated Cyclization of Bis-Michael Acceptors.

    PubMed

    Potter, Tyler J; Ellman, Jonathan A

    2016-08-05

    A Rh(III)-catalyzed C-H bond addition/primary amine-promoted cyclization of bis-Michael acceptors is reported. The C-H bond addition step occurs with high chemoselectivity, and the subsequent intramolecular Michael addition, mediated by a primary amine catalyst, sets three contiguous stereocenters with high diastereoselectivity. A broad range of directing groups and both aromatic and alkenyl C-H bonds were shown to be effective in this transformation, affording functionalized piperidines, tetrahydropyrans, and cyclohexanes.

  9. The role of intramolecular hydrogen bonds in nucleophilic addition reactions of ketenaminals

    NASA Astrophysics Data System (ADS)

    Isaev, A. N.

    2012-08-01

    Quantum-chemical calculations of the geometries and electronic structures of molecules of ketenaminals 3-(diaminomethylene)-2,4-pentanedione and dimethyl-2-(diaminomethylene)-malonate and calculations of the structures of intermediates in the reaction of the nucleophilic addition of the ketenaminals to the acetonitrile molecule are performed by B3LYP/6-31+G** method. Two possible scenarios of the process are shown, depending on the mutual orientation of reacting molecules. The nucleophilic addition proceeds in two stages. It is found that the rate-limiting stage of the process is the transfer of the proton of the intramolecular hydrogen bond in a ketenaminal molecule. The experimentally observed faster reaction of pyrimidine formation for the 3-(diaminomethylene)-2,4-pentanedione molecule relative to that for dimethyl-2-(diaminomethylene)-malonate is explained by the hydrogen bond being stronger and the barrier of proton transfer from the aminogroup to the ketogroup oxygen falling upon nucleophilic attack in the former molecule.

  10. Oxidative addition of the C-I bond on aluminum nanoclusters

    NASA Astrophysics Data System (ADS)

    Sengupta, Turbasu; Das, Susanta; Pal, Sourav

    2015-07-01

    Energetics and the in-depth reaction mechanism of the oxidative addition step of the cross-coupling reaction are studied in the framework of density functional theory (DFT) on aluminum nanoclusters. Aluminum metal in its bulk state is totally inactive towards carbon-halogen bond dissociation but selected Al nanoclusters (size ranging from 3 to 20 atoms) have shown a significantly lower activation barrier towards the oxidative addition reaction. The calculated energy barriers are lower than the gold clusters and within a comparable range with the conventional and most versatile Pd catalyst. Further investigations reveal that the activation energies and other reaction parameters are highly sensitive to the geometrical shapes and electronic structures of the clusters rather than their size, imposing the fact that comprehensive studies on aluminum clusters can be beneficial for nanoscience and nanotechnology. To understand the possible reaction mechanism in detail, the reaction pathway is investigated with the ab initio Born Oppenheimer Molecular Dynamics (BOMD) simulation and the Natural Bond Orbital (NBO) analysis. In short, our theoretical study highlights the thermodynamic and kinetic details of C-I bond dissociation on aluminum clusters for future endeavors in cluster chemistry.Energetics and the in-depth reaction mechanism of the oxidative addition step of the cross-coupling reaction are studied in the framework of density functional theory (DFT) on aluminum nanoclusters. Aluminum metal in its bulk state is totally inactive towards carbon-halogen bond dissociation but selected Al nanoclusters (size ranging from 3 to 20 atoms) have shown a significantly lower activation barrier towards the oxidative addition reaction. The calculated energy barriers are lower than the gold clusters and within a comparable range with the conventional and most versatile Pd catalyst. Further investigations reveal that the activation energies and other reaction parameters are highly

  11. Production of Recombinant Disulfide-Rich Venom Peptides for Structural and Functional Analysis via Expression in the Periplasm of E. coli

    PubMed Central

    Saez, Natalie J.; Seshadri, Radha; Lau, Ho Yee; Bende, Niraj S.; Undheim, Eivind A. B.; Rash, Lachlan D.; Mobli, Mehdi; King, Glenn F.

    2013-01-01

    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. PMID:23667680

  12. Thiol/disulfide homeostasis in asphalt workers.

    PubMed

    Yilmaz, Ömer Hınç; Bal, Ceylan; Neşelioglu, Salim; Büyükşekerci, Murat; Gündüzöz, Meşide; Eren, Funda; Tutkun, Lutfiye; Yilmaz, Fatma Meric

    2016-09-02

    The aim of this study was to investigate thiol/disulfide homeostasis in asphalt workers who are exposed to polycyclic aromatic hydrocarbons occupationally. The study was carried out in 34 nonsmoker asphalt workers. Additionally, 35 healthy nonsmoker volunteers were recruited as control group. Thiol and disulfide concentrations were determined using the novel automated measurement method. Levels of urinary 1-OH-pyrene were analyzed by liquid chromatography. Disulfide/thiol ratio was significantly higher in exposed group (p = .034). Also, a positive correlation was detected between disulfide/thiol ratio and 1-OH-pyrene values (r = .249, p = .036). Thiol/disulfide homeostasis was found to be disturbed in asphalt workers. The novel test used in this study may be useful for evaluating the oxidative status in polycyclic aromatic hydrocarbon (PAH) exposure.

  13. A degradable polydopamine coating based on disulfide-exchange reaction

    NASA Astrophysics Data System (ADS)

    Hong, Daewha; Lee, Hojae; Kim, Beom Jin; Park, Taegyun; Choi, Ji Yu; Park, Matthew; Lee, Juno; Cho, Hyeoncheol; Hong, Seok-Pyo; Yang, Sung Ho; Jung, Sun Ho; Ko, Sung-Bo; Choi, Insung S.

    2015-11-01

    Although the programmed degradation of biocompatible films finds applications in various fields including biomedical and bionanotechnological areas, coating methods have generally been limited to be substrate-specific, not applicable to any kinds of substrates. In this paper, we report a dopamine derivative, which allows for both universal coating of various substrates and stimuli-responsive film degradation, inspired by mussel-adhesive proteins. Two dopamine moieties are linked together by the disulfide bond, the cleavage of which enables the programmed film degradation. Mechanistic analysis of the degradable films indicates that the initial cleavage of the disulfide linkage causes rapid uptake of water molecules, hydrating the films, which leads to rapid degradation. Our substrate-independent coating of degradable films provides an advanced tool for drug delivery systems, tissue engineering, and anti-fouling strategies.Although the programmed degradation of biocompatible films finds applications in various fields including biomedical and bionanotechnological areas, coating methods have generally been limited to be substrate-specific, not applicable to any kinds of substrates. In this paper, we report a dopamine derivative, which allows for both universal coating of various substrates and stimuli-responsive film degradation, inspired by mussel-adhesive proteins. Two dopamine moieties are linked together by the disulfide bond, the cleavage of which enables the programmed film degradation. Mechanistic analysis of the degradable films indicates that the initial cleavage of the disulfide linkage causes rapid uptake of water molecules, hydrating the films, which leads to rapid degradation. Our substrate-independent coating of degradable films provides an advanced tool for drug delivery systems, tissue engineering, and anti-fouling strategies. Electronic supplementary information (ESI) available: Synthesis, characterization, and other additional details. See DOI: 10

  14. T47D Cells Expressing Myeloperoxidase Are Able to Process, Traffic and Store the Mature Protein in Lysosomes: Studies in T47D Cells Reveal a Role for Cys319 in MPO Biosynthesis that Precedes Its Known Role in Inter-Molecular Disulfide Bond Formation

    PubMed Central

    Laura, Richard P.; Dong, David; Reynolds, Wanda F.; Maki, Richard A.

    2016-01-01

    Among the human heme-peroxidase family, myeloperoxidase (MPO) has a unique disulfide-linked oligomeric structure resulting from multi-step processing of the pro-protein monomer (proMPO) after it exits the endoplasmic reticulum (ER). Related family members undergo some, but not all, of the processing steps involved with formation of mature MPO. Lactoperoxidase has its pro-domain proteolytically removed and is a monomer in its mature form. Eosinophil peroxidase undergoes proteolytic removal of its pro-domain followed by proteolytic separation into heavy and light chains and is a heterodimer. However, only MPO undergoes both these proteolytic modifications and then is further oligomerized into a heterotetramer by a single inter-molecular disulfide bond. The details of how and where the post-ER processing steps of MPO occur are incompletely understood. We report here that T47D breast cancer cells stably transfected with an MPO expression plasmid are able to efficiently replicate all of the processing steps that lead to formation of the mature MPO heterotetramer. MPO also traffics to the lysosome granules of T47D cells where it accumulates, allowing in-depth immunofluorescent microscopy studies of MPO trafficking and storage for the first time. Using this novel cell model we show that formation of MPO’s single inter-molecular disulfide bond can occur normally in the absence of the proteolytic events that lead to separation of the MPO heavy and light chains. We further demonstrate that Cys319, which forms MPO’s unique inter-molecular disulfide bond, is important for events that precede this step. Mutation of this residue alters the glycosylation and catalytic activity of MPO and blocks its entry into the endocytic pathway where proteolytic processing and disulfide bonding occur. Finally, using the endocytic trafficking of lysosomal hydrolases as a guide, we investigate the role of candidate receptors in the endocytic trafficking of MPO. PMID:26890638

  15. Big area additive manufacturing of high performance bonded NdFeB magnets

    SciTech Connect

    Li, Ling; Tirado, Angelica; Nlebedim, I. C.; Rios, Orlando; Post, Brian; Kunc, Vlastimil; Lowden, R. R.; Lara-Curzio, Edgar; Fredette, Robert; Ormerod, John; Lograsso, Thomas A.; Paranthaman, M. Parans

    2016-10-31

    Additive manufacturing allows for the production of complex parts with minimum material waste, offering an effective technique for fabricating permanent magnets which frequently involve critical rare earth elements. In this report, we demonstrate a novel method - Big Area Additive Manufacturing (BAAM) - to fabricate isotropic near-net-shape NdFeB bonded magnets with magnetic and mechanical properties comparable or better than those of traditional injection molded magnets. The starting polymer magnet composite pellets consist of 65 vol% isotropic NdFeB powder and 35 vol% polyamide (Nylon-12). The density of the final BAAM magnet product reached 4.8 g/cm3, and the room temperature magnetic properties are: intrinsic coercivity Hci = 688.4 kA/m, remanence Br = 0.51 T, and energy product (BH)max = 43.49 kJ/m3 (5.47 MGOe). In addition, tensile tests performed on four dog-bone shaped specimens yielded an average ultimate tensile strength of 6.60 MPa and an average failure strain of 4.18%. Scanning electron microscopy images of the fracture surfaces indicate that the failure is primarily related to the debonding of the magnetic particles from the polymer binder. As a result, the present method significantly simplifies manufacturing of near-net-shape bonded magnets, enables efficient use of rare earth elements thus contributing towards enriching the supply of critical materials.

  16. Big area additive manufacturing of high performance bonded NdFeB magnets

    DOE PAGES

    Li, Ling; Tirado, Angelica; Nlebedim, I. C.; ...

    2016-10-31

    Additive manufacturing allows for the production of complex parts with minimum material waste, offering an effective technique for fabricating permanent magnets which frequently involve critical rare earth elements. In this report, we demonstrate a novel method - Big Area Additive Manufacturing (BAAM) - to fabricate isotropic near-net-shape NdFeB bonded magnets with magnetic and mechanical properties comparable or better than those of traditional injection molded magnets. The starting polymer magnet composite pellets consist of 65 vol% isotropic NdFeB powder and 35 vol% polyamide (Nylon-12). The density of the final BAAM magnet product reached 4.8 g/cm3, and the room temperature magnetic propertiesmore » are: intrinsic coercivity Hci = 688.4 kA/m, remanence Br = 0.51 T, and energy product (BH)max = 43.49 kJ/m3 (5.47 MGOe). In addition, tensile tests performed on four dog-bone shaped specimens yielded an average ultimate tensile strength of 6.60 MPa and an average failure strain of 4.18%. Scanning electron microscopy images of the fracture surfaces indicate that the failure is primarily related to the debonding of the magnetic particles from the polymer binder. As a result, the present method significantly simplifies manufacturing of near-net-shape bonded magnets, enables efficient use of rare earth elements thus contributing towards enriching the supply of critical materials.« less

  17. Big Area Additive Manufacturing of High Performance Bonded NdFeB Magnets

    PubMed Central

    Li, Ling; Tirado, Angelica; Nlebedim, I. C.; Rios, Orlando; Post, Brian; Kunc, Vlastimil; Lowden, R. R.; Lara-Curzio, Edgar; Fredette, Robert; Ormerod, John; Lograsso, Thomas A.; Paranthaman, M. Parans

    2016-01-01

    Additive manufacturing allows for the production of complex parts with minimum material waste, offering an effective technique for fabricating permanent magnets which frequently involve critical rare earth elements. In this report, we demonstrate a novel method - Big Area Additive Manufacturing (BAAM) - to fabricate isotropic near-net-shape NdFeB bonded magnets with magnetic and mechanical properties comparable or better than those of traditional injection molded magnets. The starting polymer magnet composite pellets consist of 65 vol% isotropic NdFeB powder and 35 vol% polyamide (Nylon-12). The density of the final BAAM magnet product reached 4.8 g/cm3, and the room temperature magnetic properties are: intrinsic coercivity Hci = 688.4 kA/m, remanence Br = 0.51 T, and energy product (BH)max = 43.49 kJ/m3 (5.47 MGOe). In addition, tensile tests performed on four dog-bone shaped specimens yielded an average ultimate tensile strength of 6.60 MPa and an average failure strain of 4.18%. Scanning electron microscopy images of the fracture surfaces indicate that the failure is primarily related to the debonding of the magnetic particles from the polymer binder. The present method significantly simplifies manufacturing of near-net-shape bonded magnets, enables efficient use of rare earth elements thus contributing towards enriching the supply of critical materials. PMID:27796339

  18. Big Area Additive Manufacturing of High Performance Bonded NdFeB Magnets.

    PubMed

    Li, Ling; Tirado, Angelica; Nlebedim, I C; Rios, Orlando; Post, Brian; Kunc, Vlastimil; Lowden, R R; Lara-Curzio, Edgar; Fredette, Robert; Ormerod, John; Lograsso, Thomas A; Paranthaman, M Parans

    2016-10-31

    Additive manufacturing allows for the production of complex parts with minimum material waste, offering an effective technique for fabricating permanent magnets which frequently involve critical rare earth elements. In this report, we demonstrate a novel method - Big Area Additive Manufacturing (BAAM) - to fabricate isotropic near-net-shape NdFeB bonded magnets with magnetic and mechanical properties comparable or better than those of traditional injection molded magnets. The starting polymer magnet composite pellets consist of 65 vol% isotropic NdFeB powder and 35 vol% polyamide (Nylon-12). The density of the final BAAM magnet product reached 4.8 g/cm(3), and the room temperature magnetic properties are: intrinsic coercivity Hci = 688.4 kA/m, remanence Br = 0.51 T, and energy product (BH)max = 43.49 kJ/m(3) (5.47 MGOe). In addition, tensile tests performed on four dog-bone shaped specimens yielded an average ultimate tensile strength of 6.60 MPa and an average failure strain of 4.18%. Scanning electron microscopy images of the fracture surfaces indicate that the failure is primarily related to the debonding of the magnetic particles from the polymer binder. The present method significantly simplifies manufacturing of near-net-shape bonded magnets, enables efficient use of rare earth elements thus contributing towards enriching the supply of critical materials.

  19. Big Area Additive Manufacturing of High Performance Bonded NdFeB Magnets

    NASA Astrophysics Data System (ADS)

    Li, Ling; Tirado, Angelica; Nlebedim, I. C.; Rios, Orlando; Post, Brian; Kunc, Vlastimil; Lowden, R. R.; Lara-Curzio, Edgar; Fredette, Robert; Ormerod, John; Lograsso, Thomas A.; Paranthaman, M. Parans

    2016-10-01

    Additive manufacturing allows for the production of complex parts with minimum material waste, offering an effective technique for fabricating permanent magnets which frequently involve critical rare earth elements. In this report, we demonstrate a novel method - Big Area Additive Manufacturing (BAAM) - to fabricate isotropic near-net-shape NdFeB bonded magnets with magnetic and mechanical properties comparable or better than those of traditional injection molded magnets. The starting polymer magnet composite pellets consist of 65 vol% isotropic NdFeB powder and 35 vol% polyamide (Nylon-12). The density of the final BAAM magnet product reached 4.8 g/cm3, and the room temperature magnetic properties are: intrinsic coercivity Hci = 688.4 kA/m, remanence Br = 0.51 T, and energy product (BH)max = 43.49 kJ/m3 (5.47 MGOe). In addition, tensile tests performed on four dog-bone shaped specimens yielded an average ultimate tensile strength of 6.60 MPa and an average failure strain of 4.18%. Scanning electron microscopy images of the fracture surfaces indicate that the failure is primarily related to the debonding of the magnetic particles from the polymer binder. The present method significantly simplifies manufacturing of near-net-shape bonded magnets, enables efficient use of rare earth elements thus contributing towards enriching the supply of critical materials.

  20. A Novel Multiscale Design of Interfaces for Polymeric Composites and Bonded Joints using Additive Manufacturing

    DTIC Science & Technology

    2016-09-13

    AFRL-AFOSR-VA-TR-2016-0317 A Novel Multiscale Design of Interfaces for Polymeric Composites and Bonded Joints using Additive Manufacturing Pavana...PROGRAM ELEMENT NUMBER 5d. PROJECT NUMBER 5e. TASK NUMBER 5f. WORK UNIT NUMBER 6. AUTHOR(S) 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8...PERFORMING ORGANIZATION REPORT NUMBER 10. SPONSOR/MONITOR’S ACRONYM(S) 11. SPONSOR/MONITOR’S REPORT NUMBER(S) 9. SPONSORING/MONITORING AGENCY NAME(S) AND

  1. Direct observation of disulfide isomerization in a single protein

    NASA Astrophysics Data System (ADS)

    Alegre-Cebollada, Jorge; Kosuri, Pallav; Rivas-Pardo, Jaime Andrés; Fernández, Julio M.

    2011-11-01

    Photochemical uncaging techniques use light to release active molecules from otherwise inert compounds. Here we expand this class of techniques by demonstrating the mechanical uncaging of a reactive species within a single protein. We proved this novel technique by capturing the regiospecific reaction between a thiol and a vicinal disulfide bond. We designed a protein that includes a caged cysteine and a buried disulfide. The mechanical unfolding of this protein in the presence of an external nucleophile frees the single reactive cysteine residue, which now can cleave the target disulfide via a nucleophilic attack on either one of its two sulfur atoms. This produces two different and competing reaction pathways. We used single-molecule force spectroscopy to monitor the cleavage of the disulfides, which extends the polypeptide by a magnitude unambiguously associated with each reaction pathway. This allowed us to measure, for the first time, the kinetics of disulfide-bond isomerization in a protein.

  2. Pt and Hf Additions to NiAl Bond Coats and Their Effect on the Lifetime of Thermal Barrier Coatings

    NASA Technical Reports Server (NTRS)

    Nesbitt, J. A.; Gleeson, B.; Sordelet, D.; Barrett, C. A.

    2003-01-01

    The lifetimes of thermal barrier coatings (TBC's) with various NiAlPt(HfZr) bond coats were determined by cyclic oxidation testing at 1163 C (2125 F). The bond coats were sprayed from powders by low pressure plasma spraying onto Rene N5 superalloy substrates. Yttria stabilized zirconia (8YSZ) top coats were applied by air plasma spraying. Surprisingly, there was not a strong correlation between TBC lifetime and Pt or Hf content although Zr additions decreased lifetimes. TBC failure morphologies and bond coat microstructures were examined and are discussed with respect to the bond coat compositions.

  3. Effect of Hf Additions to Pt Aluminide Bond Coats on EB-PVD TBC Life

    NASA Technical Reports Server (NTRS)

    Nesbitt, James; Nagaraj, Ben; Williams, Jeffrey

    2000-01-01

    Small Hf additions were incorporated into a Pt aluminide coating during chemical vapor deposition (CVD) on single crystal RENE N5 substrates. Standard yttria-stabilized zirconia top coats were subsequently deposited onto the coated substrates by electron beam-physical vapor deposition (EB-PVD). The coated substrates underwent accelerated thermal cycle testing in a furnace at a temperature in excess of 1121 C (2050 F) (45 minute hot exposure, 15 minute cool to approximately 121 C (250 F)) until the thermal barrier coating (TBC) failed by spallation. Incorporating Hf in the bond coat increased the TBC life by slightly more than three times that of a baseline coating without added Hf. Scanning electron microscopy of the spalled surfaces indicated that the presence of the Hf increased the adherence of the thermally grown alumina to the Pt aluminide bond coat. The presence of oxide pegs growing into the coating from the thermally grown alumina may also partially account for the improved TBC life by creating a near-surface layer with a graded coefficient of thermal expansion.

  4. Microstructural Characterization of Bonding Interfaces in Aluminum 3003 Blocks Fabricated by Ultrasonic Additive Manufacturing

    SciTech Connect

    Schick, D. E.; Babu, Sudarsanam Suresh; Lippold, John C; Hahnlen, R.M.; Dapino, M.J.; Dehoff, Ryan R; Collins, P.

    2010-01-01

    Ultrasonic additive manufacturing (UAM) is a process by which hybrid and near-netshaped products can be manufactured from thin metallic tapes. One of the main concerns of UAM is the development of anisotropic mechanical properties. In this work, the microstructures in the bond regions are characterized with optical and electron microscopy. Recrystallization and grain growth across the interface are proposed as a mechanism for the bond formation. The presence of voids or unbonded areas, which reduce the load-bearing cross section and create a stress intensity factor, is attributed to the transfer of the sonotrode texture to the new foil layer. This results in large peaks and valleys that are not filled in during processing. Tensile testing revealed the weld interface strength was 15% of the bulk foil. Shear tests of the weld interfaces showed almost 50% of the bulk shear strength of the material. Finally, optical microscopy of the fracture surfaces from the tensile tests revealed 34% of the interface area was unbonded.

  5. Protein disulfide isomerase homolog TrPDI2 contributing to cellobiohydrolase production in Trichoderma reesei.

    PubMed

    Wang, Guokun; Lv, Pin; He, Ronglin; Wang, Haijun; Wang, Lixian; Zhang, Dongyuan; Chen, Shulin

    2015-09-01

    The majority of the cysteine residues in the secreted proteins form disulfide bonds via protein disulfide isomerase (PDI)-mediated catalysis, stabilizing the enzyme activity. The role of PDI in cellulase production is speculative, as well as the possibility of PDI as a target for improving enzyme production efficiency of Trichoderma reesei, a widely used producer of enzyme for the production of lignocellulose-based biofuels and biochemicals. Here, we report that a PDI homolog, TrPDI2 in T. reesei exhibited a 36.94% and an 11.81% similarity to Aspergillus niger TIGA and T. reesei PDI1, respectively. The capability of TrPDI2 to recover the activity of reduced and denatured RNase by promoting refolding verified its protein disulfide isomerase activity. The overexpression of Trpdi2 increased the secretion and the activity of CBH1 at the early stage of cellulase induction. In addition, both the expression level and redox state of TrPDI2 responded to cellulase induction in T. reesei, providing sustainable oxidative power to ensure cellobiohydrolase maturation and production. The results suggest that TrPDI2 may contribute to cellobiohydrolase secretion by enhancing the capability of disulfide bond formation, which is essential for protein folding and maturation.

  6. Hydrogen Bonding Cluster-Enabled Addition of Sulfonic Acids to Haloalkynes: Access to Both (E)- and (Z)-Alkenyl Sulfonates.

    PubMed

    Zeng, Xiaojun; Liu, Shiwen; Shi, Zhenyu; Xu, Bo

    2016-10-07

    We developed an efficient synthesis of alkenyl sulfonates via hydrogen bonding cluster-enabled addition of sulfonic acids to haloalkynes. The reactivity of sulfonic acids could be significantly enhanced in the presence of strong hydrogen bonding donors. This metal-free method results in good chemical yields for a wide range of haloalkyne substrates and demonstrates good functional group tolerance. What is more, we can control the stereoselectivity of addition (cis vs trans) by varying the steric bulk of the sulfonic acid.

  7. A Potent, Versatile Disulfide-Reducing Agent from Aspartic Acid

    PubMed Central

    2013-01-01

    Dithiothreitol (DTT) is the standard reagent for reducing disulfide bonds between and within biological molecules. At neutral pH, however, >99% of DTT thiol groups are protonated and thus unreactive. Herein, we report on (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), a dithiol that can be synthesized from l-aspartic acid in a few high-yielding steps that are amenable to a large-scale process. DTBA has thiol pKa values that are ∼1 unit lower than those of DTT and forms a disulfide with a similar E°′ value. DTBA reduces disulfide bonds in both small molecules and proteins faster than does DTT. The amino group of DTBA enables its isolation by cation-exchange and facilitates its conjugation. These attributes indicate that DTBA is a superior reagent for reducing disulfide bonds in aqueous solution. PMID:22353145

  8. Improving the bond strength between steel rebar and concrete by ozone treatment of rebar and polymer addition to concrete

    SciTech Connect

    Fu, X.; Chung, D.D.L.

    1997-05-01

    Ozone treatment of steel rebar, together with latex addition (20% by weight of cement) to concrete, resulted in a 39% increase in the shear bond strength between rebar and concrete, compared to a 25% increase resulted from either ozone treatment alone or latex addition alone. Ozone treatment and latex addition resulted in similarly small increases in the contact electrical resistivity between rebar and concrete. Methylcellulose addition (0.4% by weight of cement) to concrete gave slightly less bond strength increase than the latex addition, but did not affect the contact resistivity.

  9. Radiation inactivation of ricin occurs with transfer of destructive energy across a disulfide bridge

    SciTech Connect

    Haigler, H.T.; Woodbury, D.J.; Kempner, E.S.

    1985-08-01

    The ionizing radiation sensitivity of ricin, a disulfide-linked heterodimeric protein, was studied as a model to determine the ability of disulfide bonds to transmit destructive energy. The radiation-dependent loss of A chain enzymatic activity after irradiation of either intact ricin or ricin in which the interchain disulfide bond was disrupted gave target sizes corresponding to the molecular size of dimeric ricin or monomeric A chain, respectively. These results clearly show that a disulfide bond can transmit destructive energy between protein subunits.

  10. Effect of adhesion promoting monomer addition to MMA-TBBO resin on bonding to pure palladium.

    PubMed

    Minami, Hiroyuki; Murahara, Sadaaki; Muraguchi, Koichi; Sakoguchi, Kenji; Suzuki, Shiro; Tanaka, Takuo

    2013-01-01

    This study evaluated the effects of combined use of metal primers and modified monomers on the bonding of MMA-TBBO resins to pure palladium (Pd). Bonding surface was polished with 600-grit silicon carbide paper and primed with one of these four metal primers: V-Primer, M. L. Primer, Metaltite, or Alloy Primer. Four monomers, including three modified ones, were added to MMATBBO resin. One was a methyl methacrylate monomer containing no adhesion promoting monomers, while the other two modified monomers contained the functional monomer of either V-Primer or Alloy Primer. Bonded specimens were prepared by incremental build-up of MMA-TBBO resin on primed Pd surfaces. Shear bond strengths were measured after thermal cycling. Bonding to Pd was significantly improved when modified monomer containing the functional monomer of Alloy Primer was used in combination with M. L. Primer or Metaltite applied on the bonding surface.

  11. Highly enantioselective direct Michael addition of nitroalkanes to nitroalkenes catalyzed by amine-thiourea bearing multiple hydrogen-bonding donors.

    PubMed

    Dong, Xiu-Qin; Teng, Huai-Long; Wang, Chun-Jiang

    2009-03-19

    A highly diastereoselective and enantioselective Michael addition of nitroalkanes to nitroalkenes has been achieved by chiral bifunctional amine-thiourea catalyst bearing multiple hydrogen-bonding donors. This catalytic system performs well over a broad scope of substrates, furnishing various 1,3-dinitro compounds in high diastereoselectivity (up to 98:2) and excellent enantioselectivity (up to 99% ee) under mild conditions. Multiple hydrogen bonding donors play a significant role in accelerating reactions, improving diastereoselectivities and enantioselectivities.

  12. Stable Gold(III) Catalysts by Oxidative Addition of a Carbon-Carbon Bond

    PubMed Central

    Wu, Chung-Yeh; Horibe, Takahiro; Jacobsen, Christian Borch

    2014-01-01

    Whereas low-valent late transition metal catalysis has become indispensible for chemical synthesis, homogeneous high-valent transition metal catalysis is underdeveloped, mainly due to the reactivity of high-valent transition metal complexes and the challenges associated with synthesizing them. In this manuscript, we report a mild carbon-carbon bond cleavage reaction by a Au(I) complex that generates a stable Au(III) cationic complex. Complementary to the well-established soft and carbophilic Au(I) catalyst, this Au(III) complex exhibits hard, oxophilic Lewis acidity. This is exemplified by catalytic activation of α,β-unsaturated aldehydes towards selective conjugate additions as well as activation of an unsaturated aldehyde-allene for a [2 + 2] cycloaddition reaction. The origin of the regioselectivity and catalytic activity was elucidated by X-ray crystallographic analysis of an isolated Au(III)-activated cinnamaldehyde intermediate. The concepts revealed in this study provide a strategy for accessing high-valent transition metal catalysis from readily available precursors. PMID:25612049

  13. Stable gold(III) catalysts by oxidative addition of a carbon-carbon bond

    NASA Astrophysics Data System (ADS)

    Wu, Chung-Yeh; Horibe, Takahiro; Jacobsen, Christian Borch; Toste, F. Dean

    2015-01-01

    Low-valent late transition-metal catalysis has become indispensable to chemical synthesis, but homogeneous high-valent transition-metal catalysis is underdeveloped, mainly owing to the reactivity of high-valent transition-metal complexes and the challenges associated with synthesizing them. Here we report a carbon-carbon bond cleavage at ambient conditions by a Au(I) complex that generates a stable Au(III) cationic complex. In contrast to the well-established soft and carbophilic Au(I) catalyst, this Au(III) complex exhibits hard, oxophilic Lewis acidity. For example, we observed catalytic activation of α,β-unsaturated aldehydes towards selective conjugate additions as well as activation of an unsaturated aldehyde-allene for a [2 + 2] cycloaddition reaction. The origin of the regioselectivity and catalytic activity was elucidated by X-ray crystallographic analysis of an isolated Au(III)-activated cinnamaldehyde intermediate. The concepts revealed suggest a strategy for accessing high-valent transition-metal catalysis from readily available precursors.

  14. Energetics of tert-butoxyl addition reaction to norbornadiene: a method for estimating the pi-bond strength of a carbon-carbon double bond.

    PubMed

    Nunes, Paulo M; Estácio, Sílvia G; Lopes, Gustavo T; Agapito, Filipe; Santos, Rui C; Costa Cabral, Benedito J; Borges dos Santos, Rui M; Martinho Simões, José A

    2009-06-11

    The energetics of tert-butoxyl radical addition reaction to norbornadiene was investigated by time-resolved photoacoustic calorimetry (TR-PAC). The result, together with the C-O bond dissociation enthalpy (BDE) in the addition product, allowed us to calculate the pi-bond dissociation enthalpy in norbornadiene. Quantum chemistry (QC) methods were also used to obtain several enthalpies of reaction of the addition of oxygen-centered radicals to alkenes. The pi-bond dissociation enthalpies in these molecules were calculated by a procedure similar to that used in the case of norbornadiene and were compared with the pi-BDE values obtained by the method proposed by Benson. These two different approaches yield similar values for the pi-BDEs in alkenes, indicating that the addition method proposed in the present study is a valid way to derive that quantity. The influence of strain in the pi-BDEs of cyclic alkenes was investigated and allowed us to justify the difference between the pi-BDE in norbornene and norbornadiene. Finally, the thermochemistry of the addition and abstraction reactions involving these two molecules and tert-butoxyl radical was analyzed.

  15. Disulfide-Functionalized Diblock Copolymer Worm Gels.

    PubMed

    Warren, Nicholas J; Rosselgong, Julien; Madsen, Jeppe; Armes, Steven P

    2015-08-10

    Two strategies for introducing disulfide groups at the outer surface of RAFT-synthesized poly(glycerol monomethacrylate)-poly(2-hydroxypropyl methacrylate) (PGMA-PHPMA, or Gx-Hy for brevity) diblock copolymer worms are investigated. The first approach involved statistical copolymerization of GMA with a small amount of disulfide dimethacrylate (DSDMA, or D) comonomer to afford a G54-D0.50 macromolecular chain transfer agent (macro-CTA); this synthesis was conducted in relatively dilute solution in order to ensure mainly intramolecular cyclization and hence the formation of linear chains. Alternatively, a new disulfide-based bifunctional RAFT agent (DSDB) was used to prepare a G45-S-S-G45 (or (G45-S)2) macro-CTA. A binary mixture of a non-functionalized G55 macro-CTA was utilized with each of these two disulfide-based macro-CTAs in turn for the RAFT aqueous dispersion polymerization of 2-hydroxypropyl methacrylate (HPMA). By targeting a PHPMA DP of 130 and systematically varying the molar ratio of the two macro-CTAs, a series of disulfide-functionalized diblock copolymer worm gels were obtained. For both formulations, oscillatory rheology studies confirmed that higher disulfide contents led to stronger gels, presumably as a result of inter-worm covalent bond formation via disulfide/thiol exchange. Using the DSDB-based macro-CTA led to the strongest worm gels, and this formulation also proved to be more effective in suppressing the thermosensitive behavior that is observed for the nondisulfide-functionalized control worm gel. However, macroscopic precipitation occurred when the proportion of DSDB-based macro-CTA was increased to 50 mol %, whereas the DSDMA-based macro-CTA could be utilized at up to 80 mol %. Finally, the worm gel modulus could be reduced to that of a nondisulfide-containing worm gel by reductive cleavage of the inter-worm disulfide bonds using excess tris(2-carboxyethyl)phosphine (TCEP) to yield thiol groups. These new biomimetic worm gels are

  16. Evaluation of protein disulfide conversion in vitro using a continuous flow dialysis system.

    PubMed

    Jiang, Xinzhao Grace; Wang, Tian; Kaltenbrunner, Oliver; Chen, Kenneth; Flynn, Gregory C; Huang, Gang

    2013-01-15

    Recombinant therapeutic proteins are heterogeneous due to chemical and physical modifications. Understanding the impact of these modifications on drug safety and efficacy is critical for optimal process development and for setting reasonable specification limits. In this study, we describe the development of an in vitro continuous flow dialysis system to evaluate potential in vivo behavior of thiol adducted species and incorrectly disulfide bonded species of therapeutic proteins. The system is capable of maintaining the low-level cysteine concentrations found in human blood. Liabilities of cysteamine adducted species, incorrectly disulfide bonded species, and the correctly disulfide bonded form of an Fc-fusion protein were studied using this system. Results showed that 90% of the cysteamine adduct converted into the correctly disulfide bonded form and incorrectly disulfide bonded species in approximately 4 h under physiological conditions. Approximately 50% of incorrectly disulfide bonded species converted into the correctly bonded form in 2 days. These results provide valuable information on potential in vivo stability of the cysteamine adduct, incorrectly disulfide bonded species, and the correctly bonded form of the Fc-fusion protein. These are important considerations when evaluating the criticality of product quality attributes.

  17. Enzyme structure captures four cysteines aligned for disulfide relay

    PubMed Central

    Gat, Yair; Vardi-Kilshtain, Alexandra; Grossman, Iris; Major, Dan Thomas; Fass, Deborah

    2014-01-01

    Thioredoxin superfamily proteins introduce disulfide bonds into substrates, catalyze the removal of disulfides, and operate in electron relays. These functions rely on one or more dithiol/disulfide exchange reactions. The flavoenzyme quiescin sulfhydryl oxidase (QSOX), a catalyst of disulfide bond formation with an interdomain electron transfer step in its catalytic cycle, provides a unique opportunity for exploring the structural environment of enzymatic dithiol/disulfide exchange. Wild-type Rattus norvegicus QSOX1 (RnQSOX1) was crystallized in a conformation that juxtaposes the two redox-active di-cysteine motifs in the enzyme, presenting the entire electron-transfer pathway and proton-transfer participants in their native configurations. As such a state cannot generally be enriched and stabilized for analysis, RnQSOX1 gives unprecedented insight into the functional group environments of the four cysteines involved in dithiol/disulfide exchange and provides the framework for analysis of the energetics of electron transfer in the presence of the bound flavin adenine dinucleotide cofactor. Hybrid quantum mechanics/molecular mechanics (QM/MM) free energy simulations based on the X-ray crystal structure suggest that formation of the interdomain disulfide intermediate is highly favorable and secures the flexible enzyme in a state from which further electron transfer via the flavin can occur. PMID:24888638

  18. Rh(III)-Catalyzed Diastereoselective C–H Bond Addition/Cyclization Cascade of Enone Tethered Aldehydes

    PubMed Central

    Boerth, Jeffrey A.

    2016-01-01

    The Rh(III)-catalyzed cascade addition of a C–H bond across alkene and carbonyl π-bonds is reported. The reaction proceeds under mild reaction conditions with low catalyst loading. A range of directing groups were shown to be effective as was the functionalization of alkenyl in addition to aromatic C(sp2)–H bonds. When the enone and aldehyde electrophile were tethered together, cyclic β-hydroxy ketones with three contiguous stereocenters were obtained with high diastereoselectivity. The intermolecular three-component cascade reaction was demonstrated for both aldehyde and imine electrophiles. Moreover, the first x-ray structure of a cationic Cp*Rh(III) enolate with interatomic distances consistent with an η3-bound enolate is reported. PMID:26918112

  19. Enantioselective C-H bond addition of pyridines to alkenes catalyzed by chiral half-sandwich rare-earth complexes.

    PubMed

    Song, Guoyong; O, Wylie W N; Hou, Zhaomin

    2014-09-03

    Cationic half-sandwich scandium alkyl complexes bearing monocyclopentadienyl ligands embedded in chiral binaphthyl backbones act as excellent catalysts for the enantioselective C-H bond addition of pyridines to various 1-alkenes, leading to formation of a variety of enantioenriched alkylated pyridine derivatives in high yields and excellent enantioselectivity (up to 98:2 er).

  20. Acid-catalyzed oxidative addition of a C-H bond to a square planar d⁸ iridium complex.

    PubMed

    Hackenberg, Jason D; Kundu, Sabuj; Emge, Thomas J; Krogh-Jespersen, Karsten; Goldman, Alan S

    2014-06-25

    While the addition of C-H bonds to three-coordinate Ir(I) fragments is a central theme in the field of C-H bond activation, addition to square planar four-coordinate complexes is far less precedented. The dearth of such reactions may be attributed, at least in part, to kinetic factors elucidated in seminal work by Hoffmann. C-H additions to square planar carbonyl complexes in particular are unprecedented, in contrast to the extensive chemistry of oxidative addition of other substrates (e.g., H2, HX) to Vaska's Complex and related species. We report that Bronsted acids will catalyze the addition of the alkynyl C-H bond of phenylacetylene to the pincer complex (PCP)Ir(CO). The reaction occurs to give exclusively the trans-C-H addition product. Our proposed mechanism, based on kinetics and DFT calculations, involves initial protonation of (PCP)Ir(CO) to generate a highly active five-coordinate cationic intermediate, which forms a phenylacetylene adduct that is then deprotonated to give product.

  1. A study of Sn addition on bonding arrangement of Se-Te alloys using far infrared transmission spectroscopy

    NASA Astrophysics Data System (ADS)

    Kumar, Rajneesh; Sharma, Parikshit; Katyal, S. C.; Sharma, Pankaj; Rangra, V. S.

    2011-07-01

    Far infrared transmission spectra of Se92Te8-xSnx (x = 0, 1, 2, 3, 4, 5) glassy alloys are obtained in the spectral range 50-600 cm-1 at room temperature. The results are interpreted in terms of the vibrations of the isolated molecular units in such a way so as to preserve fourfold and twofold coordination for Sn and chalcogen atoms (Se,Te), respectively. With the addition of Sn, Far-IR spectra shift toward high frequency side and some new bands start appearing. Sn atoms appear to substitute for the selenium atoms in the outrigger sites due to large bond formation probability. Theoretical calculations of bond energy, relative probability of bond formation, force constant, and wave number were also made to justify the result.

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

    NASA Astrophysics Data System (ADS)

    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

  3. Cysteamine induces a loss of tissue somatostatin-28 when measured as somatostatin-28(15-28)-like immunoreactivity but not when assessed as somatostatin-28(1-14)-like immunoreactivity: evidence for the importance of the disulfide bond for cysteamine action

    SciTech Connect

    Patel, Y.C.; Pierzchala, I.

    1985-05-01

    The reported loss of somatostatin-14 (S-14)-like immunoreactivity (LI) by cysteamine (CSH) could be mediated through an action on the S-14 disulfide bond. If so, then in the case of somatostatin-28 (S-28) (a linear 14 amino acid N-terminally extended form of S-14), it should be possible to demonstrate with region-specific antisera, a selective effect of CSH on the disulfide bond containing C-terminal half of the molecule. To obtain evidence for thisthe authors have determined by RIAs, the effect of CSH on S-28 concentration measured separately as S-28(15-28) LI and S-28(1-14) LI in the jejunal mucosa, a tissue rich in S-28. Four hours after a single sc injection of CSH to rats, mucosal S-28(15-28) LI was reduced from 16.4 +/- 0.6 to 4.6 +/- 0.51 pmol/mg protein (P less than 0.01). By contrast, S-28(1-14) LI sustained no loss and in fact increased from 27.6 +/- 1.9 to 41.6 +/- 2.2 pmol/mg protein (P less than 0.01). On Sephadex G-50 columns (in 6 M urea) approximately 70% of S-28(15-28) LI and S-28(1-14) LI coeluted with synthetic S-28 marker. These data suggest that CSH acts on the 15-28 segment of the S-28 molecule and renders it nonimmunoreactive probably through interaction with the disulfide bond. This mechanism probably also accounts for CSH-induced S-14 loss.

  4. Insights into the Electronic Structure of Ozone and Sulfur Dioxide from Generalized Valence Bond Theory: Addition of Hydrogen Atoms.

    PubMed

    Lindquist, Beth A; Takeshita, Tyler Y; Dunning, Thom H

    2016-05-05

    Ozone (O3) and sulfur dioxide (SO2) are valence isoelectronic species, yet their properties and reactivities differ dramatically. In particular, O3 is highly reactive, whereas SO2 is chemically relatively stable. In this paper, we investigate serial addition of hydrogen atoms to both the terminal atoms of O3 and SO2 and to the central atom of these species. It is well-known that the terminal atoms of O3 are much more amenable to bond formation than those of SO2. We show that the differences in the electronic structure of the π systems in the parent triatomic species account for the differences in the addition of hydrogen atoms to the terminal atoms of O3 and SO2. Further, we find that the π system in SO2, which is a recoupled pair bond dyad, facilitates the addition of hydrogen atoms to the sulfur atom, resulting in stable HSO2 and H2SO2 species.

  5. Recycling of used commercial phosphate-bonded investments with additional mono-ammonium phosphate.

    PubMed

    Zhang, Zutai; Tamaki, Yukimichi; Hotta, Yasuhiro; Miyazaki, Takashi

    2005-03-01

    Industrial waste is on the increase, resulting in severe environmental contamination. Against this background, this study sought to investigate the possibility of recycling used phosphate-bonded investments. Commercial phosphate-bonded investment was mixed and heated according to manufacturer's instructions, then powdered with a ball mill machine to be used as recycled investment (Code: R). Mono-ammonium phosphate (NH4H2PO4) of 2, 5, 8, 11, and 14 g were added to 100 g of R because of residual MgO, and coded as Ra, Rb, Rc, Rd, and Re respectively. Investment properties were then examined. Particle size of R was 50.66 microm and close to the original investment. It was found that more NH4H2PO4 led to larger setting expansion, smaller thermal expansion, and lower fired strength. The data of Re in these three aspects were 1.42 +/- 0.09%, 0.78 +/- 0.21%, and 8.85 +/- 0.88 MPa respectively, and differences against the original investment were significant. Ra showed significantly longer setting time than other investments. Based on the results of this study, we concluded that it was possible to set used phosphate-bonded investments by adding NH4H2PO4; in particular, Rd showed suitable expansion and acceptable mechanical properties.

  6. Cobalt(III)-Catalyzed Synthesis of Indazoles and Furans by C–H Bond Functionalization/Addition/Cyclization Cascades

    PubMed Central

    2015-01-01

    The development of operationally straightforward and cost-effective routes for the assembly of heterocycles from simple inputs is important for many scientific endeavors, including pharmaceutical, agrochemical, and materials research. In this article we describe the development of a new air-stable cationic Co(III) catalyst for convergent, one-step benchtop syntheses of N-aryl-2H-indazoles and furans by C–H bond additions to aldehydes followed by in situ cyclization and aromatization. Only a substoichiometric amount of AcOH is required as an additive that is both low-cost and convenient to handle. The syntheses of these heterocycles are the first examples of Co(III)-catalyzed additions to aldehydes, and reactions are demonstrated for a variety of aromatic, heteroaromatic, and aliphatic derivatives. The syntheses of both N-aryl-2H-indazoles and furans have been performed on 20 mmol scales and should be readily applicable to larger scales. The reported heterocycle syntheses also demonstrate the use of directing groups that have not previously been applied to Co(III)-catalyzed C–H bond functionalizations. Additionally, the synthesis of furans demonstrates the first example of Co(III)-catalyzed functionalization of alkenyl C–H bonds. PMID:25494296

  7. Cobalt(III)-catalyzed synthesis of indazoles and furans by C-H bond functionalization/addition/cyclization cascades.

    PubMed

    Hummel, Joshua R; Ellman, Jonathan A

    2015-01-14

    The development of operationally straightforward and cost-effective routes for the assembly of heterocycles from simple inputs is important for many scientific endeavors, including pharmaceutical, agrochemical, and materials research. In this article we describe the development of a new air-stable cationic Co(III) catalyst for convergent, one-step benchtop syntheses of N-aryl-2H-indazoles and furans by C-H bond additions to aldehydes followed by in situ cyclization and aromatization. Only a substoichiometric amount of AcOH is required as an additive that is both low-cost and convenient to handle. The syntheses of these heterocycles are the first examples of Co(III)-catalyzed additions to aldehydes, and reactions are demonstrated for a variety of aromatic, heteroaromatic, and aliphatic derivatives. The syntheses of both N-aryl-2H-indazoles and furans have been performed on 20 mmol scales and should be readily applicable to larger scales. The reported heterocycle syntheses also demonstrate the use of directing groups that have not previously been applied to Co(III)-catalyzed C-H bond functionalizations. Additionally, the synthesis of furans demonstrates the first example of Co(III)-catalyzed functionalization of alkenyl C-H bonds.

  8. CRADA/NFE-15-05761 Report: Additive Manufacturing of Isotropic NdFeB Bonded Permanent Magnets

    SciTech Connect

    Paranthaman, M. Parans

    2016-07-18

    The technical objective of this technical collaboration phase I proposal is to fabricate net shape isotropic NdFeB bonded magnets utilizing additive manufacturing technologies at the ORNL MDF. The goal is to form complex shapes of thermoplastic and/or thermoset bonded magnets without expensive tooling and with minimal wasted material. Two additive manufacturing methods; the binder jet process; and big area additive manufacturing (BAAM) were used. Binder jetting produced magnets with the measured density of the magnet of 3.47 g/cm3, close to 46% relative to the NdFeB single crystal density of 7.6 g/cm3 were demonstrated. Magnetic measurements indicate that there is no degradation in the magnetic properties. In addition, BAAM was used to fabricate isotropic near-net-shape NdFeB bonded magnets with magnetic and mechanical properties comparable or better than those of traditional injection molded magnets. The starting polymer magnet composite pellets consist of 65 vol% isotropic NdFeB powder and 35 vol% polyamide (Nylon-12). The density of the final BAAM magnet product reached 4.8 g/cm3, and the room temperature magnetic properties are: Intrinsic coercivity Hci = 8.65 kOe, Remanence Br = 5.07 kG, and energy product (BH)max = 5.47 MGOe (43.50 kJ/m3). This study provides a new pathway for preparing near-net shape bonded magnets for various magnetic applications.

  9. Semienzymatic cyclization of disulfide-rich peptides using Sortase A.

    PubMed

    Jia, Xinying; Kwon, Soohyun; Wang, Ching-I Anderson; Huang, Yen-Hua; Chan, Lai Y; Tan, Chia Chia; Rosengren, K Johan; Mulvenna, Jason P; Schroeder, Christina I; Craik, David J

    2014-03-07

    Disulfide-rich cyclic peptides have generated great interest in the development of peptide-based therapeutics due to their exceptional stability toward chemical, enzymatic, or thermal attack. In particular, they have been used as scaffolds onto which bioactive epitopes can be grafted to take advantage of the favorable biophysical properties of disulfide-rich cyclic peptides. To date, the most commonly used method for the head-to-tail cyclization of peptides has been native chemical ligation. In recent years, however, enzyme-mediated cyclization has become a promising new technology due to its efficiency, safety, and cost-effectiveness. Sortase A (SrtA) is a bacterial enzyme with transpeptidase activity. It recognizes a C-terminal penta-amino acid motif, LPXTG, and cleaves the amide bond between Thr and Gly to form a thioacyl-linked intermediate. This intermediate undergoes nucleophilic attack by an N-terminal poly-Gly sequence to form an amide bond between the Thr and N-terminal Gly. Here, we demonstrate that sortase A can successfully be used to cyclize a variety of small disulfide-rich peptides, including the cyclotide kalata B1, α-conotoxin Vc1.1, and sunflower trypsin inhibitor 1. These peptides range in size from 14 to 29 amino acids and contain three, two, or one disulfide bond, respectively, within their head-to-tail cyclic backbones. Our findings provide proof of concept for the potential broad applicability of enzymatic cyclization of disulfide-rich peptides with therapeutic potential.

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

    PubMed

    Trabjerg, Esben; Jakobsen, Rasmus U; Mysling, Simon; Christensen, Søren; Jørgensen, Thomas J D; Rand, Kasper D

    2015-09-01

    Analysis of disulfide-bonded proteins by hydrogen/deuterium exchange mass spectrometry (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 solution (e.g., tris(2-carboxyethyl)phosphine (TCEP)). The chemical reduction, however, is severely limited under quenched conditions due to a narrow time window as well as low pH and temperature. Here, we demonstrate the real-world applicability of integrating electrochemical reduction into an online HDX-MS workflow. We have optimized 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 and applied square wave potential, as well as the type of labeling and quench buffer) were investigated, and the optimized workflow increased the sequence coverage of NGF from 46% with chemical reduction to 99%, when electrochemical reduction was applied. Additionally, the optimized workflow also enabled a similar high sequence coverage of 96% and 87% for the heavy and light chain of the IgG1-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 is poised to decrease the complexity of sample handling and increase the versatility of the HDX-MS technique.

  11. Additives-biological activities of tin-containing polymers bonded to noncarbons

    SciTech Connect

    Carraher, C.; Butler, C.; Foster, V.; Pandya, B.; Sterling, D.

    1993-12-31

    Organotin-containing polymers serve as effective additives to a silicon-based sealant, latex coating and talc exhibiting inhibition to tested bacteria and fungi. These polymers are candidates for uses a paint additives, sealant and caulk additives and within talc as an epidermal treatment.

  12. Relationships between the elastic and fracture properties of boronitrene and molybdenum disulfide and those of graphene.

    PubMed

    Hess, Peter

    2017-02-10

    A consistent set of 2D elastic and fracture properties of hexagonal boron nitride (h-BN) monolayers (boronitrene) and molybdenum disulfide (MoS2) nanosheets is derived. Reported literature values for Young's moduli and fracture strengths, based on experiments and DFT calculations, were used to estimate the line or edge energy with a local 2D bond-breaking model. Consistent information was obtained for intrinsic fracture properties. The basic mechanical properties of boronitrene are roughly 25% lower than the corresponding graphene values. This is consistent with the tensile bond force model, and the lower ionic-covalent bonding energy of sp(2)-hybridized B-N bonds in comparison with sp(2)-hybridized carbon bonds. While the intrinsic stiffness and strength of MoS2 correlate with the strength of its constituent chemical bonds, DFT calculations of the line or edge energy scale with roughly two times the Mo-S bonding energy, whereas the 2D bond-breaking model yields a correlation similar to that found for h-BN. Additional failure properties such as the fracture toughness and strain energy release rate were determined. Together with the intrinsic strengths a Griffith plot of the effective strength of defective h-BN and MoS2 versus the square root of half the defect size of single defects such as (multi)vacancies and micro-cracks exhibits a slope similar to that of the graphene plot.

  13. Relationships between the elastic and fracture properties of boronitrene and molybdenum disulfide and those of graphene

    NASA Astrophysics Data System (ADS)

    Hess, Peter

    2017-02-01

    A consistent set of 2D elastic and fracture properties of hexagonal boron nitride (h-BN) monolayers (boronitrene) and molybdenum disulfide (MoS2) nanosheets is derived. Reported literature values for Young’s moduli and fracture strengths, based on experiments and DFT calculations, were used to estimate the line or edge energy with a local 2D bond-breaking model. Consistent information was obtained for intrinsic fracture properties. The basic mechanical properties of boronitrene are roughly 25% lower than the corresponding graphene values. This is consistent with the tensile bond force model, and the lower ionic-covalent bonding energy of sp2-hybridized B-N bonds in comparison with sp2-hybridized carbon bonds. While the intrinsic stiffness and strength of MoS2 correlate with the strength of its constituent chemical bonds, DFT calculations of the line or edge energy scale with roughly two times the Mo-S bonding energy, whereas the 2D bond-breaking model yields a correlation similar to that found for h-BN. Additional failure properties such as the fracture toughness and strain energy release rate were determined. Together with the intrinsic strengths a Griffith plot of the effective strength of defective h-BN and MoS2 versus the square root of half the defect size of single defects such as (multi)vacancies and micro-cracks exhibits a slope similar to that of the graphene plot.

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

    SciTech Connect

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

    2006-09-01

    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{sub 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

  15. Disulfide cross-linking influences symbiotic activities of nodule peptide NCR247.

    PubMed

    Shabab, Mohammed; Arnold, Markus F F; Penterman, Jon; Wommack, Andrew J; Bocker, Hartmut T; Price, Paul A; Griffitts, Joel S; Nolan, Elizabeth M; Walker, Graham C

    2016-09-06

    Interactions of rhizobia with legumes establish the chronic intracellular infection that underlies symbiosis. Within nodules of inverted repeat-lacking clade (IRLC) legumes, rhizobia differentiate into nitrogen-fixing bacteroids. This terminal differentiation is driven by host nodule-specific cysteine-rich (NCR) peptides that orchestrate the adaptation of free-living bacteria into intracellular residents. Medicago truncatula encodes a family of >700 NCR peptides that have conserved cysteine motifs. NCR247 is a cationic peptide with four cysteines that can form two intramolecular disulfide bonds in the oxidized forms. This peptide affects Sinorhizobium meliloti transcription, translation, and cell division at low concentrations and is antimicrobial at higher concentrations. By preparing the three possible disulfide-cross-linked NCR247 regioisomers, the reduced peptide, and a variant lacking cysteines, we performed a systematic study of the effects of intramolecular disulfide cross-linking and cysteines on the activities of an NCR peptide. The relative activities of the five NCR247 variants differed strikingly among the various bioassays, suggesting that the NCR peptide-based language used by plants to control the development of their bacterial partners during symbiosis is even greater than previously recognized. These patterns indicate that certain NCR bioactivities require cysteines whereas others do not. The results also suggest that NCR247 may exert some of its effects within the cell envelope whereas other activities occur in the cytoplasm. BacA, a membrane protein that is critical for symbiosis, provides protection against all bactericidal forms of NCR247. Oxidative folding protects NCR247 from degradation by the symbiotically relevant metalloprotease HrrP (host range restriction peptidase), suggesting that disulfide bond formation may additionally stabilize NCR peptides during symbiosis.

  16. Theoretical study of the formation of naphthalene from the radical/π-bond addition between single-ring aromatic hydrocarbons.

    PubMed

    Comandini, Andrea; Brezinsky, Kenneth

    2011-06-09

    The experimental investigations performed in the 1960s on the o-benzyne + benzene reaction as well as the more recent studies on reactions involving π-electrons highlight the importance of π-bonding for different combustion processes related to PAH's and soot formation. In the present investigation radical/π-bond addition reactions between single-ring aromatic compounds have been proposed and computationally investigated as possible pathways for the formation of two-ring fused compounds, such as naphthalene, which serve as precursors to soot formation. The computationally generated optimized structures for the stationary points were obtained with uB3LYP/6-311+G(d,p) calculations, while the energies of the optimized complexes were refined using the uCCSD(T) method and the cc-pVDZ basis set. The computations have addressed the relevance of a number of radical/π-bond addition reactions including the singlet benzene + o-benzyne reaction, which leads to formation of naphthalene and acetylene through fragmentation of the benzobicyclo[2,2,2]octatriene intermediate. For this reaction, the high-pressure limit rate constants for the individual elementary reactions involved in the overall process were evaluated using transition state theory analysis. Other radical/π-bond addition reactions studied were between benzene and triplet o-benzyne, between benzene and phenyl radical, and between phenyl radicals, for all of which potential energy surfaces were produced. On the basis of the results of these reaction studies, it was found necessary to propose and subsequently confirm additional, alternative pathways for the formation of the types of PAH compounds found in combustion systems. The potential energy surface for one reaction in particular, the phenyl + phenyl addition, is shown to contain a low-energy channel leading to formation of naphthalene that is energetically comparable to the other examined conventional pathways leading to formation of biphenyl compounds. This

  17. 46 CFR 153.1040 - Carbon disulfide.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 5 2012-10-01 2012-10-01 false Carbon disulfide. 153.1040 Section 153.1040 Shipping... § 153.1040 Carbon disulfide. (a) No person may load, carry, or discharge carbon disulfide unless the... charge of a carbon disulfide transfer operation shall ensure that carbon disulfide is discharged only...

  18. 46 CFR 153.1040 - Carbon disulfide.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 5 2011-10-01 2011-10-01 false Carbon disulfide. 153.1040 Section 153.1040 Shipping... § 153.1040 Carbon disulfide. (a) No person may load, carry, or discharge carbon disulfide unless the... charge of a carbon disulfide transfer operation shall ensure that carbon disulfide is discharged only...

  19. 46 CFR 153.1040 - Carbon disulfide.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 5 2010-10-01 2010-10-01 false Carbon disulfide. 153.1040 Section 153.1040 Shipping... § 153.1040 Carbon disulfide. (a) No person may load, carry, or discharge carbon disulfide unless the... charge of a carbon disulfide transfer operation shall ensure that carbon disulfide is discharged only...

  20. 46 CFR 153.1040 - Carbon disulfide.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 5 2014-10-01 2014-10-01 false Carbon disulfide. 153.1040 Section 153.1040 Shipping... § 153.1040 Carbon disulfide. (a) No person may load, carry, or discharge carbon disulfide unless the... charge of a carbon disulfide transfer operation shall ensure that carbon disulfide is discharged only...

  1. 46 CFR 153.1040 - Carbon disulfide.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 5 2013-10-01 2013-10-01 false Carbon disulfide. 153.1040 Section 153.1040 Shipping... § 153.1040 Carbon disulfide. (a) No person may load, carry, or discharge carbon disulfide unless the... charge of a carbon disulfide transfer operation shall ensure that carbon disulfide is discharged only...

  2. Assessment of disulfide and hinge modifications in monoclonal antibodies.

    PubMed

    Moritz, Bernd; Stracke, Jan Olaf

    2016-12-16

    During the last years there was a substantial increase in the use of antibodies and related proteins as therapeutics. The emphasis of the pharmaceutical industry is on IgG1, IgG2, and IgG4 antibodies, which are therefore in the focus of this article. In order to ensure appropriate quality control of such biopharmaceuticals, deep understanding of their chemical degradation pathways and the resulting impact on potency, pharmacokinetics, and safety is required. Criticality of modifications may be specific for individual antibodies and has to be assessed for each molecule. However, some modifications of conserved structure elements occur in all or at least most IgGs. In these cases, criticality assessment may be applicable to related molecules or molecule formats. The relatively low dissociation energy of disulfide bonds and the high flexibility of the hinge region frequently lead to modifications and cleavages. Therefore, the hinge region and disulfide bonds require specific consideration during quality assessment of mAbs. In this review, available literature knowledge on underlying chemical reaction pathways of modifications, analytical methods for quantification and criticality are discussed. The hinge region is prone to cleavage and is involved in pathways that lead to thioether bond formation, cysteine racemization, and iso-Asp (Asp, aspartic acid) formation. Disulfide or sulfhydryl groups were found to be prone to reductive cleavage, trisulfide formation, cysteinylation, glutathionylation, disulfide bridging to further light chains, and disulfide scrambling. With regard to potency, disulfide cleavage, hinge cleavage, disulfide bridging to further light chains, and cysteinylation were found to influence antigen binding and fragment crystallizable (Fc) effector functionalities. Renal clearance of small fragments may be faster, whereas clearance of larger fragments appears to depend on their neonatal Fc receptor (FcRn) functionality, which in turn may be impeded by

  3. Wear characteristics of bonded solid film lubricant under high load condition

    NASA Technical Reports Server (NTRS)

    Hiraoka, Naofumi; Sasaki, Akira; Kawashima, Noritsugu; Honda, Toshio

    1991-01-01

    Wear properties of phenolic resin bonded molybdenum disulfide film lubricant were studied. In-vacuo journal bearing tests were performed to evaluate the wear-life of this film lubricant. The wear-life depends on substrate materials and on sliding velocity. Pretreated substrate surfaces were examined to reveal the reasons for these results. Additionally, investigations on film wear mechanisms were made.

  4. Less is More: Design of a Highly Stable Disulfide-Deleted Mutant of Analgesic Cyclic α-Conotoxin Vc1.1

    PubMed Central

    Yu, Rilei; Seymour, Victoria A. L.; Berecki, Géza; Jia, Xinying; Akcan, Muharrem; Adams, David J.; Kaas, Quentin; Craik, David J.

    2015-01-01

    Cyclic α-conotoxin Vc1.1 (cVc1.1) is an orally active peptide with analgesic activity in rat models of neuropathic pain. It has two disulfide bonds, which can have three different connectivities, one of which is the native and active form. In this study we used computational modeling and nuclear magnetic resonance to design a disulfide-deleted mutant of cVc1.1, [C2H,C8F]cVc1.1, which has a larger hydrophobic core than cVc1.1 and, potentially, additional surface salt bridge interactions. The new variant, hcVc1.1, has similar structure and serum stability to cVc1.1 and is highly stable at a wide range of pH and temperatures. Remarkably, hcVc1.1 also has similar selectivity to cVc1.1, as it inhibited recombinant human α9α10 nicotinic acetylcholine receptor-mediated currents with an IC50 of 13 μM and rat N-type (Cav2.2) and recombinant human Cav2.3 calcium channels via GABAB receptor activation, with an IC50 of ~900 pM. Compared to cVc1.1, the potency of hcVc1.1 is reduced three-fold at both analgesic targets, whereas previous attempts to replace Vc1.1 disulfide bonds by non-reducible dicarba linkages resulted in at least 30-fold decreased activity. Because it has only one disulfide bond, hcVc1.1 is not subject to disulfide bond shuffling and does not form multiple isomers during peptide synthesis. PMID:26290113

  5. Steps in reductive activation of the disulfide-generating enzyme Ero1p

    PubMed Central

    Heldman, Nimrod; Vonshak, Ohad; Sevier, Carolyn S; Vitu, Elvira; Mehlman, Tevie; Fass, Deborah

    2010-01-01

    Ero1p is the primary catalyst of disulfide bond formation in the yeast endoplasmic reticulum (ER). Ero1p contains a pair of essential disulfide bonds that participate directly in the electron transfer pathway from substrate thiol groups to oxygen. Remarkably, elimination of certain other Ero1p disulfides by mutation enhances enzyme activity. In particular, the C150A/C295A Ero1p mutant exhibits increased thiol oxidation in vitro and in vivo and interferes with redox homeostasis in yeast cells by hyperoxidizing the ER. Inhibitory disulfides of Ero1p are thus important for enzyme regulation. To visualize the differences between de-regulated and wild-type Ero1p, we determined the crystal structure of Ero1p C150A/C295A. The structure revealed local changes compared to the wild-type enzyme around the sites of mutation, but no conformational transitions within 25 Å of the active site were observed. To determine how the C150—C295 disulfide nonetheless participates in redox regulation of Ero1p, we analyzed using mass spectrometry the changes in Ero1p disulfide connectivity as a function of time after encounter with reducing substrates. We found that the C150—C295 disulfide sets a physiologically appropriate threshold for enzyme activation by guarding a key neighboring disulfide from reduction. This study illustrates the diverse and interconnected roles that disulfides can play in redox regulation of protein activity. PMID:20669236

  6. Do Vicinal Disulfide Bridges Mediate Functionally Important Redox Transformations in Proteins?

    PubMed Central

    de Araujo, Aline Dantas; Herzig, Volker; Windley, Monique J.; Dziemborowicz, Sławomir; Mobli, Mehdi; Nicholson, Graham M.

    2013-01-01

    Abstract Vicinal disulfide bridges, in which a disulfide bond is formed between adjacent cysteine residues, constitute an unusual but expanding class of potential allosteric disulfides. Although vicinal disulfide rings (VDRs) are relatively uncommon, they have proven to be functionally critical in almost all proteins in which they have been discovered. However, it has proved difficult to test whether these sterically constrained disulfides participate in functionally important redox transformations. We demonstrate that chemical replacement of VDRs with dicarba or diselenide bridges can be used to assess whether VDRs function as allosteric disulfides. Our approach leads to the hypothesis that not all VDRs participate in functionally important redox reactions. Antioxid. Redox Signal. 19, 1976–1980. PMID:23646911

  7. Preliminary assessment of metal-porcelain bonding strength of CoCrW alloy after 3wt.% Cu addition.

    PubMed

    Lu, Yanjin; Zhao, Chaoqian; Ren, Ling; Guo, Sai; Gan, Yiliang; Yang, Chunguang; Wu, Songquan; Lin, Junjie; Huang, Tingting; Yang, Ke; Lin, Jinxin

    2016-06-01

    In this work, a novel Cu-bearing CoCrW alloy fabricated by selective laser melting for dental application has been studied. For its successful application, the bonding strength of metal-porcelain is essential to be systematically investigated. Therefore, the aim of this study was to evaluate the metal-porcelain bonding strength of CoCrWCu alloy by three-point bending test, meanwhile the Ni-free CoCrW alloy was used as control. The oxygen content was investigated by an elemental analyzer; X-ray photoelectron spectroscopy (XPS) was used to analyze the surface chemical composition of CoCrW based alloy after preoxidation treatment; the fracture mode was investigated by X-ray energy spectrum analysis (EDS) and scanning electron microscope (SEM). Result from the oxygen content analysis showed that the content of oxygen dramatically increased after the Cu addition. And the XPS suggested that Co-oxidation, Cr2O3, CrO2, WO3, Cu2O and CuO existed on the preoxidated surface of the CoCrWCu alloy; the three-point bending test showed that the bonding strength of the CoCrWCu alloy was 43.32 MPa, which was lower than that of the CoCrW group of 47.65 MPa. However, the average metal-porcelain bonding strength is significantly higher than the minimum value in the ISO 9693 standard. Results from the SEM images and EDS indicated that the fracture mode of CoCrWCu-porcelain was mixed between cohesive and adhesive. Based on the results obtained in this study, it can be indicated that the Cu-bearing CoCrW alloy fabricated by the selective laser melting is a promising candidate for use in dental application.

  8. Rattusin structure reveals a novel defensin scaffold formed by intermolecular disulfide exchanges

    PubMed Central

    Min, Hye Jung; Yun, Hyosuk; Ji, Sehyeon; Rajasekaran, Ganesan; Kim, Jae Il; Kim, Jeong-Sun; Shin, Song Yub; Lee, Chul Won

    2017-01-01

    Defensin peptides are essential for innate immunity in humans and other living systems, as they provide protection against infectious pathogens and regulate the immune response. Here, we report the solution structure of rattusin (RTSN), an α-defensin-related peptide, which revealed a novel C2-symmetric disulfide-linked dimeric structure. RTSN was synthesized by solid-phase peptide synthesis (SPPS) and refolded by air oxidation in vitro. Dimerization of the refolded RTSN (r-RTSN) resulted from five intermolecular disulfide (SS) bond exchanges formed by ten cysteines within two protomer chains. The SS bond pairings of r-RTSN were determined by mass analysis of peptide fragments cleaved by trypsin digestion. In addition to mass analysis, nuclear magnetic resonance (NMR) experiments for a C15S mutant and r-RTSN confirmed that the intermolecular SS bond structure of r-RTSN showed an I-V’, II-IV’, III-III’, IV-II’, V-I’ arrangement. The overall structure of r-RTSN exhibited a cylindrical array, similar to that of β-sandwich folds, with a highly basic surface. Furthermore, fluorescence spectroscopy results suggest that r-RTSN exerts bactericidal activity by damaging membrane integrity. Collectively, these results provide a novel structural scaffold for designing highly potent peptide-based antibiotics suitable for use under various physiological conditions. PMID:28345637

  9. Rare-earth-catalyzed C-H bond addition of pyridines to olefins.

    PubMed

    Guan, Bing-Tao; Hou, Zhaomin

    2011-11-16

    An efficient and general protocol for the ortho-alkylation of pyridines via C-H addition to olefins has been developed, using cationic half-sandwich rare-earth catalysts, which provides an atom-economical method for the synthesis of alkylated pyridine derivatives. A wide range of pyridine and olefin substrates including α-olefins, styrenes, and conjugated dienes are compatible with the catalysts.

  10. Additives

    NASA Technical Reports Server (NTRS)

    Smalheer, C. V.

    1973-01-01

    The chemistry of lubricant additives is discussed to show what the additives are chemically and what functions they perform in the lubrication of various kinds of equipment. Current theories regarding the mode of action of lubricant additives are presented. The additive groups discussed include the following: (1) detergents and dispersants, (2) corrosion inhibitors, (3) antioxidants, (4) viscosity index improvers, (5) pour point depressants, and (6) antifouling agents.

  11. Peel bond strength of resilient liner modified by the addition of antimicrobial agents to denture base acrylic resin

    PubMed Central

    ALCÂNTARA, Cristiane S.; de MACÊDO, Allana F.C.; GURGEL, Bruno C.V.; JORGE, Janaina H.; NEPPELENBROEK, Karin H.; URBAN, Vanessa M.

    2012-01-01

    In order to prolong the clinical longevity of resilient denture relining materials and reduce plaque accumulation, incorporation of antimicrobial agents into these materials has been proposed. However, this addition may affect their properties. Objective This study evaluated the effect of the addition of antimicrobial agents into one soft liner (Soft Confort, Dencril) on its peel bond strength to one denture base (QC 20, Dentsply). Material and Methods Acrylic specimens (n=9) were made (75x10x3 mm) and stored in distilled water at 37ºC for 48 h. The drug powder concentrations (nystatin 500,000U - G2; nystatin 1,000,000U - G3; miconazole 125 mg - G4; miconazole 250 mg - G5; ketoconazole 100 mg - G6; ketoconazole 200 mg - G7; chlorhexidine diacetate 5% - G8; and 10% chlorhexidine diacetate - G9) were blended with the soft liner powder before the addition of the soft liner liquid. A group (G1) without any drug incorporation was used as control. Specimens (n=9) (75x10x6 mm) were plasticized according to the manufacturers' instructions and stored in distilled water at 37ºC for 24 h. Relined specimens were then submitted to a 180-degree peel test at a crosshead speed of 10 mm/min. Data (MPa) were analyzed by analysis of variance (α=0.05) and the failure modes were visually classified. Results No significant difference was found among experimental groups (p=0.148). Cohesive failure located within the resilient material was predominantly observed in all tested groups. Conclusions Peel bond strength between the denture base and the modified soft liner was not affected by the addition of antimicrobial agents. PMID:23329241

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

    SciTech Connect

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

    2011-06-01

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

  13. Partial De Novo Sequencing and Unusual CID Fragmentation of a 7 kDa, Disulfide-Bridged Toxin

    NASA Astrophysics Data System (ADS)

    Medzihradszky, Katalin F.; Bohlen, Christopher J.

    2012-05-01

    A 7 kDa toxin isolated from the venom of the Texas coral snake ( Micrurus tener tener) was subjected to collision-induced dissociation (CID) and electron-transfer dissociation (ETD) analyses both before and after reduction at low pH. Manual and automated approaches to de novo sequencing are compared in detail. Manual de novo sequencing utilizing the combination of high accuracy CID and ETD data and an acid-related cleavage yielded the N-terminal half of the sequence from the reduced species. The intact polypeptide, containing 3 disulfide bridges produced a series of unusual fragments in ion trap CID experiments: abundant internal amino acid losses were detected, and also one of the disulfide-linkage positions could be determined from fragments formed by the cleavage of two bonds. In addition, internal and c-type fragments were also observed.

  14. Densification of Reaction Bonded Silicon Nitride with the Addition of Fine Si Powder Effects on the Sinterability and Mechanical Properties

    SciTech Connect

    Lee, Sea-Hoon; Cho, Chun-Rae; Park, Young-Jo; Ko, Jae-Woong; Kim, Hai-Doo; Lin, Hua-Tay; Becher, Paul F

    2013-01-01

    The densification behavior and strength of sintered reaction bonded silicon nitrides (SRBSN) that contain Lu2O3-SiO2 additives were improved by the addition of fine Si powder. Dense specimens (relative density: 99.5%) were obtained by gas-pressure sintering (GPS) at 1850oC through the addition of fine Si. In contrast, the densification of conventional specimens did not complete at 1950oC. The fine Si decreased the onset temperature of shrinkage and increased the shrinkage rate because the additive helped the compaction of green bodies and induced the formation of fine Si3N4 particles after nitridation and sintering at and above 1600oC. The amount of residual SiO2 within the specimens was not strongly affected by adding fine Si powder because most of the SiO2 layer that had formed on the fine Si particles decomposed during nitridation. The maximum strength and fracture toughness of the specimens were 991 MPa and 8.0 MPa m1/2, respectively.

  15. Magnetic properties and coercivity mechanism of isotropic HDDR NdFeB bonded magnets with Co and Dy addition

    NASA Astrophysics Data System (ADS)

    Chen, W.; Gao, R. W.; Zhu, M. G.; Pan, W.; Li, W.; Li, X. M.; Han, G. B.; Feng, W. C.; Wang, B.

    2003-04-01

    Isotropic NdDyFeCoB bonded magnets with high coercivity of 1.59 MA/m and low temperature coefficient of remanence of -0.056%/ K (in the temperature range 298-428 K) were prepared successfully by controlling the HDDR process and adjusting the compositions. The influence of Co and Dy additions on the magnetic properties and the magnetization reversal process in magnet was investigated. The high coercivity in (Nd 0.8Dy 0.2) 13(Fe 0.875Co 0.125) 81B 6 HDDR magnet can be attributed to its unique microstructure and the enhancement of anisotropy field of 2:14:1 phase by substitution of Nd by Dy.

  16. Rh(III)-catalyzed addition of alkenyl C-H bond to isocyanates and intramolecular cyclization: direct synthesis 5-ylidenepyrrol-2(5H)-ones.

    PubMed

    Hou, Wei; Zhou, Bing; Yang, Yaxi; Feng, Huijin; Li, Yuanchao

    2013-04-19

    The rhodium-catalyzed addition of an alkenyl C-H bond to isocyanates via sp(2) C-H bond activation followed by an intramolecular cyclization is described. This atom-economic and catalytic reaction affords a simple and straightforward access to biologically relevant 5-ylidene pyrrol-2(5H)-ones and can be carried out under mild and neutral conditions in the absence of any additives and environmentally hazardous waste production.

  17. Effect of additive metals, Sn, Ga, and In in Ag-Pd-Au-Cu alloys on initial bond strength of 4-META adhesive cement to these alloys.

    PubMed

    Goto, Shin-ichi; Churnjitapirom, Pornkiat; Miyagawa, Yukio; Ogura, Hideo

    2008-09-01

    The purpose of this study was to investigate the effects of three additives, Sn, Ga, and In, as well as the main constituents, Pd and Cu, of Ag-Pd-Au-Cu alloys on the initial bond strength of 4-META adhesive cement to these alloys. The Ag-Pd-Au-Cu alloys consisted of 20%, 30% or 40% Pd, and 10%, 15% or 20% Cu, 20% Au, and Ag as balance. Besides, additive metals (Sn, Ga, and In) of 2% and 4% were added to these compositions. The addition of three additives, in general, increased the initial bond strength of the cement in comparison to the mother compositions (0% additives), although the degrees of effectiveness of the three additives were different and varied with their contents. Among these additives, a remarkable increase in bond strength was observed with the addition of In. The increase in Cu content, in many cases, resulted in an increase in bond strength at high Pd contents (30% and 40%), but a decrease at low Pd content (20%) in some cases. The positive effects of the three additives and Cu could be due to the formation of a suitable oxide layer for strong bonding with 4-META.

  18. Bond-Specific Dissociation Following Excitation Energy Transfer for Distance Constraint Determination in the Gas Phase

    PubMed Central

    2015-01-01

    Herein, we report chemistry that enables excitation energy transfer (EET) to be accurately measured via action spectroscopy on gaseous ions in an ion trap. It is demonstrated that EET between tryptophan or tyrosine and a disulfide bond leads to excited state, homolytic fragmentation of the disulfide bond. This phenomenon exhibits a tight distance dependence, which is consistent with Dexter exchange transfer. The extent of fragmentation of the disulfide bond can be used to determine the distance between the chromophore and disulfide bond. The chemistry is well suited for the examination of protein structure in the gas phase because native amino acids can serve as the donor/acceptor moieties. Furthermore, both tyrosine and tryptophan exhibit unique action spectra, meaning that the identity of the donating chromophore can be easily determined in addition to the distance between donor/acceptor. Application of the method to the Trpcage miniprotein reveals distance constraints that are consistent with a native-like fold for the +2 charge state in the gas phase. This structure is stabilized by several salt bridges, which have also been observed to be important previously in proteins that retain native-like structures in the gas phase. The ability of this method to measure specific distance constraints, potentially at numerous positions if combined with site-directed mutagenesis, significantly enhances our ability to examine protein structure in the gas phase. PMID:25174489

  19. Bond-specific dissociation following excitation energy transfer for distance constraint determination in the gas phase.

    PubMed

    Hendricks, Nathan G; Lareau, Nichole M; Stow, Sarah M; McLean, John A; Julian, Ryan R

    2014-09-24

    Herein, we report chemistry that enables excitation energy transfer (EET) to be accurately measured via action spectroscopy on gaseous ions in an ion trap. It is demonstrated that EET between tryptophan or tyrosine and a disulfide bond leads to excited state, homolytic fragmentation of the disulfide bond. This phenomenon exhibits a tight distance dependence, which is consistent with Dexter exchange transfer. The extent of fragmentation of the disulfide bond can be used to determine the distance between the chromophore and disulfide bond. The chemistry is well suited for the examination of protein structure in the gas phase because native amino acids can serve as the donor/acceptor moieties. Furthermore, both tyrosine and tryptophan exhibit unique action spectra, meaning that the identity of the donating chromophore can be easily determined in addition to the distance between donor/acceptor. Application of the method to the Trpcage miniprotein reveals distance constraints that are consistent with a native-like fold for the +2 charge state in the gas phase. This structure is stabilized by several salt bridges, which have also been observed to be important previously in proteins that retain native-like structures in the gas phase. The ability of this method to measure specific distance constraints, potentially at numerous positions if combined with site-directed mutagenesis, significantly enhances our ability to examine protein structure in the gas phase.

  20. A Primary Role for Disulfide Formation in the Productive Folding of Prokaryotic Cu,Zn-superoxide Dismutase*

    PubMed Central

    Sakurai, Yasuyuki; Anzai, Itsuki; Furukawa, Yoshiaki

    2014-01-01

    Enzymatic activation of Cu,Zn-superoxide dismutase (SOD1) requires not only binding of a catalytic copper ion but also formation of an intramolecular disulfide bond. Indeed, the disulfide bond is completely conserved among all species possessing SOD1; however, it remains obscure how disulfide formation controls the enzymatic activity of SOD1. Here, we show that disulfide formation is a primary event in the folding process of prokaryotic SOD1 (SodC) localized to the periplasmic space. Escherichia coli SodC was found to attain β-sheet structure upon formation of the disulfide bond, whereas disulfide-reduced SodC assumed little secondary structure even in the presence of copper and zinc ions. Moreover, reduction of the disulfide bond made SodC highly susceptible to proteolytic degradation. We thus propose that the thiol-disulfide status in SodC controls the intracellular stability of this antioxidant enzyme and that the oxidizing environment of the periplasm is required for the enzymatic activation of SodC. PMID:24917671

  1. Semienzymatic Cyclization of Disulfide-rich Peptides Using Sortase A*

    PubMed Central

    Jia, Xinying; Kwon, Soohyun; Wang, Ching-I Anderson; Huang, Yen-Hua; Chan, Lai Y.; Tan, Chia Chia; Rosengren, K. Johan; Mulvenna, Jason P.; Schroeder, Christina I.; Craik, David J.

    2014-01-01

    Disulfide-rich cyclic peptides have generated great interest in the development of peptide-based therapeutics due to their exceptional stability toward chemical, enzymatic, or thermal attack. In particular, they have been used as scaffolds onto which bioactive epitopes can be grafted to take advantage of the favorable biophysical properties of disulfide-rich cyclic peptides. To date, the most commonly used method for the head-to-tail cyclization of peptides has been native chemical ligation. In recent years, however, enzyme-mediated cyclization has become a promising new technology due to its efficiency, safety, and cost-effectiveness. Sortase A (SrtA) is a bacterial enzyme with transpeptidase activity. It recognizes a C-terminal penta-amino acid motif, LPXTG, and cleaves the amide bond between Thr and Gly to form a thioacyl-linked intermediate. This intermediate undergoes nucleophilic attack by an N-terminal poly-Gly sequence to form an amide bond between the Thr and N-terminal Gly. Here, we demonstrate that sortase A can successfully be used to cyclize a variety of small disulfide-rich peptides, including the cyclotide kalata B1, α-conotoxin Vc1.1, and sunflower trypsin inhibitor 1. These peptides range in size from 14 to 29 amino acids and contain three, two, or one disulfide bond, respectively, within their head-to-tail cyclic backbones. Our findings provide proof of concept for the potential broad applicability of enzymatic cyclization of disulfide-rich peptides with therapeutic potential. PMID:24425873

  2. Cleavage of ether, ester, and tosylate C(sp3)-O bonds by an iridium complex, initiated by oxidative addition of C-H bonds. Experimental and computational studies.

    PubMed

    Kundu, Sabuj; Choi, Jongwook; Wang, David Y; Choliy, Yuriy; Emge, Thomas J; Krogh-Jespersen, Karsten; Goldman, Alan S

    2013-04-03

    A pincer-ligated iridium complex, (PCP)Ir (PCP = κ(3)-C6H3-2,6-[CH2P(t-Bu)2]2), is found to undergo oxidative addition of C(sp(3))-O bonds of methyl esters (CH3-O2CR'), methyl tosylate (CH3-OTs), and certain electron-poor methyl aryl ethers (CH3-OAr). DFT calculations and mechanistic studies indicate that the reactions proceed via oxidative addition of C-H bonds followed by oxygenate migration, rather than by direct C-O addition. Thus, methyl aryl ethers react via addition of the methoxy C-H bond, followed by α-aryloxide migration to give cis-(PCP)Ir(H)(CH2)(OAr), followed by iridium-to-methylidene hydride migration to give (PCP)Ir(CH3)(OAr). Methyl acetate undergoes C-H bond addition at the carbomethoxy group to give (PCP)Ir(H)[κ(2)-CH2OC(O)Me] which then affords (PCP-CH2)Ir(H)(κ(2)-O2CMe) (6-Me) in which the methoxy C-O bond has been cleaved, and the methylene derived from the methoxy group has migrated into the PCP Cipso-Ir bond. Thermolysis of 6-Me ultimately gives (PCP)Ir(CH3)(κ(2)-O2CR), the net product of methoxy group C-O oxidative addition. Reaction of (PCP)Ir with species of the type ROAr, RO2CMe or ROTs, where R possesses β-C-H bonds (e.g., R = ethyl or isopropyl), results in formation of (PCP)Ir(H)(OAr), (PCP)Ir(H)(O2CMe), or (PCP)Ir(H)(OTs), respectively, along with the corresponding olefin or (PCP)Ir(olefin) complex. Like the C-O bond oxidative additions, these reactions also proceed via initial activation of a C-H bond; in this case, C-H addition at the β-position is followed by β-migration of the aryloxide, carboxylate, or tosylate group. Calculations indicate that the β-migration of the carboxylate group proceeds via an unusual six-membered cyclic transition state in which the alkoxy C-O bond is cleaved with no direct participation by the iridium center.

  3. Disulfide cross-linking influences symbiotic activities of nodule peptide NCR247

    PubMed Central

    Shabab, Mohammed; Arnold, Markus F. F.; Penterman, Jon; Wommack, Andrew J.; Bocker, Hartmut T.; Price, Paul A.; Griffitts, Joel S.; Nolan, Elizabeth M.; Walker, Graham C.

    2016-01-01

    Interactions of rhizobia with legumes establish the chronic intracellular infection that underlies symbiosis. Within nodules of inverted repeat-lacking clade (IRLC) legumes, rhizobia differentiate into nitrogen-fixing bacteroids. This terminal differentiation is driven by host nodule-specific cysteine-rich (NCR) peptides that orchestrate the adaptation of free-living bacteria into intracellular residents. Medicago truncatula encodes a family of >700 NCR peptides that have conserved cysteine motifs. NCR247 is a cationic peptide with four cysteines that can form two intramolecular disulfide bonds in the oxidized forms. This peptide affects Sinorhizobium meliloti transcription, translation, and cell division at low concentrations and is antimicrobial at higher concentrations. By preparing the three possible disulfide–cross-linked NCR247 regioisomers, the reduced peptide, and a variant lacking cysteines, we performed a systematic study of the effects of intramolecular disulfide cross-linking and cysteines on the activities of an NCR peptide. The relative activities of the five NCR247 variants differed strikingly among the various bioassays, suggesting that the NCR peptide-based language used by plants to control the development of their bacterial partners during symbiosis is even greater than previously recognized. These patterns indicate that certain NCR bioactivities require cysteines whereas others do not. The results also suggest that NCR247 may exert some of its effects within the cell envelope whereas other activities occur in the cytoplasm. BacA, a membrane protein that is critical for symbiosis, provides protection against all bactericidal forms of NCR247. Oxidative folding protects NCR247 from degradation by the symbiotically relevant metalloprotease HrrP (host range restriction peptidase), suggesting that disulfide bond formation may additionally stabilize NCR peptides during symbiosis. PMID:27551097

  4. Acid-denatured Green Fluorescent Protein (GFP) as model substrate to study the chaperone activity of protein disulfide isomerase.

    PubMed

    Mares, Rosa E; Meléndez-López, Samuel G; Ramos, Marco A

    2011-01-01

    Green fluorescent protein (GFP) has been widely used in several molecular and cellular biology applications, since it is remarkably stable in vitro and in vivo. Interestingly, native GFP is resistant to the most common chemical denaturants; however, a low fluorescence signal has been observed after acid-induced denaturation. Furthermore, this acid-denatured GFP has been used as substrate in studies of the folding activity of some bacterial chaperones and other chaperone-like molecules. Protein disulfide isomerase enzymes, a family of eukaryotic oxidoreductases that catalyze the oxidation and isomerization of disulfide bonds in nascent polypeptides, play a key role in protein folding and it could display chaperone activity. However, contrasting results have been reported using different proteins as model substrates. Here, we report the further application of GFP as a model substrate to study the chaperone activity of protein disulfide isomerase (PDI) enzymes. Since refolding of acid-denatured GFP can be easily and directly monitored, a simple micro-assay was used to study the effect of the molecular participants in protein refolding assisted by PDI. Additionally, the effect of a well-known inhibitor of PDI chaperone activity was also analyzed. Because of the diversity their functional activities, PDI enzymes are potentially interesting drug targets. Since PDI may be implicated in the protection of cells against ER stress, including cancer cells, inhibitors of PDI might be able to enhance the efficacy of cancer chemotherapy; furthermore, it has been demonstrated that blocking the reductive cleavage of disulfide bonds of proteins associated with the cell surface markedly reduces the infectivity of the human immunodeficiency virus. Although several high-throughput screening (HTS) assays to test PDI reductase activity have been described, we report here a novel and simple micro-assay to test the chaperone activity of PDI enzymes, which is amenable for HTS of PDI

  5. Sintered-reaction Bonded Silicon Nitride Densified by a Gas Pressure Sintering Process Effects of Rare Earth Oxide Sintering Additives

    SciTech Connect

    Lee, S. H.; Ko, J. W.; Park, Y. J.; Kim, H. D.; Lin, Hua-Tay; Becher, Paul F

    2012-01-01

    Reaction-bonded silicon nitrides containing rare-earth oxide sintering additives were densified by gas pressure sintering. The sintering behavior, microstructure and mechanical properties of the resultant specimens were analyzed. For that purpose, Lu2O3-SiO2 (US), La2O3-MgO (AM) and Y2O3-Al2O3 (YA) additive systems were selected. Among the tested compositions, densification of silicon nitride occurred at the lowest temperature when using the La2O3-MgO system. Since the Lu2O3-SiO2 system has the highest melting temperature, full densification could not be achieved after sintering at 1950oC. However, the system had a reasonably high bending strength of 527 MPa at 1200oC in air and a high fracture toughness of 9.2 MPa m1/2. The Y2O3-Al2O3 system had the highest room temperature bending strength of 1.2 GPa

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

    PubMed Central

    Martínez-Oliván, Juan; Fraga, Hugo; Arias-Moreno, Xabier; Ventura, Salvador; Sancho, Javier

    2015-01-01

    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. PMID:26168158

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

    PubMed Central

    Safavi-Hemami, Helena; Li, Qing; Jackson, Ronneshia L.; Song, Albert S.; Boomsma, Wouter; Bandyopadhyay, Pradip K.; Gruber, Christian W.; Purcell, Anthony W.; Yandell, Mark; Olivera, Baldomero M.

    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 conotoxin-specific PDIs, significantly and differentially accelerate the kinetics of disulfide-bond formation of several conotoxins. Our results are consistent with a unique biological scenario associated with protein folding: The diversification of a family of foldases can be correlated with the rapid evolution of an unprecedented diversity of disulfide-rich structural domains expressed by venomous marine snails in the superfamily Conoidea. PMID:26957604

  8. Raman spectroscopy study on the ν1-2ν2 Fermi resonance of liquid carbon disulfide in binary solutions: effect of the weak hydrogen bond formation on the Fermi resonance.

    PubMed

    Li, DongFei; Sun, Shang; Sun, ChengLin; Jiang, XiuLan; Gao, ShuQin; Li, ZuoWei

    2012-10-01

    We have measured the Raman spectra of liquid CS(2) at different volume concentrations in CHCl(3) and CH(2)Cl(2) solutions. With decreasing the volume concentration of CS(2), a noticeable growth in the 2ν(2) band frequency was observed, while the ν(1) band location remained practically unchanged. This asymmetric wavenumber shift phenomenon of the Fermi doublet ν(1) and 2ν(2) of CS(2) has been ascribed to weak, non-conventional hydrogen bonds formed between the CS(2) and the solvent molecules. These weak hydrogen bonds were also responsible for significant decreases in the C-H bond symmetric stretching vibration band frequencies of CHCl(3) and CH(2)Cl(2). The values of the ν(1)-2ν(2) FR parameters of CS(2) in CH(2)Cl(2) and CHCl(3) at different volume concentrations were calculated according to the FR theory. The magnitude of the FR coupling coefficient W of CS(2) increases upon dilution with CH(2)Cl(2) and CHCl(3), indicating that the vibrational anharmonicity is relatively sensitive to variations in the weak hydrogen bonding. Compared with the changing tendencies of Fermi coupling coefficient W of CS(2) in CH(2)Cl(2) and CHCl(3) at different volume concentrations, we discussed the effect of the weak hydrogen bond formation on the FR and the asymmetric wavenumber shift phenomenon of the Fermi doublet ν(1) and 2ν(2) of CS(2).

  9. Method for removal of asphaltene depositions with amine-activated disulfide oil

    SciTech Connect

    Sharp, S.P.

    1983-04-12

    A method for treating and removing unwanted asphaltene deposits from oil and gas wells, surface equipment, flow lines, and pore spaces of oil-baring formations comprises treatment with an amine -activated aliphatic disulfide oil as an asphaltene solvent. In a preferred aspect, the aliphatic disulfide oil is a dialkyl disulfide oil and is activated by the addition of 10 weight percent of diethylamine. In a specific use, the activated disulfide oil is used to remove asphaltene deposits from an oilbearing formation and a producing well penetrating the formation.

  10. Structures of the Ets Protein DNA-binding Domains of Transcription Factors Etv1, Etv4, Etv5, and Fev: DETERMINANTS OF DNA BINDING AND REDOX REGULATION BY DISULFIDE BOND FORMATION.

    PubMed

    Cooper, Christopher D O; Newman, Joseph A; Aitkenhead, Hazel; Allerston, Charles K; Gileadi, Opher

    2015-05-29

    Ets transcription factors, which share the conserved Ets DNA-binding domain, number nearly 30 members in humans and are particularly involved in developmental processes. Their deregulation following changes in expression, transcriptional activity, or by chromosomal translocation plays a critical role in carcinogenesis. Ets DNA binding, selectivity, and regulation have been extensively studied; however, questions still arise regarding binding specificity outside the core GGA recognition sequence and the mode of action of Ets post-translational modifications. Here, we report the crystal structures of Etv1, Etv4, Etv5, and Fev, alone and in complex with DNA. We identify previously unrecognized features of the protein-DNA interface. Interactions with the DNA backbone account for most of the binding affinity. We describe a highly coordinated network of water molecules acting in base selection upstream of the GGAA core and the structural features that may account for discrimination against methylated cytidine residues. Unexpectedly, all proteins crystallized as disulfide-linked dimers, exhibiting a novel interface (distant to the DNA recognition helix). Homodimers of Etv1, Etv4, and Etv5 could be reduced to monomers, leading to a 40-200-fold increase in DNA binding affinity. Hence, we present the first indication of a redox-dependent regulatory mechanism that may control the activity of this subset of oncogenic Ets transcription factors.

  11. Identifying the presence of a disulfide linkage in peptides by the selective elimination of hydrogen disulfide from collisionally activated alkali and alkaline earth metal complexes.

    PubMed

    Kim, Hugh I; Beauchamp, J L

    2008-01-30

    We report a new method for identifying disulfide linkages in peptides using mass spectrometry. This is accomplished by collisional activation of singly charged cationic alkali and alkaline earth metal complexes, which results in the highly selective elimination of hydrogen disulfide (H2S2). Complexes of peptides possessing disulfide bonds with sodium and alkaline earth metal are generated using electrospray ionization (ESI). Isolation followed by collision induced dissociation (CID) of singly charged peptide complexes results in selective elimination of H2S2 to leave newly formed dehydroalanine residues in the peptide. Further activation of the product yields sequence information in the region previously short circuited by the disulfide bond. For example, singly charged magnesium and calcium ion bound complexes of [Lys8]-vasopressin exhibit selective elimination of H2S2 via low-energy CID. Further isolation of the product followed by CID yields major b- and z-type fragments revealing the peptide sequence in the region between the newly formed dehydroalanine residues. Numerous model peptides provide mechanistic details for the selective elimination of H2S2. The process is initiated starting with a metal stabilized enolate anion at Cys, followed by cleavage of the S-C bond. An examination of the peptic digest of insulin provides an example of the application of the selective elimination of H2S2 for the identification of peptides with disulfide linkages. The energetics and mechanisms of H2S2 elimination from model compounds are investigated using density functional theory (DFT) calculations.

  12. PTR-MS measurements and analysis of models for the calculation of Henry's law constants of monosulfides and disulfides.

    PubMed

    Schuhfried, Erna; Biasioli, Franco; Aprea, Eugenio; Cappellin, Luca; Soukoulis, Christos; Ferrigno, Antonella; Märk, Tilmann D; Gasperi, Flavia

    2011-04-01

    Sulfides are known for their strong odor impact even at very low concentrations. Here, we report Henry's law constants (HLCs) measured at the nanomolar concentration range in water for monosulfides (dimethylsulfide, ethylmethylsulfide, diethylsulfide, allylmethylsulfide) and disulfides (dimethyldisulfide, diethylsulfide, dipropylsulfide) using a dynamic stripping technique coupled to Proton Transfer Reaction-Mass Spectrometry (PTR-MS). The experimental data were compared with literature values and to vapor/solubility calculations and their consistency was confirmed employing the extra-thermodynamic enthalpy-entropy compensation effect. Our experimental data are compatible with reported literature values, and they are typically lower than averaged experimental literature values by about 10%. Critical comparison with other freely available models (modeled vapor/solubility; group and bond additivity methods; Linear Solvation Energy Relationship; SPARC) was performed to validate their applicability to monosulfides and disulfides. Evaluation of theoretical models reveals a large deviation from our measured values by up to four times (in units of Matm(-1)). Two group contribution models were adjusted in view of the new data, and HLCs for a list of sulfur compounds were calculated. Based on our findings we recommend the evaluation and adaption of theoretical models for monosulfides and disulfides to lower values of solubility and higher values of fugacity.

  13. Biodegradable poly(disulfide)s derived from RAFT polymerization: monomer scope, glutathione degradation, and tunable thermal responses.

    PubMed

    Phillips, Daniel J; Gibson, Matthew I

    2012-10-08

    Telechelic, RAFT (reversible addition-fragmentation chain transfer)-derived macromonomers with a pyridyl disulfide end-group were converted into high molecular weight, disulfide-linked polymers using a polycondensation, step-growth procedure. The applicability of the method to polycondense a library of macromonomers with different functionalities including (meth)acrylates and acrylamides was investigated. Side-chain sterics were found to be important as nonlinear poly(ethylene glycol) analogues, which proved incompatible with this synthetic methodology, as were methacrylates due to their pendant methyl group. This method was used to incorporate disulfide bonds into poly(N-isopropylacrylamide), pNIPAM, precursors to give dual-responsive (thermo- and redox) materials. These polymers were shown to selectively degrade in the presence of intracellular concentrations of glutathione but be stable at low concentrations. Due to the molecular weight-dependent cloud point of pNIPAM, the lower critical solution temperature behavior could be switched off by a glutathione gradient without a temperature change: an isothermal transition.

  14. Hydrogen/deuterium exchange and mass spectrometric analysis of a protein containing multiple disulfide bonds: Solution structure of recombinant macrophage colony stimulating factor-beta (rhM-CSFbeta).

    PubMed

    Yan, Xuguang; Zhang, Heidi; Watson, Jeffrey; Schimerlik, Michael I; Deinzer, Max L

    2002-09-01

    Studies with the homodimeric recombinant human macrophage colony-stimulating factor beta (rhM-CSFbeta), show for the first time that a large number (9) of disulfide linkages can be reduced after amide hydrogen/deuterium (H/D) exchange, and the protein digested and analyzed successfully for the isotopic composition by electrospray mass spectrometry. Analysis of amide H/D after exchange-in shows that in solution the conserved four-helix bundle of (rhM-CSFbeta) has fast and moderately fast exchangeable sections of amide hydrogens in the alphaA helix, and mostly slow exchanging sections of amide hydrogens in the alphaB, alphaC, and alphaD helices. Most of the amide hydrogens in the loop between the beta1 and beta4 sheets exhibited fast or moderately fast exchange, whereas in the amino acid 63-67 loop, located at the interface of the two subunits, the exchange was slow. Solvent accessibility as measured by H/D exchange showed a better correlation with the average depth of amide residues calculated from reported X-ray crystallographic data for rhM-CSFalpha than with the average B-factor. The rates of H/D exchange in rhM-CSFbeta appear to correlate well with the exposed surface calculated for each amino acid residue in the crystal structure except for the alphaD helix. Fast hydrogen isotope exchange throughout the segment amino acids 150-221 present in rhM-CSFbeta, but not rhM-CSFalpha, provides evidence that the carboxy-terminal region is unstructured. It is, therefore, proposed that the anomalous behavior of the alphaD helix is due to interaction of the carboxy-terminal tail with this helical segment.

  15. Thiol/disulfide redox states in signaling and sensing

    PubMed Central

    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

  16. Effects of Platinum Additions and Sulfur Impurities on the Microstructure and Scale Adhesion Behavior of Single-Phase CVD Aluminide Bond Coatings

    SciTech Connect

    Cooley, K.M.; Haynes, J.A.; Lee, W.Y.; Pint, B.A.; Wright, I.G.; Zhang, Y.

    1999-02-28

    The adhesion of alumina scales to aluminide bond coats is a life-limiting factor for some advanced thermal barrier coating systems. This study investigated the effects of aluminide bond coat sulfur and platinum contents on alumina scale adhesion and coating microstructural evolution during isothermal and cyclic oxidation testing at 1150 C. Low-sulfur NiAl and NiPtAl bond coats were fabricated by chemical vapor deposition (CVD). Lowering the sulfur contents of CVD NiAl bond coatings significantly improved scale adhesion, but localized scale spallation eventually initiated along coating grain boundaries. Further improvements in scale adhesion were obtained with Pt additions. The observed influences of Pt additions included: (1) mitigation of the detrimental effects of high sulfur levels, (2) drastic reductions in void growth along the scale-metal interface, (3) alteration of the oxide-metal interface morphology, and (4) elimination of Ta-rich oxides in the Al{sub 2}O{sub 3} scales during thermal cycling. The results of this study also suggested that the microstructure (especially the grain size) of CVD aluminide bond coatings plays a significant role in scale adhesion.

  17. 46 CFR Appendix F to Subpart C of... - Optional Rider for Additional NVOCC Financial Responsibility for Group Bonds [Optional Rider to...

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 9 2011-10-01 2011-10-01 false Optional Rider for Additional NVOCC Financial Responsibility for Group Bonds F Appendix F to Subpart C of Part 515 Shipping FEDERAL MARITIME COMMISSION... Against Ocean Transportation Intermediaries Pt. 515, Subpt. C, App. F Appendix F to Subpart C of Part...

  18. 46 CFR Appendix F to Subpart C of... - Optional Rider for Additional NVOCC Financial Responsibility for Group Bonds [Optional Rider to...

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 9 2010-10-01 2010-10-01 false Optional Rider for Additional NVOCC Financial Responsibility for Group Bonds F Appendix F to Subpart C of Part 515 Shipping FEDERAL MARITIME COMMISSION... Against Ocean Transportation Intermediaries Pt. 515, Subpt. C, App. F Appendix F to Subpart C of Part...

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

    PubMed

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

    2011-06-01

    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.

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

    SciTech Connect

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

    2011-12-31

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

  1. Tissue factor de-encryption, thrombus formation, and thiol-disulfide exchange.

    PubMed

    Chen, Vivien M Y

    2013-02-01

    Tissue factor (TF) by forming a complex with factor VIIa (FVIIa) initiates blood coagulation. It was traditionally believed that the separation of FVIIa in circulation from subendothelial TF was the main control that was preventing spontaneous initiation of thrombosis and that circulating cells and endothelium did not express TF protein at rest in healthy individuals. However, TF has been detected in healthy human plasma and animal models of thrombosis, which indicate that TF in circulation can contribute to thrombin generation and fibrin formation after an activation event. Circulating TF-and indeed, most of the TF on the cell surface-is "encrypted" or coagulation inactive. The de-encryption step involves exposure of phosphatidylserine (PS), but PS exposure alone is insufficient for full TF activity. Allosteric disulfide bonds control protein function by mediating conformal change through the formation and breaking of disulfide bonds. TF contains a typical surface exposed allosteric bond in the membrane proximal fibronectin type III domain. Thiol-disulfide exchange involving this disulfide is implicated in TF activation with the formation of the disulfide bond corresponding with the active conformation of TF and free thiol or thiol-modified forms corresponding with encryption. Although the exact mechanism by which TF de-encryption occurs remains a subject of debate, thiol blockade and inhibition of oxidoreductases show an important role for thiol-disulfide reactions in platelet-independent pathways of coagulation in vitro and in vivo. In particular, redox active extracellular protein disulfide isomerase is involved in the earliest stages of thrombus initiation and has proven to be a potential target for antithrombotic drug development.

  2. Blastomyces Virulence Adhesin-1 Protein Binding to Glycosaminoglycans Is Enhanced by Protein Disulfide Isomerase

    PubMed Central

    Beaussart, Audrey; Brandhorst, Tristan

    2015-01-01

    ABSTRACT Blastomyces adhesin-1 (BAD-1) protein mediates the virulence of the yeast Blastomyces dermatitidis, in part by binding host lung tissue, the extracellular matrix, and cellular receptors via glycosaminoglycans (GAGs), such as heparan sulfate. The tandem repeats that make up over 90% of BAD-1 appear in their native state to be tightly folded into an inactive conformation, but recent work has shown that they become activated and adhesive upon reduction of a disulfide linkage. Here, atomic force microscopy (AFM) of a single BAD-1 molecule interacting with immobilized heparin revealed that binding is enhanced upon treatment with protein disulfide isomerase and dithiothreitol (PDI/DTT). PDI/DTT treatment of BAD-1 induced a plateau effect in atomic force signatures that was consistent with sequential rupture of tandem binding domains. Inhibition of PDI in murine macrophages blunted BAD-1 binding to heparin in vitro. Based on AFM, we found that a short Cardin-Weintraub sequence paired with a WxxWxxW sequence in the first, degenerate repeat at the N terminus of BAD-1 was sufficient to initiate heparin binding. Removal of half of the 41 BAD-1 tandem repeats led to weaker adhesion, illustrating their role in enhanced binding. Mass spectroscopy of the tandem repeat revealed that the PDI-induced interaction with heparin is characterized by ruptured disulfide bonds and that cysteine thiols remain reduced. Further binding studies showed direct involvement of thiols in heparin ligation. Thus, we propose that the N-terminal domain of BAD-1 governs the initial association with host GAGs and that proximity to GAG-associated host PDI catalyzes activation of additional binding motifs conserved within the tandem repeats, leading to enhanced avidity and availability of reduced thiols. PMID:26396244

  3. Disulfide Trapping for Modeling and Structure Determination of Receptor:Chemokine Complexes

    PubMed Central

    Kufareva, Irina; Gustavsson, Martin; Holden, Lauren G.; Qin, Ling; Zheng, Yi; Handel, Tracy M.

    2016-01-01

    Despite the recent breakthrough advances in GPCR crystallography, structure determination of protein-protein complexes involving chemokine receptors and their endogenous chemokine ligands remains challenging. Here we describe disulfide trapping, a methodology for generating irreversible covalent binary protein complexes from unbound protein partners by introducing two cysteine residues, one per interaction partner, at selected positions within their interaction interface. Disulfide trapping can serve at least two distinct purposes: (i) stabilization of the complex to assist structural studies, and/or (ii) determination of pairwise residue proximities to guide molecular modeling. Methods for characterization of disulfide-trapped complexes are described and evaluated in terms of throughput, sensitivity, and specificity towards the most energetically favorable cross-links. Due to abundance of native disulfide bonds at receptor:chemokine interfaces, disulfide trapping of their complexes can be associated with intramolecular disulfide shuffling and result in misfolding of the component proteins; because of this, evidence from several experiments is typically needed to firmly establish a positive disulfide crosslink. An optimal pipeline that maximizes throughput and minimizes time and costs by early triage of unsuccessful candidate constructs is proposed. PMID:26921956

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

    PubMed Central

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

    2006-01-01

    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 310–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 cost of losing a disulfide bond by augmenting and optimizing weaker yet nonetheless effective non-covalent interactions. PMID:16929098

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

    PubMed

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

    2006-09-01

    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 alpha-dendrotoxin and bovine pancreatic trypsin inhibitor (BPTI). While conkunitzin-S1 and alpha-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)-beta-beta-alpha 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 alpha-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 cost of losing a disulfide bond by augmenting and optimizing weaker yet nonetheless effective non-covalent interactions.

  6. Non-innocent additives in a palladium(II)-catalyzed C-H bond activation reaction: insights into multimetallic active catalysts.

    PubMed

    Anand, Megha; Sunoj, Raghavan B; Schaefer, Henry F

    2014-04-16

    The role of a widely employed additive (AgOAc) in a palladium acetate-catalyzed ortho-C-H bond activation reaction has been examined using the M06 density functional theory. A new hetero-bimetallic Pd-(μ-OAc)3-Ag is identified as the most likely active species. This finding could have far-reaching implications with respect to the notion of the active species in palladium catalysis in the presence of other metal salt additives.

  7. Mapping of domains on HIV envelope protein mediating association with calnexin and protein-disulfide isomerase.

    PubMed

    Papandréou, Marie-Jeanne; Barbouche, Rym; Guieu, Régis; Rivera, Santiago; Fantini, Jacques; Khrestchatisky, Michel; Jones, Ian M; Fenouillet, Emmanuel

    2010-04-30

    The cell catalysts calnexin (CNX) and protein-disulfide isomerase (PDI) cooperate in establishing the disulfide bonding of the HIV envelope (Env) glycoprotein. Following HIV binding to lymphocytes, cell-surface PDI also reduces Env to induce the fusogenic conformation. We sought to define the contact points between Env and these catalysts to illustrate their potential as therapeutic targets. In lysates of Env-expressing cells, 15% of the gp160 precursor, but not gp120, coprecipitated with CNX, whereas only 0.25% of gp160 and gp120 coprecipitated with PDI. Under in vitro conditions, which mimic the Env/PDI interaction during virus/cell contact, PDI readily associated with Env. The domains of Env interacting in cellulo with CNX or in vitro with PDI were then determined using anti-Env antibodies whose binding site was occluded by CNX or PDI. Antibodies against domains V1/V2, C2, and the C terminus of V3 did not bind CNX-associated Env, whereas those against C1, V1/V2, and the CD4-binding domain did not react with PDI-associated Env. In addition, a mixture of the latter antibodies interfered with PDI-mediated Env reduction. Thus, Env interacts with intracellular CNX and extracellular PDI via discrete, largely nonoverlapping, regions. The sites of interaction explain the mode of action of compounds that target these two catalysts and may enable the design of further new competitive agents.

  8. Mapping of Domains on HIV Envelope Protein Mediating Association with Calnexin and Protein-disulfide Isomerase*

    PubMed Central

    Papandréou, Marie-Jeanne; Barbouche, Rym; Guieu, Régis; Rivera, Santiago; Fantini, Jacques; Khrestchatisky, Michel; Jones, Ian M.; Fenouillet, Emmanuel

    2010-01-01

    The cell catalysts calnexin (CNX) and protein-disulfide isomerase (PDI) cooperate in establishing the disulfide bonding of the HIV envelope (Env) glycoprotein. Following HIV binding to lymphocytes, cell-surface PDI also reduces Env to induce the fusogenic conformation. We sought to define the contact points between Env and these catalysts to illustrate their potential as therapeutic targets. In lysates of Env-expressing cells, 15% of the gp160 precursor, but not gp120, coprecipitated with CNX, whereas only 0.25% of gp160 and gp120 coprecipitated with PDI. Under in vitro conditions, which mimic the Env/PDI interaction during virus/cell contact, PDI readily associated with Env. The domains of Env interacting in cellulo with CNX or in vitro with PDI were then determined using anti-Env antibodies whose binding site was occluded by CNX or PDI. Antibodies against domains V1/V2, C2, and the C terminus of V3 did not bind CNX-associated Env, whereas those against C1, V1/V2, and the CD4-binding domain did not react with PDI-associated Env. In addition, a mixture of the latter antibodies interfered with PDI-mediated Env reduction. Thus, Env interacts with intracellular CNX and extracellular PDI via discrete, largely nonoverlapping, regions. The sites of interaction explain the mode of action of compounds that target these two catalysts and may enable the design of further new competitive agents. PMID:20202930

  9. Interface confined hydrogen evolution reaction in zero valent metal nanoparticles-intercalated molybdenum disulfide

    NASA Astrophysics Data System (ADS)

    Chen, Zhongxin; Leng, Kai; Zhao, Xiaoxu; Malkhandi, Souradip; Tang, Wei; Tian, Bingbing; Dong, Lei; Zheng, Lirong; Lin, Ming; Yeo, Boon Siang; Loh, Kian Ping

    2017-02-01

    Interface confined reactions, which can modulate the bonding of reactants with catalytic centres and influence the rate of the mass transport from bulk solution, have emerged as a viable strategy for achieving highly stable and selective catalysis. Here we demonstrate that 1T'-enriched lithiated molybdenum disulfide is a highly powerful reducing agent, which can be exploited for the in-situ reduction of metal ions within the inner planes of lithiated molybdenum disulfide to form a zero valent metal-intercalated molybdenum disulfide. The confinement of platinum nanoparticles within the molybdenum disulfide layered structure leads to enhanced hydrogen evolution reaction activity and stability compared to catalysts dispersed on carbon support. In particular, the inner platinum surface is accessible to charged species like proton and metal ions, while blocking poisoning by larger sized pollutants or neutral molecules. This points a way forward for using bulk intercalated compounds for energy related applications.

  10. Interface confined hydrogen evolution reaction in zero valent metal nanoparticles-intercalated molybdenum disulfide

    PubMed Central

    Chen, Zhongxin; Leng, Kai; Zhao, Xiaoxu; Malkhandi, Souradip; Tang, Wei; Tian, Bingbing; Dong, Lei; Zheng, Lirong; Lin, Ming; Yeo, Boon Siang; Loh, Kian Ping

    2017-01-01

    Interface confined reactions, which can modulate the bonding of reactants with catalytic centres and influence the rate of the mass transport from bulk solution, have emerged as a viable strategy for achieving highly stable and selective catalysis. Here we demonstrate that 1T′-enriched lithiated molybdenum disulfide is a highly powerful reducing agent, which can be exploited for the in-situ reduction of metal ions within the inner planes of lithiated molybdenum disulfide to form a zero valent metal-intercalated molybdenum disulfide. The confinement of platinum nanoparticles within the molybdenum disulfide layered structure leads to enhanced hydrogen evolution reaction activity and stability compared to catalysts dispersed on carbon support. In particular, the inner platinum surface is accessible to charged species like proton and metal ions, while blocking poisoning by larger sized pollutants or neutral molecules. This points a way forward for using bulk intercalated compounds for energy related applications. PMID:28230105

  11. A degradable polydopamine coating based on disulfide-exchange reaction.

    PubMed

    Hong, Daewha; Lee, Hojae; Kim, Beom Jin; Park, Taegyun; Choi, Ji Yu; Park, Matthew; Lee, Juno; Cho, Hyeoncheol; Hong, Seok-Pyo; Yang, Sung Ho; Jung, Sun Ho; Ko, Sung-Bo; Choi, Insung S

    2015-12-21

    Although the programmed degradation of biocompatible films finds applications in various fields including biomedical and bionanotechnological areas, coating methods have generally been limited to be substrate-specific, not applicable to any kinds of substrates. In this paper, we report a dopamine derivative, which allows for both universal coating of various substrates and stimuli-responsive film degradation, inspired by mussel-adhesive proteins. Two dopamine moieties are linked together by the disulfide bond, the cleavage of which enables the programmed film degradation. Mechanistic analysis of the degradable films indicates that the initial cleavage of the disulfide linkage causes rapid uptake of water molecules, hydrating the films, which leads to rapid degradation. Our substrate-independent coating of degradable films provides an advanced tool for drug delivery systems, tissue engineering, and anti-fouling strategies.

  12. Oxidation of kinetically trapped thiols by protein disulfide isomerase.

    PubMed

    Walker, K W; Gilbert, H F

    1995-10-17

    The formation of a stabilized structure during oxidative protein folding can severely retard disulfide formation if the structure must be disrupted to gain access to buried cysteines. These kinetic traps can slow protein folding and disulfide bond formation to the extent that unassisted folding is too slow to be kinetically competent in the cell. Protein disulfide isomerase (PDI) facilitates the oxidation of a kinetically trapped state of RTEM-1 beta-lactamase in which two cysteines that form the single disulfide bond in the native protein are buried and approximately 500-fold less reactive than exposed cysteines. Under second-order conditions, PDI-dependent oxidation of reduced, folded beta-lactamase is 500-fold faster than GSSG-dependent oxidation. The rate difference observed between PDI and GSSG can be accounted for by the 520-fold higher kinetic reactivity of PDI as an oxidant. Noncovalent interactions between PDI (35 microM) and beta-lactamase increase the reactivity or unfolding of beta-lactamase in the steady-state by less than 3-fold. At high concentrations of PDI or alkylating agents, the reaction of beta-lactamase cysteines approaches a constant rate, limited by the spontaneous unfolding of the protein (kunfold = 0.024 +/- 0.005 min-1). PDI does not substantially increase the rate of beta-lactamase unfolding; however, once beta-lactamase spontaneously unfolds, PDI at concentrations greater than 44 +/- 4 microM, oxidizes the unfolded substrate before it can refold (kfold = 1.5 +/- 0.2 min-1).(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Direct Syn Addition of Two Silicon Atoms to a C≡C Triple Bond by Si-Si Bond Activation: Access to Reactive Disilylated Olefins.

    PubMed

    Ahmad, Maha; Gaumont, Annie-Claude; Durandetti, Muriel; Maddaluno, Jacques

    2017-02-20

    A catalytic intramolecular silapalladation of alkynes affords, in good yields and stereoselectively, syn-disilylated heterocycles of different chemical structure and size. When applied to silylethers, this reaction leads to vinylic silanols that undergo a rhodium-catalyzed addition to activated olefins, providing the oxa-Heck or oxa-Michael products, depending on the reaction conditions.

  14. Lid mobility in lipase SMG1 validated using a thiol/disulfide redox potential probe.

    PubMed

    Guo, Shaohua; Popowicz, Grzegorz Maria; Li, Daoming; Yuan, Dongjuan; Wang, Yonghua

    2016-05-01

    Most lipases possess a lid domain above the catalytic site that is responsible for their activation. Lipase SMG1 from Malassezia globose CBS 7966 (Malassezia globosa LIP1), is a mono- and diacylglycerol lipase with an atypical loop-like lid domain. Activation of SMG1 was proposed to be solely through a gating mechanism involving two residues (F278 and N102). However, through disulfide bond cross-linking of the lid, this study shows that full activation also requires mobility of the lid domain, contrary to a previous proposal. The newly introduced disulfide bond makes lipase SMG1 eligible as a ratiometric thiol/disulfide redox potential probe, when it is coupled with chromogenic substrates. This redox-switch lipase could also be of potential use in cascade biocatalysis.

  15. Depolarized light scattering in dilute solutions of alkanes: A comparison of the bond additive and interacting atom approximations to the molecular polarizability

    SciTech Connect

    Keyes, T.; Evans, G.T.; Ladanyi, B.M.

    1981-04-01

    The molecular polarizability of a few small alkane (4--10 bond) chains has been represented by (1) an interacting atom model (IAM), wherein the atoms are treated as isotropic point polarizabilities interacting by the dipole tensor; and (2) the bond additive approximation (BAA) in which each bond is assigned an axially symmetric polarizability tensor, and the total molecular polarizability is the sum of the individual bond values. For selected values of the gauche--trans energy difference (0.3 kcal/mole), the calculated mean anisotropy per backbone atom /N increases linearly with N for the IAM and is essentially independent of N in the BAA. Orientational correlation functions have been determined for several second rank tensors characterizing the flexible chains using a modified version of Fixman's Brownian dynamics programs. The orientational correlation functions displayed an effective nonanalytic decay for short times merging into an exponential for long times. Single particle correlation times for the IAM increased more rapidly with N than did those of the BAA. Relaxation of the end-to-end vector (actually its second rank analog) was found to be the slowest process, followed by the IAM and the BAA polarizabilities, and finally the fastest was a local rotational mode.

  16. Development of MDP-based one-step self-etch adhesive--effect of additional 4-META on bonding performance.

    PubMed

    Iwai, Hitoshi; Fujita, Kou; Iwai, Hirotoshi; Ikemi, Takuji; Goto, Haruhiko; Aida, Masahiro; Nishiyama, Norihiro

    2013-01-01

    We designed three experimental 10-methacryloyloxydecyl dihydrogen phosphate (MDP)-based one-step (EX) adhesives consisting of MDP, urethane dimethacrylate, and triethylene glycol dimethacrylate adhesives with different water contents (98.4, 196.8, and 294.4 mg/g), and 4-methacryloyloxyethyl trimellitic anhydride (4-META) or 2-hydroxyethyl methacrylate (HEMA)-containing onestep adhesive. The effect of the amount of MDP-calcium (MDP-Ca) salt produced through demineralization of enamel and dentin on the bonding performance was examined. The efficacy of 4-META and HEMA was then discussed. When the amount of water in EX adhesive was increased, the production amount of MDP-Ca salt of enamel increased, but not the dentin. The enamel bond strength slightly increased with increasing the production amount of MDP-Ca salt, in contrast to the dentin. However, addition of 4-META in the EX adhesive (water content=98.4 mg/g) increased both bond strengths, although the production amounts of MDP-Ca salt significantly decreased. The 4-META enhances both bond strengths more effectively than the HEMA.

  17. Theoretical study of the thermal decomposition of dimethyl disulfide.

    PubMed

    Vandeputte, Aäron G; Reyniers, Marie-Françoise; Marin, Guy B

    2010-10-07

    Despite its use in a wide variety of industrially important thermochemical processes, little is known about the thermal decomposition mechanism of dimethyl disulfide (DMDS). To obtain more insight, the radical decomposition mechanism of DMDS is studied theoretically and a kinetic model is developed accounting for the formation of all the decomposition products observed in the experimental studies available in literature. Thermochemical data and rate coefficients are obtained using the high-level CBS-QB3 composite method. Among five methods tested (BMK/6-311G(2d,d,p), MPW1PW91/6-311G(2d,d,p), G3, G3B3, and CBS-QB3), the CBS-QB3 method was found to reproduce most accurately the experimental standard enthalpies of formation for a set of 17 small organosulfur compounds and the bond dissociation energies for a set of 10 sulfur bonds. Enthalpies of formation were predicted within 4 kJ mol(-1) while the mean absolute deviation on the bond dissociation enthalpies amounts to 7 kJ mol(-1). From the theoretical study, a new reaction path is identified for the formation of carbon disulfide via dithiirane (CH(2)S(2)). A reaction mechanism was constructed containing 36 reactions among 25 species accounting for the formation of all the decomposition products reported in literature. High-pressure limit rate coefficients for the 36 reactions in the reaction mechanism are presented. The kinetic model is able to grasp the experimental observations. With the recombination of thiyl radicals treated as being in the low-pressure limit, the experimentally reported first-order rate coefficients for the decomposition of DMDS are reproduced within 1 order of magnitude, while the observed product selectivities of most compounds are reproduced satisfactory. Simulations indicate that at high conversions most of the carbon disulfide forms according to the newly identified reaction path involving the formation of dithiirane.

  18. Effect of Addition of Curcumin Nanoparticles on Antimicrobial Property and Shear Bond Strength of Orthodontic Composite to Bovine Enamel

    PubMed Central

    Sodagar, Ahmad; Bahador, Abbas; Pourhajibagher, Maryam; Ahmadi, Badreddin; Baghaeian, Pedram

    2016-01-01

    Objectives: This study sought to assess the effect of curcumin nanoparticles (curcNPs) on antimicrobial property and shear bond strength (SBS) of orthodontic composite to bovine enamel. Materials and Methods: In this in vitro, experimental study, 1%, 5% and 10% curcNPs were added to Transbond XT composite. Stainless steel brackets were bonded to 48 sound bovine incisors in four groups (n=12) using composite containing 0% (control), 1%, 5% and 10% curcNPs. The bracket-tooth SBS was measured by a universal testing machine. The adhesive remnant index (ARI) score was calculated after debonding using a stereomicroscope. Also, 180 discs were fabricated of the four composites; 108 were subjected to eluted component test, 36 were used for disc diffusion test and 36 were used for biofilm test to assess their antimicrobial activity against Streptococcus mutans, Streptococcus sanguinis and Lactobacillus acidophilus. Results: The highest and lowest SBS belonged to control and 10% curcNP groups, respectively. The difference in SBS was significant among the four groups (P=0.008). The SBS of control group was significantly higher than that of 10% curcNPs (P=0.006). The four groups were not significantly different in terms of ARI score (P>0.05). Growth inhibition zones were not seen in any group. In biofilm test, the colony counts of all bacteria significantly decreased by an increase in percentage of curcNPs. Colony count significantly decreased only at 30 days. Conclusions: At 1% concentration, curcNPs have significant antimicrobial activity against cariogenic bacteria with no adverse effect on SBS. However, insolubility of curcNPs remains a major drawback. PMID:28127332

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

    PubMed

    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.

  20. Stereochemical Control of Enzymatic Carbon-Carbon Bond-Forming Michael-Type Additions by "Substrate Engineering".

    PubMed

    Miao, Yufeng; Tepper, Pieter G; Geertsema, Edzard M; Poelarends, Gerrit J

    2016-11-01

    The enzyme 4-oxalocrotonate tautomerase (4-OT) promiscuously catalyzes the Michael-type addition of acetaldehyde to β-nitrostyrene derivatives to yield chiral γ-nitroaldehydes, which are important precursors for pharmaceutically active γ-aminobutyric acids. In this study, we investigated the effect of different substituents at the aromatic ring of the Michael acceptor on the catalytic efficiency and stereoselectivity of the 4-OT-catalyzed acetaldehyde addition reactions. Highly enantioenriched (R)- and (S)-γ-nitroaldehydes and 4-substituted chroman-2-ol could be obtained in good to excellent yields by applying different substituents at appropriate positions of the aromatic substrate. Stereochemical control of these enzymatic Michael-type additions by "substrate engineering" allowed the enantioselective synthesis of valuable γ-aminobutyric acid precursors. In addition, the results suggest a novel enzymatic synthesis route towards precursors for chromans and derivatives, which are valuable scaffolds for preparing biologically active natural products.

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

    PubMed

    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.

  2. Green polymer chemistry: Synthesis of poly(disulfide) polymers and networks

    NASA Astrophysics Data System (ADS)

    Rosenthal-Kim, Emily Quinn

    The disulfide group is unique in that it presents a covalent bond that is easily formed and cleaved under certain biological conditions. While the ease of disulfide bond cleavage is often harnessed as a method of biodegradation, the ease of disulfide bond formation as a synthetic strategy is often overlooked. The objective this research was to synthesize poly(disulfide) polymers and disulfide crosslinked networks from a green chemistry approach. The intent of the green chemistry approach was to take advantage of the mild conditions applicable to disulfide bond synthesis from thiols. With anticipated use as biomaterials, it was also desired that the polymer materials could be degraded under biological conditions. Here, a new method of poly(disulfide) polymer synthesis is introduced which was inspired by the reaction conditions and reagents found in Nature. Ambient temperatures and aqueous mixtures were used in the new method. Hydrogen peroxide, one of the Nature's most powerful oxidizing species was used as the oxidant in the new polymerization reaction. The dithiol monomer, 3,6-dioxa-1,8-octanedithiol was first solubilized in triethylamine, which activated the thiol groups and made the monomer water soluble. At room temperature, the organic dithiol/amine solution was then mixed with dilute aqueous hydrogen peroxide (3% by weight) to make the poly(disulfide) polymers. The presence of a two phase system (organic and aqueous phases) was critical to the polymerization reaction. As the reaction progresses, a third, polymer phase appeared. At ambient temperatures and above, this phase separated from the reaction mixture and the polymer product was easily removed from the reaction solution. These polymers reach Mn > 250,000 g/mol in under two hours. Molecular weight distributions were between 1.5 and 2.0. Reactions performed in an ice bath which remain below room temperature contain high molecular weight polymers with Mn ≈ 120,000 g/mol and have a molecular weight

  3. Formation of C-C Bonds via Ruthenium Catalyzed Transfer Hydrogenation: Carbonyl Addition from the Alcohol or Aldehyde Oxidation Level.

    PubMed

    Shibahara, Fumitoshi; Krische, Michael J

    2008-01-01

    Under the conditions of ruthenium catalyzed transfer hydrogenation employing isopropanol as terminal reductant, π-unsaturated compounds (1,3-dienes, allenes, 1,3-enynes and alkynes) reductively couple to aldehydes to furnish products of carbonyl addition. In the absence of isopropanol, π-unsaturated compounds couple directly from the alcohol oxidation level to form identical products of carbonyl addition. Such "alcohol-unsaturate C-C couplings" enable carbonyl allylation, propargylation and vinylation from the alcohol oxidation level in the absence of stoichiometric organometallic reagents or metallic reductants. Thus, direct catalytic C-H functionalization of alcohols at the carbinol carbon is achieved.

  4. General Approach To Determine Disulfide Connectivity in Cysteine-Rich Peptides by Sequential Alkylation on Solid Phase and Mass Spectrometry.

    PubMed

    Albert, Anastasia; Eksteen, J Johannes; Isaksson, Johan; Sengee, Myagmarsuren; Hansen, Terkel; Vasskog, Terje

    2016-10-04

    Within the field of bioprospecting, disulfide-rich peptides are a promising group of compounds that has the potential to produce important leads for new pharmaceuticals. The disulfide bridges stabilize the tertiary structure of the peptides and often make them superior drug candidates to linear peptides. However, determination of disulfide connectivity in peptides with many disulfide bridges has proven to be laborious and general methods are lacking. This study presents a general approach for structure elucidation of disulfide-rich peptides. The method features sequential reduction and alkylation of a peptide on solid phase combined with sequencing of the fully alkylated peptide by tandem mass spectrometry. Subsequently, the disulfide connectivity is assigned on the basis of the determined alkylation pattern. The presented method is especially suitable for peptides that are prone to disulfide scrambling or are unstable in solution with partly reduced bridges. Additionally, the use of small amounts of peptide in the lowest nmol range makes the method ideal for structure elucidation of unknown peptides from the bioprospecting process. This study successfully demonstrates the new method for seven different peptides with two to four disulfide bridges. Two peptides with previous contradicting publications, μ-conotoxin KIIA and hepcidin-25, are included, and their disulfide connectivity is confirmed in accordance with the latest published results.

  5. Addition of quadricyclane to C[sub 60]: Easy access to fullerene derivatives bearing a reactive double bond in the side chain

    SciTech Connect

    Prato, M. ); Maggini, M.; Scorrano, G. ); Lucchini, V. )

    1993-07-02

    The reaction of C[sub 60] with quadricyclane gives a stable 6,6 adduct which has been spectroscopically characterized. The double bond of the [2.2.1]bicycloheptene moiety reacts readily with electrophiles (e.g., PhSCl). Soon after the isolation and characterization of fullerene C[sub 60], the electrophilic character of this carbon cluster was disclosed by both experimental and theoretical results. Additions of several electrophiles to C[sub 60] have also been reported, but the conditions necessary for these reactions to occur led often to inseparable mixtures of products of multiple addition. In order to allow a controlled addition of electrophiles and to enrich the chemistry of functionalization, C[sub 60] has to be structurally modified. Herein the authors report a simple cycloaddition approach to a stable and characterizable C[sub 60] derivative, in which the incorporated olefinic moiety shows high reactivity toward electrophiles. 3 figs.

  6. Water interaction and bond strength to dentin of dye-labelled adhesive as a function of the addition of rhodamine B

    PubMed Central

    WANG, Linda; BIM, Odair; LOPES, Adolfo Coelho de Oliveira; FRANCISCONI-DOS-RIOS, Luciana Fávaro; MAENOSONO, Rafael Massunari; D’ALPINO, Paulo Henrique Perlatti; HONÓRIO, Heitor Marques; ATTA, Maria Teresa

    2016-01-01

    ABSTRACT Objective This study investigated the effect of the fluorescent dye rhodamine B (RB) for interfacial micromorphology analysis of dental composite restorations on water sorption/solubility (WS/WSL) and microtensile bond strength to dentin (µTBS) of a 3-step total etch and a 2-step self-etch adhesive system. Material and Methods The adhesives Adper Scotchbond Multi-Purpose (MP) and Clearfil SE Bond (SE) were mixed with 0.1 mg/mL of RB. For the WS/WSL tests, cured resin disks (5.0 mm in diameter x 0.8 mm thick) were prepared and assigned into four groups (n=10): MP, MP-RB, SE, and SE-RB. For µTBS assessment, extracted human third molars (n=40) had the flat occlusal dentin prepared and assigned into the same experimental groups (n=10). After the bonding and restoration procedures, specimens were sectioned in rectangular beams, stored in water and tested after seven days or after 12 months. The failure mode of fractured specimens was qualitatively evaluated under optical microscope (x40). Data from WS/WSL and µTBS were assessed by one-way and three-way ANOVA, respectively, and Tukey’s test (α=5%). Results RB increased the WSL of MP and SE. On the other hand, WS of both MP and SE was not affected by the addition of RB. No significance in µTBS between MP and MP-RB for seven days or one year was observed, whereas for SE a decrease in the µTBS means occurred in both storage times. Conclusions RB should be incorporated into non-simplified DBSs with caution, as it can interfere with their physical-mechanical properties, leading to a possible misinterpretation of bonded interface. PMID:27556201

  7. Vanadium nitride functionalization and denitrogenation by carbon disulfide and dioxide.

    PubMed

    Brask, Justin K; Durà-Vilà, Víctor; Diaconescu, Paula L; Cummins, Christopher C

    2002-04-21

    A dramatic difference in behavior is observed for the dithiocarbamate and carbamate complexes [Ar(But)N]3V(NCE2)Na(THF)2(E = S or O, respectively), prepared from the corresponding nitride species ([Ar(But)N]3V identical to NNa)2 by way of a nucleophilic addition reaction involving carbon disulfide or dioxide, and is rationalized with the aid of DFT calculations.

  8. Hapten-directed spontaneous disulfide shuffling: a universal technology for site-directed covalent coupling of payloads to antibodies.

    PubMed

    Dengl, Stefan; Hoffmann, Eike; Grote, Michael; Wagner, Cornelia; Mundigl, Olaf; Georges, Guy; Thorey, Irmgard; Stubenrauch, Kay-Gunnar; Bujotzek, Alexander; Josel, Hans-Peter; Dziadek, Sebastian; Benz, Joerg; Brinkmann, Ulrich

    2015-05-01

    Humanized hapten-binding IgGs were designed with an accessible cysteine close to their binding pockets, for specific covalent payload attachment. Individual analyses of known structures of digoxigenin (Dig)- and fluorescein (Fluo) binding antibodies and a new structure of a biotin (Biot)-binder, revealed a "universal" coupling position (52(+2)) in proximity to binding pockets but without contributing to hapten interactions. Payloads that carry a free thiol are positioned on the antibody and covalently linked to it via disulfides. Covalent coupling is achieved and driven toward complete (95-100%) payload occupancy by spontaneous redox shuffling between antibody and payload. Attachment at the universal position works with different haptens, antibodies, and payloads. Examples are the haptens Fluo, Dig, and Biot combined with various fluorescent or peptidic payloads. Disulfide-bonded covalent antibody-payload complexes do not dissociate in vitro and in vivo. Coupling requires the designed cysteine and matching payload thiol because payload or antibody without the Cys/thiol are not linked (<5% nonspecific coupling). Hapten-mediated positioning is necessary as hapten-thiol-payload is only coupled to antibodies that bind matching haptens. Covalent complexes are more stable in vivo than noncovalent counterparts because digoxigeninylated or biotinylated fluorescent payloads without disulfide-linkage are cleared more rapidly in mice (approximately 50% reduced 48 hour serum levels) compared with their covalently linked counterparts. The coupling technology is applicable to many haptens and hapten binding antibodies (confirmed by automated analyses of the structures of 140 additional hapten binding antibodies) and can be applied to modulate the pharmacokinetics of small compounds or peptides. It is also suitable to link payloads in a reduction-releasable manner to tumor- or tissue-targeting delivery vehicles.

  9. Mutations in domain a′ of protein disulfide isomerase affect the folding pathway of bovine pancreatic ribonuclease A

    PubMed Central

    Ruoppolo, Margherita; Orrù, Stefania; Talamo, Fabio; Ljung, Johanna; Pirneskoski, Annamari; Kivirikko, Kari I.; Marino, Gennaro; Koivunen, Peppi

    2003-01-01

    Protein disulfide isomerase (PDI, EC 5.3.4.1), an enzyme and chaperone, catalyses disulfide bond formation and rearrangements in protein folding. It is also a subunit in two proteins, the enzyme collagen prolyl 4-hydroxylase and the microsomal triglyceride transfer protein. It consists of two catalytically active domains, a and a′, and two inactive ones, b and b′, all four domains having the thioredoxin fold. Domain b′ contains the primary peptide binding site, but a′ is also critical for several of the major PDI functions. Mass spectrometry was used here to follow the folding pathway of bovine pancreatic ribonuclease A (RNase A) in the presence of three PDI mutants, F449R, Δ455–457, and abb′, and the individual domains a and a′. The first two mutants contained alterations in the last α helix of domain a′, while the third lacked the entire domain a′. All mutants produced genuine, correctly folded RNase A, but the appearance rate of 50% of the product, as compared to wild-type PDI, was reduced 2.5-fold in the case of PDI Δ455–457, 7.5-fold to eightfold in the cases of PDI F449R and PDI abb′, and over 15-fold in the cases of the individual domains a and a′. In addition, PDI F449R and PDI abb′ affected the distribution of folding intermediates. Domains a and a′ catalyzed the early steps in the folding but no disulfide rearrangements, and therefore the rate observed in the presence of these individual domains was similar to that of the spontaneous process. PMID:12717017

  10. Linearity and additivity in cluster-induced sputtering: A molecular-dynamics study of van der Waals bonded systems

    SciTech Connect

    Anders, Christian; Urbassek, Herbert M.; Johnson, Robert E.

    2004-10-15

    Using molecular-dynamics simulation, we study sputtering of a condensed-gas solid induced by the impact of atomic clusters with sizes 1{<=}n{<=}10{sup 4}. Above a nonlinear onset regime, we find a linear increase of the sputter yield Y with the total energy E of the bombarding cluster. The fitting coefficients in the linear regime depend only on the cluster size n such that for fixed bombardment energy, sputtering decreases with increasing cluster size n. We find that to a good approximation the sputter yield in this regime obeys an additivity rule in cluster size n such that doubling the cluster size at the same cluster velocity amounts to doubling the sputter yield. The sputter-limiting energy {epsilon}{sub s} is introduced which separates erosion ({epsilon}>{epsilon}{sub s}) from growth ({epsilon}<{epsilon}{sub s}) under cluster impact.

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

    PubMed

    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.

  12. TXNDC5, a newly discovered disulfide isomerase with a key role in cell physiology and pathology.

    PubMed

    Horna-Terrón, Elena; Pradilla-Dieste, Alberto; Sánchez-de-Diego, Cristina; Osada, Jesús

    2014-12-17

    Thioredoxin domain-containing 5 (TXNDC5) is a member of the protein disulfide isomerase family, acting as a chaperone of endoplasmic reticulum under not fully characterized conditions As a result, TXNDC5 interacts with many cell proteins, contributing to their proper folding and correct formation of disulfide bonds through its thioredoxin domains. Moreover, it can also work as an electron transfer reaction, recovering the functional isoform of other protein disulfide isomerases, replacing reduced glutathione in its role. Finally, it also acts as a cellular adapter, interacting with the N-terminal domain of adiponectin receptor. As can be inferred from all these functions, TXNDC5 plays an important role in cell physiology; therefore, dysregulation of its expression is associated with oxidative stress, cell ageing and a large range of pathologies such as arthritis, cancer, diabetes, neurodegenerative diseases, vitiligo and virus infections. Its implication in all these important diseases has made TXNDC5 a susceptible biomarker or even a potential pharmacological target.

  13. Experimental investigations of a partial Ru-O bond during the metal-ligand bifunctional addition in Noyori-type enantioselective ketone hydrogenation.

    PubMed

    Takebayashi, Satoshi; Dabral, Nupur; Miskolzie, Mark; Bergens, Steven H

    2011-06-29

    The transition state for the metal-ligand bifunctional addition step in Noyori's enantioselective ketone hydrogenation was investigated using intramolecular trapping experiments. The bifunctional addition between the Ru dihydride trans-[Ru((R)-BINAP)(H)(2)((R,R)-dpen)] and the hydroxy ketone 4-HOCH(2)C(6)H(4)(CO)CH(3) at -80 °C exclusively formed the corresponding secondary ruthenium alkoxide trans-[Ru((R)-BINAP)(H)(4-HOCH(2)C(6)H(4)CH(CH(3))O)((R,R)-dpen)]. Combined with the results of control experiments, this observation provides strong evidence for the formation of a partial Ru-O bond in the transition state.

  14. The disulfide oxidoreductase SdbA is active in Streptococcus gordonii using a single C-terminal cysteine of the CXXC motif.

    PubMed

    Davey, Lauren; Cohen, Alejandro; LeBlanc, Jason; Halperin, Scott A; Lee, Song F

    2016-01-01

    Recently, we identified a novel disulfide oxidoreductase, SdbA, in the oral bacterium Streptococcus gordonii. Disulfide oxidoreductases form disulfide bonds in nascent proteins using a CXXC catalytic motif. Typically, the N-terminal cysteine interacts with substrates, whereas the C-terminal cysteine is buried and only reacts with the first cysteine of the motif. In this study, we investigated the SdbA C(86) P(87) D(88) C(89) catalytic motif. In vitro, SdbA single cysteine variants at the N or C-terminal position (SdbAC86P and SdbAC89A ) were active but displayed different susceptibility to oxidation, and N-terminal cysteine was prone to sulfenylation. In S. gordonii, mutants with a single N-terminal cysteine were inactive and formed unstable disulfide adducts with other proteins. Activity was partially restored by inactivation of pyruvate oxidase, a hydrogen peroxide generator. Presence of the C-terminal cysteine alone (in the SdbAC86P variant) could complement the ΔsdbA mutant and restore disulfide bond formation in recombinant and natural protein substrates. These results provide evidence that certain disulfide oxidoreductases can catalyze disulfide bond formation using a single cysteine of the CXXC motif, including the buried C-terminal cysteine.

  15. GeoFold: Topology-based protein unfolding pathways capture the effects of engineered disulfides on kinetic stability

    PubMed Central

    Ramakrishnan, Vibin; Srinivasan, Sai Praveen; Salem, Saeed M; Matthews, Suzanne J; Colón, Wilfredo; Zaki, Mohammed; Bystroff, Christopher

    2011-01-01

    Protein unfolding is modeled as an ensemble of pathways, where each step in each pathway is the addition of one topologically possible conformational degree of freedom. Starting with a known protein structure, GeoFold hierarchically partitions (cuts) the native structure into substructures using revolute joints and translations. The energy of each cut and its activation barrier are calculated using buried solvent accessible surface area, side chain entropy, hydrogen bonding, buried cavities, and backbone degrees of freedom. A directed acyclic graph is constructed from the cuts, representing a network of simultaneous equilibria. Finite difference simulations on this graph simulate native unfolding pathways. Experimentally observed changes in the unfolding rates for disulfide mutants of barnase, T4 lysozyme, dihydrofolate reductase, and factor for inversion stimulation were qualitatively reproduced in these simulations. Detailed unfolding pathways for each case explain the effects of changes in the chain topology on the folding energy landscape. GeoFold is a useful tool for the inference of the effects of disulfide engineering on the energy landscape of protein unfolding. PMID:22189917

  16. Structure of Coenzyme A-Disulfide Reductase from Staphylococcus aureus at 1.54 Angstrom Resolution

    SciTech Connect

    Mallett,T.; Wallen, J.; Karplus, P.; Sakai, H.; Tsukihara, T.; Claiborne, A.

    2006-01-01

    Coenzyme A (CoASH) replaces glutathione as the major low molecular weight thiol in Staphylococcus aureus; it is maintained in the reduced state by coenzyme A-disulfide reductase (CoADR), a homodimeric enzyme similar to NADH peroxidase but containing a novel Cys43-SSCoA redox center. The crystal structure of S. aureus CoADR has been solved using multiwavelength anomalous dispersion data and refined at a resolution of 1.54 {angstrom}. The resulting electron density maps define the Cys43-SSCoA disulfide conformation, with Cys43-S{gamma} located at the flavin si face, 3.2 {angstrom} from FAD-C4aF, and the CoAS- moiety lying in an extended conformation within a cleft at the dimer interface. A well-ordered chloride ion is positioned adjacent to the Cys43-SSCoA disulfide and receives a hydrogen bond from Tyr361'-OH of the complementary subunit, suggesting a role for Tyr361' as an acid-base catalyst during the reduction of CoAS-disulfide. Tyr419'-OH is located 3.2 {angstrom} from Tyr361'-OH as well and, based on its conservation in known functional CoADRs, also appears to be important for activity. Identification of residues involved in recognition of the CoAS-disulfide substrate and in formation and stabilization of the Cys43-SSCoA redox center has allowed development of a CoAS-binding motif. Bioinformatics analyses indicate that CoADR enzymes are broadly distributed in both bacterial and archaeal kingdoms, suggesting an even broader significance for the CoASH/CoAS-disulfide redox system in prokaryotic thiol/disulfide homeostasis.

  17. Assignment of disulfide-linked peptides using automatic a1 ion recognition.

    PubMed

    Huang, Sheng Yu; Wen, Chien Hsien; Li, Ding Tzai; Hsu, Jue Liang; Chen, Chinpan; Shi, Fong Ku; Lin, Yueh Yi

    2008-12-01

    We present a novel approach for the assignment of peptides containing disulfide linkages. Dimethyl labeling is introduced to generate labeled peptides which exhibit enhanced a1 ion signals during MS/MS fragmentation. For disulfide-linked peptides, multiple a1 ions can be observed due to multiple N-termini. This distinct feature allows sieving out the disulfide-linked peptides; meanwhile, the N-terminal amino acids can be identified. With such information, the number of possible peptide combinations involved in a disulfide bond dramatically narrows down. Furthermore, we developed a computational algorithm to perform target a1 ion screening followed by molecular weight matching of cysteine-containing peptides with specific amino acids at the N-termini. Once the protein sequence and the peak list from a LC-MS/MS survey scan of labeled peptides are imported, the identities of disulfide-linked peptides can be readily obtained. The presented approach is simple and straightforward, offering a valuable tool for protein structural characterization.

  18. Transfer of molybdenum disulfide to various metals

    NASA Technical Reports Server (NTRS)

    Barton, G. C.; Pepper, S. V.

    1977-01-01

    Sliding friction experiments were conducted with molybdenum disulfide single crystals in contact with sputter cleaned surfaces of copper, nickel, gold, and 304 stainless steel. Transfer of the molybdenum disulfide to the metals was monitored with Auger electron spectroscopy. Results of the investigation indicate molybdenum disulfide transfers to all clean metal surfaces after a single pass over the metal surface with film thickness observed to increase with repeated passes over the same surfaces. Large particle transfer occurs when the orientation of the crystallites is other than basal. This is frequently accompanied by abrasion of the metal. Adhesion of molybdenum disulfide films occurred readily to copper and nickel, less readily to 304 stainless steel, and even less effectively to the gold, which indicates a chemical effect.

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

    SciTech Connect

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

    2012-04-27

    Highlights: Black-Right-Pointing-Pointer Disulfide-rich peptide aptamer inhibits IL-6-dependent cell proliferation. Black-Right-Pointing-Pointer Disulfide bond of peptide aptamer is essential for its affinity to IL-6R. Black-Right-Pointing-Pointer 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.

  20. Role of disulfide bridges in the activity and stability of a cold-active alpha-amylase.

    PubMed

    Siddiqui, Khawar Sohail; Poljak, Anne; Guilhaus, Michael; Feller, Georges; D'Amico, Salvino; Gerday, Charles; Cavicchioli, Ricardo

    2005-09-01

    The cold-adapted alpha-amylase from Pseudoalteromonas haloplanktis unfolds reversibly and cooperatively according to a two-state mechanism at 30 degrees C and unfolds reversibly and sequentially with two transitions at temperatures below 12 degrees C. To examine the role of the four disulfide bridges in activity and conformational stability of the enzyme, the eight cysteine residues were reduced with beta-mercaptoethanol or chemically modified using iodoacetamide or iodoacetic acid. Matrix-assisted laser desorption-time of flight mass spectrometry analysis confirmed that all of the cysteines were modified. The iodoacetamide-modified enzyme reversibly folded/unfolded and retained approximately one-third of its activity. Removal of all disulfide bonds resulted in stabilization of the least stable region of the enzyme (including the active site), with a concomitant decrease in activity (increase in activation enthalpy). Disulfide bond removal had a greater impact on enzyme activity than on stability (particularly the active-site region). The functional role of the disulfide bridges appears to be to prevent the active site from developing ionic interactions. Overall, the study demonstrated that none of the four disulfide bonds are important in stabilizing the native structure of enzyme, and instead, they appear to promote a localized destabilization to preserve activity.

  1. Respective contributions of polar vs enthalpy effects in the addition/fragmentation of mercaptobenzoxazole-derived thiyl radicals and analogues to double bonds.

    PubMed

    Lalevée, J; Allonas, X; Morlet-Savary, F; Fouassier, J P

    2006-10-19

    The formation and the reactivity of three selected sulfur-centered radicals formed from mercaptobenzoxazole, mercaptobenzimidazole, and mercaptobenzothiazole toward four double bonds (methyl acrylate, acrylonitrile, vinyl ether, and vinyl acetate) are investigated. The reversibility of the addition/fragmentation reaction in these widely used photoinitiating systems of radical polymerization was studied, for the first time, through the measurement of the corresponding rate constants by time-resolved laser spectroscopy. The combination of these results with quantum mechanical calculations clearly evidences that, contrary to previous studies on other aryl thiyl radicals, the addition rate constants (ka) are governed here by the polar effects associated with the very high electrophilic character of these radicals. However, interestingly, the back-fragmentation reaction (k-a) is mainly influenced by the enthalpy effects as supported by the relationship between the rate constants and the addition reaction enthalpy DeltaHR. The addition and fragmentation rate constants calculated from the transition state theory (TST) are in satisfactory agreement with the experimental ones. Therefore, molecular orbital (MO) calculations offered new opportunities for a better understanding of the sulfur-centered radical reactivity.

  2. Multiple disulfide bridges modulate conformational stability and flexibility in hyperthermophilic archaeal purine nucleoside phosphorylase.

    PubMed

    Bagarolo, Maria Libera; Porcelli, Marina; Martino, Elisa; Feller, Georges; Cacciapuoti, Giovanna

    2015-10-01

    5'-Deoxy-5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus is a hexameric hyperthermophilic protein containing in each subunit two pairs of disulfide bridges, a CXC motif, and one free cysteine. The contribution of each disulfide bridge to the protein conformational stability and flexibility has been assessed by comparing the thermal unfolding and the limited proteolysis of the wild-type enzyme and its variants obtained by site-directed mutagenesis of the seven cysteine residues. All variants catalyzed efficiently MTA cleavage with specific activity similar to the wild-type enzyme. The elimination of all cysteine residues caused a substantial decrease of ΔHcal (850 kcal/mol) and Tmax (39°C) with respect to the wild-type indicating that all cysteine pairs and especially the CXC motif significantly contribute to the enzyme thermal stability. Disulfide bond Cys200-Cys262 and the CXC motif weakly affected protein flexibility while the elimination of the disulfide bond Cys138-Cys205 lead to an increased protease susceptibility. Experimental evidence from limited proteolysis, differential scanning calorimetry, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions also allowed to propose a stabilizing role for the free Cys164.

  3. Binding of factor VIII to von willebrand factor is enabled by cleavage of the von Willebrand factor propeptide and enhanced by formation of disulfide-linked multimers.

    PubMed

    Bendetowicz, A V; Morris, J A; Wise, R J; Gilbert, G E; Kaufman, R J

    1998-07-15

    von Willebrand factor (vWF) is a multimeric adhesive glycoprotein with one factor VIII binding site/subunit. Prior reports suggest that posttranslational modifications of vWF, including formation of N-terminal intersubunit disulfide bonds and subsequent cleavage of the propeptide, influence availability and/or affinity of factor VIII binding sites. We found that deletion of the vWF propeptide produced a dimeric vWF molecule lacking N-terminal intersubunit disulfide bonds. This molecule bound fluorescein-labeled factor VIII with sixfold lower affinity than multimeric vWF in an equilibrium flow cytometry assay (approximate KDs, 5 nmol/L v 0.9 nmol/L). Coexpression of propeptide-deleted vWF with the vWF propeptide in trans yielded multimeric vWF that displayed increased affinity for factor VIII. Insertion of an alanine residue at the N-terminus of the mature vWF subunit destroyed binding to factor VIII, indicating that the native mature N-terminus is required for factor VIII binding. The requirement for vWF propeptide cleavage was shown by (1) a point mutation of the vWF propeptide cleavage site yielding pro-vWF that was defective in factor VIII binding and (2) correlation between efficiency of intracellular propeptide cleavage and factor VIII binding. Furthermore, in a cell-free system, addition of the propeptide-cleaving enzyme PACE/furin enabled factor VIII binding in parallel with propeptide cleavage. Our results indicate that high-affinity factor VIII binding sites are located on N-terminal disulfide-linked vWF subunits from which the propeptide has been cleaved.

  4. 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

    SciTech Connect

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

    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

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

    NASA Astrophysics Data System (ADS)

    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.

  6. Display of disulfide-rich proteins by complementary DNA display and disulfide shuffling assisted by protein disulfide isomerase.

    PubMed

    Naimuddin, Mohammed; Kubo, Tai

    2011-12-01

    We report an efficient system to produce and display properly folded disulfide-rich proteins facilitated by coupled complementary DNA (cDNA) display and protein disulfide isomerase-assisted folding. The results show that a neurotoxin protein containing four disulfide linkages can be displayed in the folded state. Furthermore, it can be refolded on a solid support that binds efficiently to its natural acetylcholine receptor. Probing the efficiency of the display proteins prepared by these methods provided up to 8-fold higher enrichment by the selective enrichment method compared with cDNA display alone, more than 10-fold higher binding to its receptor by the binding assays, and more than 10-fold higher affinities by affinity measurements. Cotranslational folding was found to have better efficiency than posttranslational refolding between the two investigated methods. We discuss the utilities of efficient display of such proteins in the preparation of superior quality proteins and protein libraries for directed evolution leading to ligand discovery.

  7. Effects of Interface Coating and Nitride Enhancing Additive on Properties of Hi-Nicalon SiC Fiber Reinforced Reaction-Bonded Silicon Nitride Composites

    NASA Technical Reports Server (NTRS)

    Bhatt, Ramakrishana T.; Hull, David R.; Eldridge, Jeffrey I.; Babuder, Raymond

    2000-01-01

    Strong and tough Hi-Nicalon SiC fiber reinforced reaction-bonded silicon nitride matrix composites (SiC/ RBSN) have been fabricated by the fiber lay-up approach. Commercially available uncoated and PBN, PBN/Si-rich PBN, and BN/SiC coated SiC Hi-Nicalon fiber tows were used as reinforcement. The composites contained approximately 24 vol % of aligned 14 micron diameter SiC fibers in a porous RBSN matrix. Both one- and two-dimensional composites were characterized. The effects of interface coating composition, and the nitridation enhancing additive, NiO, on the room temperature physical, tensile, and interfacial shear strength properties of SiC/RBSN matrix composites were evaluated. Results indicate that for all three coated fibers, the thickness of the coatings decreased from the outer periphery to the interior of the tows, and that from 10 to 30 percent of the fibers were not covered with the interface coating. In the uncoated regions, chemical reaction between the NiO additive and the SiC fiber occurs causing degradation of tensile properties of the composites. Among the three interface coating combinations investigated, the BN/SiC coated Hi-Nicalon SiC fiber reinforced RBSN matrix composite showed the least amount of uncoated regions and reasonably uniform interface coating thickness. The matrix cracking stress in SiC/RBSN composites was predicted using a fracture mechanics based crack bridging model.

  8. Collision-induced dissociation fragmentation inside disulfide C-terminal loops of natural non-tryptic peptides.

    PubMed

    Samgina, Tatiana Y; Vorontsov, Egor A; Gorshkov, Vladimir A; Artemenko, Konstantin A; Zubarev, Roman A; Ytterberg, Jimmy A; Lebedev, Albert T

    2013-07-01

    Collision-induced dissociation (CID) spectra of long non-tryptic peptides are usually quite complicated and rather difficult to interpret. Disulfide bond formed by two cysteine residues at C-terminus of frog skin peptides precludes one to determine sequence inside the forming loop. Thereby, chemical modification of S-S bonds is often used in "bottom up" sequencing approach. However, low-energy CID spectra of natural non-tryptic peptides with C-terminal disulfide cycle demonstrate an unusual fragmentation route, which may be used to elucidate the "hidden" C-terminal sequence. Low charge state protonated molecules experience peptide bond cleavage at the N-terminus of C-terminal cysteine. The forming isomeric acyclic ions serve as precursors for a series of b-type ions revealing sequence inside former disulfide cycle. The reaction is preferable for peptides with basic lysine residues inside the cycle. It may also be activated by acidic protons of Asp and Glu residues neighboring the loop. The observed cleavages may be quite competitive, revealing the sequence inside disulfide cycle, although S-S bond rupture does not occur in this case.

  9. Tris(3-hydroxypropyl)phosphine (THPP): A mild, air-stable reagent for the rapid, reductive cleavage of small-molecule disulfides.

    PubMed

    McNulty, James; Krishnamoorthy, Venkatesan; Amoroso, Dino; Moser, Michael

    2015-10-01

    Tris(3-hydroxypropyl)phosphine (THPP) is demonstrated to be a versatile, water-soluble and air-stable reducing agent, allowing for the rapid, irreversible reductive cleavage of disulfide bonds in both aqueous and buffered aqueous-organic media. The reagent shows exceptional stability at biological pH under which condition it permits the rapid reduction of a wide range of differentially functionalized small-molecule disulfides.

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

    PubMed

    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.

  11. Stabilization of HIV-1 gp120-CD4 Receptor Complex through Targeted Interchain Disulfide Exchange*

    PubMed Central

    Cerutti, Nichole; Mendelow, Barry V.; Napier, Grant B.; Papathanasopoulos, Maria A.; Killick, Mark; Khati, Makobetsa; Stevens, Wendy; Capovilla, Alexio

    2010-01-01

    HIV-1 enters cells via interaction between the trimeric envelope (Env) glycoprotein gp120/gp41 and the host cell surface receptor molecule CD4. The requirement of CD4 for viral entry has rationalized the development of recombinant CD4-based proteins as competitive viral attachment inhibitors and immunotherapeutic agents. In this study, we describe a novel recombinant CD4 protein designed to bind gp120 through a targeted disulfide-exchange mechanism. According to structural models of the gp120-CD4 receptor complex, substitution of Ser60 on the CD4 domain 1 α-helix with Cys positions a thiol in proximity of the gp120 V1/V2 loop disulfide (Cys126–Cys196), satisfying the stereochemical and geometric conditions for redox exchange between CD4 Cys60 and gp120 Cys126, and the consequent formation of an interchain disulfide bond. In this study, we provide experimental evidence for this effect by describing the expression, purification, refolding, receptor binding and antiviral activity analysis of a recombinant two-domain CD4 variant containing the S60C mutation (2dCD4-S60C). We show that 2dCD4-S60C binds HIV-1 gp120 with a significantly higher affinity than wild-type protein under conditions that facilitate disulfide exchange and that this translates into a corresponding increase in the efficacy of CD4-mediated viral entry inhibition. We propose that targeted redox exchange between conserved gp120 disulfides and nucleophilic moieties positioned strategically on CD4 (or CD4-like scaffolds) conceptualizes a new strategy in the development of high affinity HIV-1 Env ligands, with important implications for therapy and vaccine development. More generally, this chalcogen substitution approach provides a general means of stabilizing receptor-ligand complexes where the structural and biophysical conditions for disulfide exchange are satisfied. PMID:20538591

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

    PubMed

    Becker, Julien; Maes, Francis; Wehenkel, Louis

    2013-01-01

    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 [Formula: see text

  13. The disulfide relay of the intermembrane space of mitochondria: an oxygen-sensing system?

    PubMed

    Bihlmaier, Karl; Mesecke, Nikola; Kloeppel, Christine; Herrmann, Johannes M

    2008-12-01

    The intermembrane space of mitochondria contains many proteins that lack classical mitochondrial targeting sequences. Instead, these proteins often show characteristic patterns of cysteine residues that are critical for their accumulation in the organelle. Import of these proteins is catalyzed by two essential components, Mia40 and Erv1. Mia40 is a protein in the intermembrane space that directly binds newly imported proteins via disulfide bonds. By reorganization of these bonds, intramolecular disulfide bonds are formed in the imported proteins, which are thereby released from Mia40 into the intermembrane space. Because folded proteins are unable to traverse the import pore of the outer membrane, this leads to a permanent location of these proteins within the mitochondria. During this reaction, Mia40 becomes reduced and needs to be re-oxidized to regain its activity. Oxidation of Mia40 is carried out by Erv1, a conserved flavine adenine dinucleotide (FAD)-binding sulfhydryl oxidase. Erv1 directly interacts with Mia40 and shuttles electrons from reduced Mia40 to oxidized cytochrome c, from whence they flow through cytochrome oxidase to molecular oxygen. The connection of the disulfide relay with the respiratory chain not only significantly increases the efficiency of the oxidase activity, but also prevents the formation of potentially deleterious hydrogen peroxide. The oxidative activity of Erv1 strongly depends on the oxygen concentration in mitochondria. Erv1, therefore, may function as a molecular switch that adapts mitochondrial activities to the oxygen levels in the cell.

  14. Characterization of disulfide-linked heterodimers containing apolipoprotein D in human plasma lipoproteins.

    PubMed

    Blanco-Vaca, F; Via, D P; Yang, C Y; Massey, J B; Pownall, H J

    1992-12-01

    Human plasma apolipoprotein (apo) D is a glycoprotein with an apparent molecular weight of 29,000 M(r). It is present, mainly, in high density lipoproteins (HDL) and very high density lipoproteins (VHDL). Western blot analysis of HDL and VHDL using rabbit antibodies to human apoD revealed major immunoreactive bands at 29,000 and 38,000 M(r), with minor bands ranging from 50,000 to and 80,000 M(r). Only the 29,000 M(r) band corresponding to apoD remained when the electrophoresis was conducted under reducing conditions, demonstrating that apoD is cross-linked to other proteins via disulfide bonds. The broad pattern of immunoreactivity was also observed under nonreducing conditions when the blood was collected into a solution of sulfhydryl-trapping reagents, or when these reagents were added to the isolated lipoproteins. These results indicated that the disulfide bonds were not the result of disulfide exchange during the experimental procedures. On the basis of amino acid sequencing and reactions to antibodies, the 38,000 M(r) band was identified as an apoD-apoA-II heterodimer. The apoD-apoA-II was also demonstrated in plasma. In both HDL and plasma, the apoD-apoA-II heterodimer constituted the major form of apoD. Disulfide-linked heterodimers of apoD and apoB-100 were also found in low and very low density lipoproteins, and in whole plasma. It is concluded that a fraction of human apoD, like other cysteine-containing apolipoproteins, exists as a disulfide-linked heterodimer with other apolipoproteins in all major human lipoprotein fractions.

  15. Facile and Promising Method for Michael Addition of Indole and Pyrrole to Electron-Deficient trans-β-Nitroolefins Catalyzed by a Hydrogen Bond Donor Catalyst Feist's Acid and Preliminary Study of Antimicrobial Activity

    PubMed Central

    Al Majid, Abdullah M. A.; Islam, Mohammad Shahidul; Barakat, Assem; Al-Agamy, Mohamed H. M.; Naushad, Mu.

    2014-01-01

    The importance of cooperative hydrogen-bonding effects has been demonstrated using novel 3-methylenecyclopropane-1,2-dicarboxylic acid (Feist's acid (FA)) as hydrogen bond donor catalysts for the addition of indole and pyrrole to trans-β-nitrostyrene derivatives. Because of the hydrogen bond donor (HBD) ability, Feist's acid (FA) has been introduced as a new class of hydrogen bond donor catalysts for the activation of nitroolefin towards nucleophilic substitution reaction. It has effectively catalyzed the Michael addition of indoles and pyrrole to β-nitroolefins under optimum reaction condition to furnish the corresponding Michael adducts in good to excellent yields (up to 98%). The method is general, atom-economical, convenient, and eco-friendly and could provide excellent yields and regioselectivities. Some newly synthesized compounds were for examined in vitro antimicrobial activity and their preliminary results are reported. PMID:24574906

  16. Facile and promising method for michael addition of indole and pyrrole to electron-deficient trans-β-nitroolefins catalyzed by a hydrogen bond donor catalyst Feist's acid and preliminary study of antimicrobial activity.

    PubMed

    Al Majid, Abdullah M A; Islam, Mohammad Shahidul; Barakat, Assem; Al-Agamy, Mohamed H M; Naushad, Mu

    2014-01-01

    The importance of cooperative hydrogen-bonding effects has been demonstrated using novel 3-methylenecyclopropane-1,2-dicarboxylic acid (Feist's acid (FA)) as hydrogen bond donor catalysts for the addition of indole and pyrrole to trans-β-nitrostyrene derivatives. Because of the hydrogen bond donor (HBD) ability, Feist's acid (FA) has been introduced as a new class of hydrogen bond donor catalysts for the activation of nitroolefin towards nucleophilic substitution reaction. It has effectively catalyzed the Michael addition of indoles and pyrrole to β-nitroolefins under optimum reaction condition to furnish the corresponding Michael adducts in good to excellent yields (up to 98%). The method is general, atom-economical, convenient, and eco-friendly and could provide excellent yields and regioselectivities. Some newly synthesized compounds were for examined in vitro antimicrobial activity and their preliminary results are reported.

  17. Reversible methanol addition to copper Schiff base complexes: a kinetic, structural and spectroscopic study of reactions at azomethine C[double bond, length as m-dash]N bonds.

    PubMed

    Zhang, Wuyu; Saraei, Nina; Nie, Hanlin; Vaughn, John R; Jones, Alexis S; Mashuta, Mark S; Buchanan, Robert M; Grapperhaus, Craig A

    2016-10-12

    The reversible methanolysis of an azomethine C[double bond, length as m-dash]N in a series of copper(ii) Schiff base complexes has been investigated through combined spectroscopic, structural, and kinetic studies. Pentadentate copper(ii) complexes [L1-Cu(X)]Y (L1 = 1,2-bis[(1-methyl-2-imidazolyl)methyleneamino]ethane; X = Y = ClO4(-) (1); X = Y = TfO(-) (2); X = Y = BF4(-) (3); X = H2O, Y = (ClO4(-))2 (4) spontaneously add methanol in a ligand centered reaction to yield stable, isolable hemiaminal ether product complexes 5-8. In methanol free solution, 5-8 spontaneously release alcohol to regenerate 1-4. The methanol addition reaction is first-order in methanol and first-order in complex with second-order rate constants varying from 1.1 × 10(-4) to 187 × 10(-4) M(-1) s(-1) dependent on the donor ability of the axial ligand. Rate constants for methanol elimination vary from 0.67 to 3.7 × 10(-4) s(-1) with dependence on the counterion and water content of the solvent. Equilibrium constants for methanolysis range from 1.5 to 51 M(-1). Structural comparisons of the Schiff base complexes 1-4 and the hemiaminal ether complexes 5-8 suggest methanol addition is favored by the release of ligand strain associated with three planar five-membered chelates in 1-4.

  18. Addition of Si-H and B-H bonds and redox reactivity involving low-coordinate nitrido-vanadium complexes.

    PubMed

    Thompson, Rick; Tran, Ba L; Ghosh, Soumya; Chen, Chun-Hsing; Pink, Maren; Gao, Xinfeng; Carroll, Patrick J; Baik, Mu-Hyun; Mindiola, Daniel J

    2015-03-16

    In this study we enumerate the reactivity for two molecular vanadium nitrido complexes of [(nacnac)V≡N(X)] formulation [nacnac = (Ar)NC(Me)CHC(Me)(Ar)(-), Ar = 2,6-(CHMe2)2C6H3); X(-) = OAr (1) and N(4-Me-C6H4)2 (Ntolyl2) (2)]. Density functional theory calculations and reactivity studies indicate the nitride motif to have nucleophilic character, but where the nitrogen atom can serve as a conduit for electron transfer, thus allowing the reduction of the vanadium(V) metal ion with concurrent oxidation of the incoming substrate. Silane, H2SiPh2, readily converts the nitride ligand in 1 into a primary silyl-amide functionality with concomitant two-electron reduction at the vanadium center to form the complex [(nacnac)V{N(H)SiHPh2}(OAr)] (3). Likewise, addition of the B-H bond in pinacolborane to the nitride moiety in 2 results in formation of the boryl-amide complex [(nacnac)V{N(H)B(pinacol)}(Ntolyl2)] (4). In addition to spectroscopic data, complexes 3 and 4 were also elucidated structurally by single-crystal X-ray diffraction analysis. One-electron reduction of 1 with 0.5% Na/Hg on a preparative scale allowed for the isolation and structural determination of an asymmetric bimolecular nitride radical anion complex having formula [Na]2[(nacnac)V(N)(OAr)]2 (5), in addition to room-temperature solution X-band electron paramagnetic resonance spectroscopic studies.

  19. Solvation agent for disulfide precipitates from inhibited glycol-water solutions

    NASA Technical Reports Server (NTRS)

    Taylor, M. F.

    1971-01-01

    Small additions /0.01 percent or less/ of triethanoloamine sodium sulfite adduct to mercapto benzothiazole inhibited glycol water heat transfer solutions containing disulfide precipitate produce marked reduction in amount of precipitate. Adduct is useful as additive in glycol base antifreezes and coolants.

  20. [Introduction of additional thiol groups into glucoamylase in Aspergillus awamori and their effect on the thermal stability and catalytic activity of the enzyme].

    PubMed

    Surzhik, M A; Shmidt, A E; Glazunov, E A; Firsov, D L; Petukhov, M G

    2014-01-01

    Five mutant forms of glucoamylase (GA) from the filamentous fungus Aspergillus awamori with artificial disulfide bonds (4D-G137A\\A14C, 6D-A14C\\Y419C\\G137A, 10D-V13C\\G396C, 11D-V13C\\G396C\\A14C\\Y419C\\G137A, and 20D-G137A\\A246C\\A14C) were constructed using computer simulation and experimentally tested for thermostability. The introduction of two additional disulfide bonds between its first and thirteenth alpha-helices and that of the loop located close to a catalytic residue--E400--made it possible to assess the effects of disulfide bridges on protein thermostability. The mutant proteins with combined amino acid substitutions G137A\\A14C, V13C\\G396C\\A14C\\Y419C\\G137A, and G137A\\A246C\\A14C showed higher thermal stability as compared to the wild-type protein. At the same time, new disulfide bridges in the mutant A14C\\Y419C\\G137A and V13C\\G396C proteins led to the destabilization of their structure and the loss of thermal stability.

  1. Delicate balance of electrostatic interactions and disulfide bridges in thermostability of firefly luciferase.

    PubMed

    Karimzadeh, Somayeh; Moradi, Maryam; Hosseinkhani, Saman

    2012-12-01

    The wild type Photinus pyralis luciferase does not have any disulfide bridge. Disulfide bridges are determinant in inherent stability of protein at moderate temperatures. Meanwhile, arginin is responsible for thermostability at higher temperatures. In this study, by concomitant introduction of disulfide bridge and a surface arginin in a mutant (A296C-A326C/I232R), the contribution of disulfide bridge introduction and surface hydrophilic residue on activity and global stability of P. pyralis luciferase is investigated. In addition to the mentioned mutant; I232R, A296C-A326C and wild type luciferases are characterized. Though addition of Arg caused stability against proteolysis but in combination with disulfide bridge resulted in decreased thermal stability compared to A296C-A326C mutant. In spite of long distance of two different mutations (A296C-A326C and I232R) from each other in the three-dimensional structure, combination of their effects on the stability of luciferase was not cumulative.

  2. Protein-thiol substitution or protein dethiolation by thiol/disulfide exchange reactions: the albumin model.

    PubMed

    Summa, Domenico; Spiga, Ottavia; Bernini, Andrea; Venditti, Vincenzo; Priora, Raffaella; Frosali, Simona; Margaritis, Antonios; Di Giuseppe, Danila; Niccolai, Neri; Di Simplicio, Paolo

    2007-11-01

    Dethiolation experiments of thiolated albumin with thionitrobenzoic acid and thiols (glutathione, cysteine, homocysteine) were carried out to understand the role of albumin in plasma distribution of thiols and disulfide species by thiol/disulfide (SH/SS) exchange reactions. During these experiments we observed that thiolated albumin underwent thiol substitution (Alb-SS-X+RSH<-->Alb-SS-R+XSH) or dethiolation (Alb-SS-X+XSH<-->Alb-SH+XSSX), depending on the different pK(a) values of thiols involved in protein-thiol mixed disulfides (Alb-SS-X). It appeared in these reactions that the compound with lower pK(a) in mixed disulfide was a good leaving group and that the pK(a) differences dictated the kind of reaction (substitution or dethiolation). Thionitrobenzoic acid, bound to albumin by mixed disulfide (Alb-TNB), underwent rapid substitution after thiol addition, forming the corresponding Alb-SS-X (peaks at 0.25-1 min). In turn, Alb-SS-X were dethiolated by the excess nonprotein SH groups because of the lower pK(a) value in mixed disulfide with respect to that of other thiols. Dethiolation of Alb-SS-X was accompanied by formation of XSSX and Alb-SH up to equilibrium levels at 35 min, which were different for each thiol. Structures by molecular simulation of thiolated albumin, carried out for understanding the role of sulfur exposure in mixed disulfides in dethiolation process, evidenced that the sulfur exposure is important for the rate but not for determining the kind of reaction (substitution or dethiolation). Our data underline the contribution of SH/SS exchanges to determine levels of various thiols as reduced and oxidized species in human plasma.

  3. Reactions of the alkoxy radicals formed following OH-addition to alpha-pinene and beta-pinene. C-C bond scission reactions.

    PubMed

    Dibble, T S

    2001-05-09

    The atmospheric degradation pathways of the atmospherically important terpenes alpha-pinene and beta-pinene are studied using density functional theory. We employ the correlation functional of Lee, Yang, and Parr and the three-parameter HF exchange functional of Becke (B3LYP) together with the 6-31G(d) basis set. The C-C bond scission reactions of the beta-hydroxyalkoxy radicals that are formed after OH addition to alpha-pinene and beta-pinene are investigated. Both of the alkoxy radicals formed from the alpha-pinene-OH adduct possess a single favored C-C scission pathway with an extremely low barrier (approximately 3 kcal/mol) leading to the formation of pinonaldehyde. Neither of these pathways produces formaldehyde, and preliminary computational results offer some support for suggestions that 1,5 or 1,6 H-shift (isomerization) reactions of alkoxy radicals contribute to formaldehyde production. In the case of the alkoxy radical formed following OH addition to the methylene group of beta-pinene, there exists two C-C scission reactions with nearly identical barrier heights (approximately 7.5 kcal/mol); one leads to known products (nopinone and formaldehyde) but the ultimate products of the competing reaction are unknown. The single C-C scission pathway of the other alkoxy radical from beta-pinene possesses a very low (approximately 4 kcal/mol) barrier. The kinetically favored C-C scission reactions of all four alkoxy radicals appear to be far faster than expected rates of reaction with O2. The rearrangement of the alpha-pinene-OH adduct, a key step in the proposed mechanism of formation of acetone from alpha-pinene, is determined to possess a barrier of 11.6 kcal/mol. This value is consistent with another computational result and is broadly consistent with the modest acetone yields observed in product yield studies.

  4. Linker Immolation Determines Cell Killing Activity of Disulfide-Linked Pyrrolobenzodiazepine Antibody-Drug Conjugates.

    PubMed

    Zhang, Donglu; Pillow, Thomas H; Ma, Yong; Cruz-Chuh, Josefa Dela; Kozak, Katherine R; Sadowsky, Jack D; Lewis Phillips, Gail D; Guo, Jun; Darwish, Martine; Fan, Peter; Chen, Jingtian; He, Changrong; Wang, Tao; Yao, Hui; Xu, Zijin; Chen, Jinhua; Wai, John; Pei, Zhonghua; Hop, Cornelis E C A; Khojasteh, S Cyrus; Dragovich, Peter S

    2016-11-10

    Disulfide bonds could be valuable linkers for a variety of therapeutic applications requiring tunable cleavage between two parts of a molecule (e.g., antibody-drug conjugates). The in vitro linker immolation of β-mercaptoethyl-carbamate disulfides and DNA alkylation properties of associated payloads were investigated to understand the determinant of cell killing potency of anti-CD22 linked pyrrolobenzodiazepine (PBD-dimer) conjugates. Efficient immolation and release of a PBD-dimer with strong DNA alkylation properties were observed following disulfide cleavage of methyl- and cyclobutyl-substituted disulfide linkers. However, the analogous cyclopropyl-containing linker did not immolate, and the associated thiol-containing product was a poor DNA alkylator. As predicted from these in vitro assessments, the related anti-CD22 ADCs showed different target-dependent cell killing activities in WSU-DLCL2 and BJAB cell lines. These results demonstrate how the in vitro immolation models can be used to help design efficacious ADCs.

  5. Thermochemical Properties and Bond Dissociation Energies for Fluorinated Methanol, CH3-xFxOH, and Fluorinated Methyl Hydroperoxides, CH3-xFxOOH: Group Additivity.

    PubMed

    Wang, Heng; Bozzelli, Joseph W

    2016-09-08

    Oxygenated fluorocarbons are routinely found in sampling of environmental soils and waters as a result of the widespread use of fluoro and chlorofluoro carbons as heat transfer fluids, inert materials, polymers, fire retardants and solvents; the influence of these chemicals on the environment is a growing concern. The thermochemical properties of these species are needed for understanding their stability and reactions in the environment and in thermal process. Structures and thermochemical properties on the mono- to trifluoromethanol, CH3-xFxOH, and fluoromethyl hydroperoxide, CH3-xFxOOH (1 ≤ x ≤ 3), are determined by CBS-QB3, CBS-APNO, and G4 calculations. Entropy, S°298, and heat capacities, Cp(T)'s (300 ≤ T/K ≤ 1500) from vibration, translation, and external rotation contributions are calculated on the basis of the vibration frequencies and structures obtained from the B3LYP/6-31+G(d,p) density functional method. Potential barriers for the internal rotations are also calculated from this method and used to calculate hindered rotor contributions to S°298 and Cp(T)'s using direct integration over energy levels of the internal rotational potentials. Standard enthalpies of formation, ΔfH°298 (units in kcal mol(-1)) are CH2FOOH (-83.7), CHF2OOH (-138.1), CF3OOH (-193.6), CH2FOO(•) (-44.9), CHF2OO(•) (-99.6), CF3OO(•) (-153.8), CH2FOH (-101.9), CHF2OH (-161.6), CF3OH (-218.1), CH2FO(•) (-49.1), CHF2O(•) (-97.8), CF3O(•) (-150.5), CH2F(•) (-7.6), CHF2(•) (-58.8), and CF3(•) (-112.6). Bond dissociation energies for the R-OOH, RO-OH, ROO-H, R-OO(•), RO-O(•), R-OH, RO-H, R-O(•), and R-H bonds are determined and compared with methyl hydroperoxide to observe the trends from added fluoro substitutions. Enthalpy of formation for the fluoro-hydrocarbon oxygen groups C/F/H2/O, C/F2/H/O, C/F3/O, are derived from the above fluorinated methanol and fluorinated hydroperoxide species for use in Benson's Group Additivity. It was determined that

  6. 46 CFR 153.520 - Special requirements for carbon disulfide.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 5 2012-10-01 2012-10-01 false Special requirements for carbon disulfide. 153.520... Equipment Special Requirements § 153.520 Special requirements for carbon disulfide. A containment system carrying carbon disulfide must meet the following: (a) Each cargo pump must be of the intank type...

  7. 40 CFR 180.467 - Carbon disulfide; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Carbon disulfide; tolerances for... § 180.467 Carbon disulfide; tolerances for residues. Tolerances are established for the nematicide, insecticide, and fungicide carbon disulfide, from the application of sodium tetrathiocarbonate, in or on...

  8. 40 CFR 180.467 - Carbon disulfide; tolerances for residues.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Carbon disulfide; tolerances for... § 180.467 Carbon disulfide; tolerances for residues. Tolerances are established for the nematicide, insecticide, and fungicide carbon disulfide, from the application of sodium tetrathiocarbonate, in or on...

  9. 46 CFR 153.520 - Special requirements for carbon disulfide.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 5 2014-10-01 2014-10-01 false Special requirements for carbon disulfide. 153.520... Equipment Special Requirements § 153.520 Special requirements for carbon disulfide. A containment system carrying carbon disulfide must meet the following: (a) Each cargo pump must be of the intank type...

  10. 40 CFR 180.467 - Carbon disulfide; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Carbon disulfide; tolerances for... § 180.467 Carbon disulfide; tolerances for residues. Tolerances are established for the nematicide, insecticide, and fungicide carbon disulfide, from the application of sodium tetrathiocarbonate, in or on...

  11. 40 CFR 180.467 - Carbon disulfide; tolerances for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Carbon disulfide; tolerances for... § 180.467 Carbon disulfide; tolerances for residues. Tolerances are established for the nematicide, insecticide, and fungicide carbon disulfide, from the application of sodium tetrathiocarbonate, in or on...

  12. 40 CFR 180.467 - Carbon disulfide; tolerances for residues.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Carbon disulfide; tolerances for... § 180.467 Carbon disulfide; tolerances for residues. Tolerances are established for the nematicide, insecticide, and fungicide carbon disulfide, from the application of sodium tetrathiocarbonate, in or on...

  13. 46 CFR 153.520 - Special requirements for carbon disulfide.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 5 2011-10-01 2011-10-01 false Special requirements for carbon disulfide. 153.520... Equipment Special Requirements § 153.520 Special requirements for carbon disulfide. A containment system carrying carbon disulfide must meet the following: (a) Each cargo pump must be of the intank type...

  14. 46 CFR 153.520 - Special requirements for carbon disulfide.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 5 2013-10-01 2013-10-01 false Special requirements for carbon disulfide. 153.520... Equipment Special Requirements § 153.520 Special requirements for carbon disulfide. A containment system carrying carbon disulfide must meet the following: (a) Each cargo pump must be of the intank type...

  15. 21 CFR 524.2101 - Selenium disulfide suspension.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Selenium disulfide suspension. 524.2101 Section... § 524.2101 Selenium disulfide suspension. (a) Specifications. The product contains 0.9-percent weight in weight (w/w) selenium disulfide (1-percent weight in volume (w/v)). (b) Sponsors. See Nos. 000061,...

  16. 21 CFR 524.2101 - Selenium disulfide suspension.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Selenium disulfide suspension. 524.2101 Section... § 524.2101 Selenium disulfide suspension. (a) Specifications. The product contains 0.9-percent weight in weight (w/w) selenium disulfide (1-percent weight in volume (w/v)). (b) Sponsors. See Nos. 000061,...

  17. 21 CFR 524.2101 - Selenium disulfide suspension.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Selenium disulfide suspension. 524.2101 Section... § 524.2101 Selenium disulfide suspension. (a) Specifications. The product contains 0.9-percent weight in weight (w/w) selenium disulfide (1-percent weight in volume (w/v)). (b) Sponsors. See Nos. 000061,...

  18. 21 CFR 524.2101 - Selenium disulfide suspension.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Selenium disulfide suspension. 524.2101 Section... § 524.2101 Selenium disulfide suspension. (a) Specifications. The product contains 0.9-percent weight in weight (w/w) selenium disulfide (1-percent weight in volume (w/v)). (b) Sponsors. See Nos. 000061,...

  19. 21 CFR 524.2101 - Selenium disulfide suspension.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Selenium disulfide suspension. 524.2101 Section... § 524.2101 Selenium disulfide suspension. (a) Specifications. The product contains 0.9-percent weight in weight (w/w) selenium disulfide (1-percent weight in volume (w/v)). (b) Sponsors. See Nos. 000061,...

  20. Dehydrofluorination of Hydrofluorocarbons by Titanium Alkylidynes via Sequential C-H/C-F Bond Activation Reactions. A Synthetic, Structural, and Mechanistic Study of 1,2-CH Bond Addition and [beta]-Fluoride Elimination

    SciTech Connect

    Fout, A.R.; Scott, J.; Miller, D.L.; Bailey, B.C.; Pink, M.; Mindiola, D.J.

    2009-01-07

    The neopentylidene-neopentyl complex (PNP)Ti=CH{sup t}Bu(CH{sub 2}{sup t}Bu) (1); (PNP{sup -} = N[2-P(CHMe{sub 2}){sub 2}-4-methylphenyl]{sub 2}) extrudes neopentane in neat fluorobenzene under mild conditions (25 C) to generate the transient titanium alkylidyne (PNP)Ti-C{sup t}Bu (A), which subsequently undergoes regioselective 1,2-CH bond addition of a fluorobenzene across the Ti-C linkage to generate (PNP)Ti=CH{sup t}Bu(o-FC{sub 6}H{sub 4}) (2). Kinetic and mechanistic studies suggest that the C-H activation process is pseudo-first-order in titanium, with the {alpha}-hydrogen abstraction being the rate-determining step and the post-rate-determining step being the C-H bond activation of fluorobenzene. At 100 C complex 2 does not equilibrate back to A and the preference for C-H activation in benzene versus fluorobenzene is 2:3, respectively. Compound 1 also reacts readily, and in most cases cleanly, with a series of hydrofluoroarenes (HAr{sub F}), to form a family of alkylidene-arylfluoride derivatives of the type (PNP)Ti=CH{sup t}Bu(Ar{sub F}). Thermolysis of the latter compounds generates the titanium alkylidene-fluoride (PNP)Ti=CH{sup t}Bu(F) (14) by a {beta}-fluoride elimination, concurrent with formation of o-benzyne. {beta}-Fluoride elimination to yield 14 occurs from 2 under elevated temperatures with k{sub average} = 4.96(16) x 10{sup -5} s{sup -1} and with activation parameters {Delta}H{sub {-+}} = 29(1) kcal/mol and {Delta}S{sub {-+}} = -3(4) cal/mol {center_dot}K. It was found that {beta}-fluoride elimination is accelerated when electron-rich groups are adjacent to the fluoride group, thus implying that a positive charge buildup at the arylfluoride ring occurs in the activated complex of 2. The alkylidene derivative (PNP)Ti=CHSiMe{sub 3}(CH{sub 2}SiMe{sub 3}) (15) also undergoes {alpha}-hydrogen abstraction to form the putative (PNP)Ti'-CSiMe{sub 3} (B) at higher temperatures (>70 C) and dehydrofluorinates the same series of HArF when the reaction

  1. ``Additive'' cooperativity of hydrogen bonds in complexes of catechol with proton acceptors in the gas phase: FTIR spectroscopy and quantum chemical calculations

    NASA Astrophysics Data System (ADS)

    Varfolomeev, Mikhail A.; Klimovitskii, Alexander E.; Abaidullina, Dilyara I.; Madzhidov, Timur I.; Solomonov, Boris N.

    2012-06-01

    Experimental study of hydrogen bond cooperativity in hetero-complexes in the gas phase was carried out by IR-spectroscopy method. Stretching vibration frequencies of Osbnd H groups in phenol and catechol molecules as well as of their complexes with nitriles and ethers were determined in the gas phase using a specially designed cell. Osbnd H groups experimental frequency shifts in the complexes of catechol induced by the formation of intermolecular hydrogen bonds are significantly higher than in the complexes of phenol due to the hydrogen bond cooperativity. It was shown that the cooperativity factors of hydrogen bonds in the complexes of catechol with nitriles and ethers in the gas phase are approximately the same. Quantum chemical calculations of the studied systems have been performed using density functional theory (DFT) methods. It was shown, that theoretically obtained cooperativity factors of hydrogen bonds in the complexes of catechol with proton acceptors are in good agreement with experimental values. Cooperative effects lead to a strengthening of intermolecular hydrogen bonds in the complexes of catechol on about 30%, despite the significant difference in the proton acceptor ability of the bases. The analysis within quantum theory of atoms in molecules was carried out for the explanation of this fact.

  2. "Additive" cooperativity of hydrogen bonds in complexes of catechol with proton acceptors in the gas phase: FTIR spectroscopy and quantum chemical calculations.

    PubMed

    Varfolomeev, Mikhail A; Klimovitskii, Alexander E; Abaidullina, Dilyara I; Madzhidov, Timur I; Solomonov, Boris N

    2012-06-01

    Experimental study of hydrogen bond cooperativity in hetero-complexes in the gas phase was carried out by IR-spectroscopy method. Stretching vibration frequencies of O-H groups in phenol and catechol molecules as well as of their complexes with nitriles and ethers were determined in the gas phase using a specially designed cell. O-H groups experimental frequency shifts in the complexes of catechol induced by the formation of intermolecular hydrogen bonds are significantly higher than in the complexes of phenol due to the hydrogen bond cooperativity. It was shown that the cooperativity factors of hydrogen bonds in the complexes of catechol with nitriles and ethers in the gas phase are approximately the same. Quantum chemical calculations of the studied systems have been performed using density functional theory (DFT) methods. It was shown, that theoretically obtained cooperativity factors of hydrogen bonds in the complexes of catechol with proton acceptors are in good agreement with experimental values. Cooperative effects lead to a strengthening of intermolecular hydrogen bonds in the complexes of catechol on about 30%, despite the significant difference in the proton acceptor ability of the bases. The analysis within quantum theory of atoms in molecules was carried out for the explanation of this fact.

  3. Oxidative addition of Pd to C-H, C-C and C-Cl bonds: Importance of relativistic effects in DFT calculations

    NASA Astrophysics Data System (ADS)

    Diefenbach, Axel; Bickelhaupt, F. Matthias

    2001-09-01

    To assess the importance of relativistic effects for the quantum chemical description of oxidative addition reactions of palladium to C-H, C-C and C-Cl bonds, we have carried out a systematic study of the corresponding reactions of CH4, C2H6 and CH3Cl with Pd-d10 using nonrelativistic (NR), quasirelativistic (QR), and zeroth-order regularly approximated (ZORA) relativistic density functional theory (DFT) at the BP86/TZ(2)P level. Relativistic effects are important according to both QR and ZORA, the former yielding similar but somewhat more pronounced effects than the latter, more reliable method: activation barriers are reduced by 6-14 kcal/mol and reaction enthalpies become 15-20 kcal/mol more exothermic if one goes from NR to ZORA. This yields, for example, 298 K activation enthalpies ΔH298≠ of -5.0 (C-H), 9.6 (C-C) and -6.0 kcal/mol (C-Cl) relative to the separate reactants at ZORA-BP86/TZ(2)P. In accordance with gas-phase experiments on reactions of Pd with alkanes, we find reaction profiles with pronounced potential wells for reactant complexes (collisionally stabilized and observed in experiments for alkanes larger than CH4) at -11.4 (CH4), -11.6 (C2H6) and -15.6 kcal/mol (CH3Cl) relative to separated reactants [ZORA-BP86/TZ(2)P]. Furthermore, we analyze the height of and the relativistic effects on the activation energies ΔE≠ in terms of the activation strain ΔEstrain≠ of and the transition-state interaction ΔEint≠ between the reactants in the activated complex, with ΔE≠=ΔEstrain≠+ΔEint≠.

  4. Diaryl Disulfides as Novel Stabilizers of Tumor Suppressor Pdcd4

    PubMed Central

    Schmid, Tobias; Blees, Johanna S.; Bajer, Magdalena M.; Wild, Janine; Pescatori, Luca; Cuzzucoli Crucitti, Giuliana; Scipione, Luigi; Costi, Roberta; Henrich, Curtis J.; Brüne, Bernhard; Colburn, Nancy H.; Di Santo, Roberto

    2016-01-01

    The translation inhibitor and tumor suppressor Pdcd4 was reported to be lost in various tumors and put forward as prognostic marker in tumorigenesis. Decreased Pdcd4 protein stability due to PI3K-mTOR-p70S6K1 dependent phosphorylation of Pdcd4 followed by β-TrCP1-mediated ubiquitination, and proteasomal destruction of the protein was characterized as a major mechanism contributing to the loss of Pdcd4 expression in tumors. In an attempt to identify stabilizers of Pdcd4, we used a luciferase-based high-throughput compatible cellular assay to monitor phosphorylation-dependent proteasomal degradation of Pdcd4 in response to mitogen stimulation. Following a screen of approximately 2000 compounds, we identified 1,2-bis(4-chlorophenyl)disulfide as a novel Pdcd4 stabilizer. To determine an initial structure-activity relationship, we used 3 additional compounds, synthesized according to previous reports, and 2 commercially available compounds for further testing, in which either the linker between the aryls was modified (compounds 2–4) or the chlorine residues were replaced by groups with different electronic properties (compounds 5 and 6). We observed that those compounds with alterations in the sulfide linker completely lost the Pdcd4 stabilizing potential. In contrast, modifications in the chlorine residues showed only minor effects on the Pdcd4 stabilizing activity. A reporter with a mutated phospho-degron verified the specificity of the compounds for stabilizing the Pdcd4 reporter. Interestingly, the active diaryl disulfides inhibited proliferation and viability at concentrations where they stabilized Pdcd4, suggesting that Pdcd4 stabilization might contribute to the anti-proliferative properties. Finally, computational modelling indicated that the flexibility of the disulfide linker might be necessary to exert the biological functions of the compounds, as the inactive compound appeared to be energetically more restricted. PMID:26982744

  5. Orthogonal halogen and hydrogen bonds involving a peptide bond model† †Electronic supplementary information (ESI) available: Experimental part, DSC, IR spectroscopic and crystallographic data. CCDC 899779–899785. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c4ce01514b Click here for additional data file. Click here for additional data file.

    PubMed Central

    Vasylyeva, Vera; Nayak, Susanta K.; Cavallo, Gabriella; Resnati, Giuseppe

    2014-01-01

    The peptide bond model N-methylacetamide self-assembles with a range of dihalotetrafluorobenzenes forming co-crystals that all show the occurrence of orthogonal hydrogen and halogen bonds. PMID:25663816

  6. Dual-degradable disulfide-containing PEI-Pluronic/DNA polyplexes: transfection efficiency and balancing protection and DNA release.

    PubMed

    Zhang, Lifen; Chen, Zhenzhen; Li, Yanfeng

    2013-01-01

    Polymeric gene-delivery vectors to achieve lack of toxicity and a balance between protection and DNA release remains a formidable challenge. Incorporating intracellular environment-responsive degradable bonds is an appreciable step toward developing safer transfection agents. In this study, novel, dual-degradable polycation copolymers (Pluronic-diacrylate [PA]-polyethyleneimine [PEI]-SS) were synthesized through the addition of low molecular weight (800 Da) PEI cross-linked with SS (PEI-SS) to PA. Three PA-PEI-SS copolymers (PA-PEI-SS1, 2, and 3) with different PEI-SS to Pluronic molar ratios were investigated and found to strongly condense plasmid DNA into positively charged nanoparticles with an average particle size of approximately 200 nm and to possess higher stability against DNase I digestion and sodium heparin. Disulfide and ester bonds of the copolymers were susceptible to intracellular redox conditions. In vitro experiments demonstrated that the PA-PEI-SS copolymers had significantly lower cytotoxicity and higher transfection efficiency in both BGC-823 and 293T cell lines than the controls of degradable PEI-SS and nondegradable 25 kDa PEI. Transfection activity was influenced by the PEI-SS content in the polymers and PA-PEI-SS1 showed the highest efficiency of the three copolymers. These studies suggest that these dual-degradable copolymers could be used as potential biocompatible gene delivery carriers.

  7. SO2−· Electron Transfer Ion/Ion Reactions with Disulfide Linked Polypeptide Ions

    PubMed Central

    Chrisman, Paul A.; Pitteri, Sharon J.; Hogan, Jason M.; McLuckey, Scott A.

    2005-01-01

    Multiply-charged peptide cations comprised of two polypeptide chains (designated A and B) bound via a disulfide linkage have been reacted with SO2−· in an electrodynamic ion trap mass spectrometer. These reactions proceed through both proton transfer (without dissociation) and electron transfer (with and without dissociation). Electron transfer reactions are shown to give rise to cleavage along the peptide backbone, loss of neutral molecules, and cleavage of the cystine bond. Disulfide bond cleavage is the preferred dissociation channel and both Chain A (or B)—S· and Chain A (or B)—SH fragment ions are observed, similar to those observed with electron capture dissociation (ECD) of disulfide-bound peptides. Electron transfer without dissociation produces [M + 2H]+· ions, which appear to be less kinetically stable than the proton transfer [M + H]+ product. When subjected to collision-induced dissociation (CID), the [M + 2H]+· ions fragment to give products that were also observed as dissociation products during the electron transfer reaction. However, not all dissociation channels noted in the electron transfer reaction were observed in the CID of the [M + 2H]+· ions. The charge state of the peptide has a significant effect on both the extent of electron transfer dissociation observed and the variety of dissociation products, with higher charge states giving more of each. PMID:15914021

  8. Mapping the accessibility of the disulfide crosslink network in the wool fiber cortex.

    PubMed

    Deb-Choudhury, Santanu; Plowman, Jeffrey E; Rao, Kelsey; Lee, Erin; van Koten, Chikako; Clerens, Stefan; Dyer, Jolon M; Harland, Duane P

    2015-02-01

    The disulfide bond network within the cortex of mammalian hair has a critical influence on the physical and mechanical characteristics of the fiber. The location, pattern, and accessibility of free and crosslinked cysteines underpin the properties of this network, but have been very difficult to map and understand, because traditional protein extraction techniques require the disruption of these disulfide bonds. Cysteine accessibility in both trichocyte keratins and keratin associated proteins (KAPs) of wool was investigated using staged labeling, where reductants and chaotropic agents were used to expose cysteines in a stepwise fashion according to their accessibility. Cysteines thus exposed were labeled with distinguishable alkylation agents. Proteomic profiling was used to map peptide modifications and thereby explore the role of KAPs in crosslinking keratins. Labeled cysteines from KAPs were detected when wool was extracted with reductant only. Among them were sequences from the end domains of KAPs, indicating that those cysteines were easily accessible in the fiber and could be involved in forming interdisulfide linkages with keratins or with other KAPs. Some of the identified peptides were from the rod domains of Types I and II keratins, with their cysteines positioned on the exposed surface of the α-helix. Peptides were also identified from keratin head and tail domains, demonstrating that they are not buried within the filament structure and, hence, have a possible role in forming disulfide linkages. From this study, a deeper understanding of the accessibility and potential reactivity of cysteine residues in the wool fiber cortex was obtained.

  9. The intracellular delivery of plasmid DNA using cationic reducible carbon nanotube - Disulfide conjugates of polyethylenimine.

    PubMed

    Nia, Azadeh Hashem; Eshghi, Hossein; Abnous, Kalil; Ramezani, Mohammad

    2017-03-30

    A series of polyethylenimine conjugates of single-walled carbon nanotube (PEI-SWNT) containing bioreducible disulfide bonds was synthesized and evaluated for their transfection efficiency. Different molecular weights of polyethylenimine (PEI) were thiolated with different mole ratio of 2-iminothiolane (2-IT). Single-walled carbon nanotube (SWNT) was first carboxylated and then three different cysteine-functionalized SWNT formulations were synthesized via introduced linkers: a) carbonyl group b) spermidine c) 1,8-diamino 3,6-dioxo octane. The final nanocarriers were fabricated upon conjugation of thiolated PEIs and thiolated SWNT via oxidative disulfide bond formation. All PEI-disulfide-SWNT conjugates were capable of DNA condensation and showed improved viability and transfection efficiency compared to PEI itself. Transfection efficiencies were up to 1500 times greater than PEI 25kDa (C/P=0.8). The results of this study suggest that the synthesized formulations based on SWNT-CO-Cysteine and PEI 1.8kDa were the most efficient carriers. Considering the decreased cytotoxicity and higher transfection levels, the conjugates bear the potential for effective delivery of genetic materials.

  10. Disulfide isomers of alpha-neurotoxins from King cobra (Ophiophagus hannah) venom.

    PubMed

    Lin, S R; Chang, L S; Chang, C C

    1999-01-08

    Two novel alpha-neurotoxins, Oh-6A and Oh-6B, isolated from the king cobra (Ophiophagus hannah) venom, consist of 70 amino acid residues with 10 cysteine residues and share the same amino acid sequences as determined by Edman degradation on the peptide fragments generated from the proteolytic hydrolysates. Their sequences share 46-53% homology with Oh-4, Oh-5, Toxin a, and Toxin b from the same venom. The finding that Oh-6A and Oh-6B had different retention times in the reversed-phase column suggested that the two toxin molecules should not have the same conformation. Selective reduction on the disulfide bond, Cys26--Cys30, at the tip of their loop II structures resulted in the production of the partially reduced derivatives eluted at the same position. Under redox conditions, the partially reduced Oh-6A and 6B exclusively converted into native Oh-6A as evidenced by HPLC analyses. This suggests that Oh-6A and Oh-6B are disulfide isomers which probably arise from cis-trans isomerization of the Cys26--Cys30 disulfide bond. Alternatively, the two toxins exhibited binding activity toward nAChR and lethal toxicity equally. It reflects that the diversity around the extra loop at the loop II structure does not exert a significant effect on the manifestation of the neurotoxicity of Oh-6A and Oh-6B.

  11. Catalytic hydrofunctionalization of alkynes through P-H bond addition: the unique role of orientation and properties of the phosphorus group in the insertion step.

    PubMed

    Ananikov, Valentine P; Makarov, Anton V; Beletskaya, Irina P

    2011-11-04

    The puzzling question of alkyne insertion into Pd-P and Pd-H bonds leading to the formation of new Pd-C, C-P, and C-H bonds was explored by theoretical calculations at the CCSD(T) and B3LYP levels of theory. The key factors responsible for selectivity of catalytic hydrofunctionalization of alkynes were resolved and studied in details for the models of hydrophosphorylation, hydrophosphinylation, and hydrophospination reactions. In contrast with the generally accepted mechanistic picture, the calculations have shown that several pathways are possible depending on the nature and geometrical arrangement of the phosphorus group. It was found that the product of alkyne insertion into the metal-hydrogen bond should be easily formed under kinetic-control conditions, while the product of alkyne insertion into the metal-phosphorus bond may be formed in certain cases under thermodynamic control. For the first time, the calculations have revealed the role of the oxygen atom in the reactivity of P=P(O)R(2) groups and the role of the interactions involving the lone pair of the P=PR(2) group in the reagent. The fundamental properties of the Pd-P, C-P, and P-H bonds were reported, and the larger bond strength upon increasing the number of oxygen atoms bound to phosphorus (P=PR(2), P(O)R(2), and P(O)(OR)(2)) have been shown. The relationship between bond energy, acidity, and reactivity of the studied phosphorus compounds has been determined.

  12. The compromise of dynamic disulfide/thiol homeostasis as a biomarker of oxidative stress in trichloroethylene exposure.

    PubMed

    Bal, C; Büyükşekerci, M; Koca, C; Ağış, E R; Erdoğan, S; Baran, P; Gündüzöz, M; Yilmaz, Öh

    2016-09-01

    In this study, we aimed to investigate disulfide/thiol homeostasis in trichloroethylene (TCE) exposure. The study was carried out in 30 nonsmoker TCE-exposed workers with a variety of occupations. Additionally, 30 healthy nonsmoker volunteers were recruited as the control group. TCE exposure was determined by measuring urinary trichloroacetic acid (TCA) concentration. Median urinary TCA levels of exposed workers (20.5 mg/L) were significantly higher than control subjects (5 mg/L). Thiol and disulfide concentrations were determined using a novel automated method. Disulfide/thiol ratio was significantly higher in the exposed group (p < 0.001). Thiol/disulfide homeostasis was found to be disturbed in TCE-exposed workers. We predict that in TCE-exposed workers this disturbance can be a therapeutic target, and the efficiency of the treatment can easily be monitored by the novel method we used.

  13. Protein disulfide isomerase is essential for viability in Saccharomyces cerevisiae.

    PubMed

    Farquhar, R; Honey, N; Murant, S J; Bossier, P; Schultz, L; Montgomery, D; Ellis, R W; Freedman, R B; Tuite, M F

    1991-12-01

    Protein disulfide isomerase (PDI) is an enzyme involved in the catalysis of disulfide bond formation in secretory and cell-surface proteins. Using an oligodeoxyribonucleotide designed to detect the conserved 'thioredoxin-like' active site of vertebrate PDIs, we have isolated a gene encoding PDI from the lower eukaryote, Saccharomyces cerevisiae. The nucleotide sequence and deduced open reading frame of the cloned gene predict a 530-amino-acid (aa) protein of Mr 59,082 and a pI of 4.1, physical properties characteristic of mammalian PDIs. Furthermore, the aa sequence shows 30-32% identity with mammalian and avian PDI sequences and has a very similar overall organisation, namely the presence of two approx. 100-aa segments, each of which is repeated, with the most significant homologies to mammalian and avian PDIs being in the regions (a, a') that contain the conserved 'thioredoxin-like' active site. The N-terminal region has the characteristics of a cleavable secretory signal sequence and the C-terminal four aa (-His-Asp-Glu-Leu) are consistent with the protein being a component of the S. cerevisiae endoplasmic reticulum. Transformants carrying multiple copies of this gene (designated PDI1) have tenfold higher levels of PDI activity and overproduce a protein of the predicted Mr. The PDI1 gene is unique in the yeast genome and encodes a single 1.8-kb transcript that is not found in stationary phase cells. Disruption of the PDI1 gene is haplo-lethal indicating that the product of this gene is essential for viability.

  14. Effect of addition of chitosan to self-etching primer: antibacterial activity and push-out bond strength to radicular dentin

    PubMed Central

    Elsaka, Shaymaa; Elnaghy, Amr

    2012-01-01

    The purpose of this study was to evaluate the antibacterial activity of a modified self-etching primer incorporating chitosan and whether this modification affected the bond strength to radicular dentin. A modified self-etching primer was prepared by adding chitosan solutions at 0.03%, 0.06%, 0.12% and 0.25% (W/W) to RealSeal selfe-tching primer. RealSeal primer without chitosan was used as the control. The antibacterial activity of the modified self-etching primer was evaluated using the direct contact test against Enterococcus faecalis. The bonding ability of the RealSeal system to radicular dentin was evaluated using the push-out bond strength test. The modes of failure were examined under a stereomicroscope. Data were analyzed using analysis of variance (ANOVA) and Tukey's test, with a P-value < 0.05 indicating statistical significance. The results showed that the antibacterial properties of the freshly prepared and aged modified self-etching primer incorporating chitosan exhibited potent antibacterial effect against Enterococcus faecalis compared with the unmodified primer. The RealSeal system with the aged modified self-etching primer incorporating chitosan showed no significant differences in the bond strength as compared with the control (P = 0.99). The findings suggest that modified self-etching primer incorporating chitosan is a promising antibacterial primer which does not adversely affect the bond strength of the RealSeal system to radicular dentin. PMID:23554762

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

    PubMed Central

    Davey, Lauren; Halperin, Scott A.; Lee, Song F.

    2016-01-01

    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. PMID:27846284

  16. The b' domain provides the principal peptide-binding site of protein disulfide isomerase but all domains contribute to binding of misfolded proteins.

    PubMed Central

    Klappa, P; Ruddock, L W; Darby, N J; Freedman, R B

    1998-01-01

    Protein disulfide isomerase (PDI) is a very efficient catalyst of folding of many disulfide-bonded proteins. A great deal is known about the catalytic functions of PDI, while little is known about its substrate binding. We recently demonstrated by cross-linking that PDI binds peptides and misfolded proteins, with high affinity but broad specificity. To characterize the substrate-binding site of PDI, we investigated the interactions of various recombinant fragments of human PDI, expressed in Escherichia coli, with different radiolabelled model peptides. We observed that the b' domain of human PDI is essential and sufficient for the binding of small peptides. In the case of larger peptides, specifically a 28 amino acid fragment derived from bovine pancreatic trypsin inhibitor, or misfolded proteins, the b' domain is essential but not sufficient for efficient binding, indicating that contributions from additional domains are required. Hence we propose that the different domains of PDI all contribute to the binding site, with the b' domain forming the essential core. PMID:9463371

  17. Ligand noninnocence of thiolate/disulfide in dinuclear copper complexes: solvent-dependent redox isomerization and proton-coupled electron transfer.

    PubMed

    Thomas, Andrew M; Lin, Bo-Lin; Wasinger, Erik C; Stack, T Daniel P

    2013-12-18

    Copper thiolate/disulfide interconversions are related to the functions of several important proteins such as human Sco1, Cu-Zn superoxide dismutase (SOD1), and mammalian zinc-bonded metallothionein. The synthesis and characterization of well-defined synthetic analogues for such interconversions are challenging yet provide important insights into the mechanisms of such redox processes. Solvent-dependent redox isomerization and proton-coupled electron transfer mimicking these interconversions are observed in two structurally related dimeric μ,η(2):η(2)-thiolato Cu(II)Cu(II) complexes by various methods, including X-ray diffraction, XAS, NMR, and UV-vis. Spectroscopic evidence shows that a solvent-dependent equilibrium exists between the dimeric μ-thiolato Cu(II)Cu(II) state and its redox isomeric μ-disulfido Cu(I)Cu(I) form. Complete formation of μ-disulfido Cu(I)Cu(I) complexes, however, only occurs after the addition of 2 equiv of protons, which promote electron transfer from thiolate to Cu(II) and formation of disulfide and Cu(I) via protonation of the coordinating ligand. Proton removal reverses this reaction. The reported unusual reductive protonation/oxidative deprotonation of the metal centers may serve as a new chemical precedent for how related proteins manage Cu ions in living organisms.

  18. Adsorption mechanisms of isoxazole and oxazole on Si(100)-2 × 1 surface: Si-N dative bond addition vs. [4+2] cycloaddition

    NASA Astrophysics Data System (ADS)

    Kumer Ghosh, Manik; Choi, Cheol Ho

    2011-12-01

    The surface reaction pathways of isoxazole and oxazole on Si(100)-2 × 1 surface were theoretically investigated. They both form a weakly bound Si-N dative bond adduct on Si(100)-2 × 1 surface. In the case of isoxazole, the barrierlessly formed Si-N adduct is the most important surface product, that cannot be easily converted into other species. On the other hand, a facile concerted [4+2]CC cycloaddition without involving the initial Si-N dative bond adduct was also found in the case of oxazole adsorption. The existence of Diels-Alder reactions is attributed to the particular arrangement of the two heteroatoms of oxazole in such a way that the two Si-C σ-bonds can be formed in a [4+2] fashion. In short, the unique geometric arrangements and electronegativity of these similar heteroatomic molecules yielded distinctively different surface reaction characteristics.

  19. Enhancing the thermal stability of avidin. Introduction of disulfide bridges between subunit interfaces.

    PubMed

    Nordlund, Henri R; Laitinen, Olli H; Uotila, Sanna T H; Nyholm, Thomas; Hytönen, Vesa P; Slotte, J Peter; Kulomaa, Markku S

    2003-01-24

    In this study we showed that tetrameric chicken avidin can be stabilized by introducing intermonomeric disulfide bridges between its subunits. These covalent bonds had no major effects on the biotin binding properties of the respective mutants. Moreover, one of the mutants (Avd-ccci) maintained its tetrameric integrity even in denaturing conditions. The new avidin forms Avd-ci and Avd-ccci, which have native --> denatured transition midpoints (T(m)) of 98.6 and 94.7 degrees C, respectively, in the absence of biotin, will find use in applications where extreme stability or minimal leakage of subunits is required. Furthermore, we showed that the intramonomeric disulfide bridges found in the wild-type avidin affect its stability. The mutant Avd-nc, in which this bridge was removed, had a lower T(m) in the absence of biotin than the wild-type avidin but showed comparable stability in the presence of biotin.

  20. A vibrational spectroscopic assignment of the disulfide bridges in recombinant bovine growth hormone and growth hormone analogs

    NASA Astrophysics Data System (ADS)

    Thamann, Thomas J.

    1999-07-01

    Disulfide stretching vibrations for bovine growth hormone (bGH) occur in a vibrational envelope centered at 540 cm -1 which spans 480-580 cm -1. A multitude of vibrational bands present in this envelope, that are not related to disulfide stretching, emphasize the need for model compounds when assigning S-S stretching modes. Raman spectroscopic data for bGH analogs, in which one or both of the two disulfide bridges have been selectively cleaved, have been used to characterize the S-S stretching envelope for the two cystine links in bGH. The Raman data for the r-bGH analogs indicate that the number of disulfide bonds present in r-bGH is determined, not by the observance of the presence or absence of a single spectral peak, but by the relative intensity of vibrational envelope from 520-560 cm -1. Cleavage of disulfide bridges in bGH results in a general decrease in vibrational spectral intensity in the 520-560 cm -1 range. This decrease in intensity is proportional to the number of cystine links severed.

  1. Structure of transition-metal cluster compounds: Use of an additional orbital resulting from the f, g character of spd bond orbitals*

    PubMed Central

    Pauling, Linus

    1977-01-01

    A general theory of the structure of complexes of the transition metals is developed on the basis of the enneacovalence of the metals and the requirements of the electroneutrality principle. An extra orbital may be provided through the small but not negligible amount of f and g character of spd bond orbitals, and an extra electron or electron pair may be accepted in this orbital for a single metal or a cluster to neutralize the positive electric charge resulting from the partial ionic character of the bonds with ligands, such as the carbonyl group. Examples of cluster compounds of cobalt, ruthenium, rhodium, osmium, and gold are discussed. PMID:16592470

  2. The Protein-disulfide Isomerase ERp57 Regulates the Steady-state Levels of the Prion Protein*

    PubMed Central

    Torres, Mauricio; Medinas, Danilo B.; Matamala, José Manuel; Woehlbier, Ute; Cornejo, Víctor Hugo; Solda, Tatiana; Andreu, Catherine; Rozas, Pablo; Matus, Soledad; Muñoz, Natalia; Vergara, Carmen; Cartier, Luis; Soto, Claudio; Molinari, Maurizio; Hetz, Claudio

    2015-01-01

    Although the accumulation of a misfolded and protease-resistant form of the prion protein (PrP) is a key event in prion pathogenesis, the cellular factors involved in its folding and quality control are poorly understood. PrP is a glycosylated and disulfide-bonded protein synthesized at the endoplasmic reticulum (ER). The ER foldase ERp57 (also known as Grp58) is highly expressed in the brain of sporadic and infectious forms of prion-related disorders. ERp57 is a disulfide isomerase involved in the folding of a subset of glycoproteins in the ER as part of the calnexin/calreticulin cycle. Here, we show that levels of ERp57 increase mainly in neurons of Creutzfeldt-Jacob patients. Using gain- and loss-of-function approaches in cell culture, we demonstrate that ERp57 expression controls the maturation and total levels of wild-type PrP and mutant forms associated with human disease. In addition, we found that PrP physically interacts with ERp57, and also with the closest family member PDIA1, but not ERp72. Furthermore, we generated a conditional knock-out mouse for ERp57 in the nervous system and detected a reduction in the steady-state levels of the mono- and nonglycosylated forms of PrP in the brain. In contrast, ERp57 transgenic mice showed increased levels of endogenous PrP. Unexpectedly, ERp57 expression did not affect the susceptibility of cells to ER stress in vitro and in vivo. This study identifies ERp57 as a new modulator of PrP levels and may help with understanding the consequences of ERp57 up-regulation observed in human disease. PMID:26170458

  3. Synthesis, structural characterization and conversion of dinuclear iron-sulfur clusters containing the disulfide ligand: [Cp*Fe(μ-η(2):η(2)-bdt)(cis-μ-η(1):η(1)-S2)FeCp*], [Cp*Fe(μ-S(C6H4S2))(cis-μ-η(1):η(1)-S2)FeCp*], and [{Cp*Fe(bdt)}2(trans-μ-η(1):η(1)-S2)].

    PubMed

    Ji, Xiaoxiao; Tong, Peng; Yang, Dawei; Wang, Baomin; Zhao, Jinfeng; Li, Yang; Qu, Jingping

    2017-03-21

    The treatment of [Cp*Fe(μ-η(2):η(4)-bdt)FeCp*] (1, Cp* = η(5)-C5Me5, bdt = benzene-1,2-dithiolate) with 1/4 equiv. of elemental sulfur (S8) gave a dinuclear iron-sulfur cluster [Cp*Fe(μ-η(2):η(2)-bdt)(cis-μ-η(1):η(1)-S2)FeCp*] (2), which contains a cis-1,2-disulfide ligand. When complex 2 further interacted with 1/8 equiv. of S8, another sulfur atom inserted into an Fe-S bond to give a rare product [Cp*Fe(μ-S(C6H4S2))(cis-μ-η(1):η(1)-S2)FeCp*] (3). Unexpectedly, a trans-1,2 disulfide-bridged diiron complex [{Cp*Fe(bdt)}2(trans-μ-η(1):η(1)-S2)] (4) was isolated from the reaction of complex 1 with 1/2 equiv. of S8, which represents a structural isomer of [2Fe-2S] ferredoxin-type clusters. In addition, cis-1,2-disulfide-bridged complex 3 can slowly convert into trans-1,2-disulfide-bridged complex 4 and the complex [Cp*Fe(μ-η(2):η(2)-S2)(cis-μ-η(1):η(1)-S2)FeCp*] (5) by self-assembly reaction at ambient temperature, which is evidenced by time-dependent (1)H NMR spectroscopy.

  4. The alkaline earth intercalates of molybdenum disulfide

    NASA Technical Reports Server (NTRS)

    Somoano, R. B.; Hadek, V.; Rembaum, A.; Samson, S.; Woollam, J. A.

    1975-01-01

    Molybdenum disulfide has been intercalated with calcium and strontium by means of the liquid ammonia technique. Chemical, X-ray, and superconductivity data are presented. The X-ray data reveal a lowering of crystal symmetry and increase of complexity of the structure upon intercalation with the alkaline earth metals. The Ca and Sr intercalates start to superconduct at 4 and 5.6 K, respectively, and show considerable anisotropy regarding the critical magnetic field.

  5. Electrochemical Performance of Titanium Disulfide and Molybdenum Disulfide Nanoplatelets

    NASA Astrophysics Data System (ADS)

    Siordia, Andrew Francisco

    Composite materials are widely used in various structural applications, including within the automotive and aerospace industries. Unidirectional composite layups have replaced other materials such as metals due to composites' high strength-to-weight ratio and durability. Finite-element (FE) models are actively being developed to model response of composite systems subjected to a variety of loads including impact loads. These FE models rely on an array of measured material properties as input for accuracy. This work focuses on an orthotropic plasticity constitutive model that has three components - deformation, damage and failure. The model relies on the material properties of the composite such as Young's modulus, Poisson's ratio, stress-strain curves in the principal and off-axis material directions, etc. This thesis focuses on two areas important to the development of the FE model - tabbing of the test specimens and data processing of the tests used to generate the required stress-strain curves. A comparative study has been performed on three candidate adhesives using double lap-shear testing to determine their effectiveness in composite specimen tabbing. These tests determined the 3M DP460 epoxy performs best in shear. The Loctite Superglue with 80% the ultimate stress of the 3M DP460 epoxy is acceptable when test specimens have to be ready for testing within a few hours. JB KwikWeld is not suitable for tabbing. In addition, the Experimental Data Processing (EDP) program has been improved for use in post-processing raw data from composites test. EDP has improved to allow for complete processing with the implementation of new weighted least squares smoothing options, curve averaging techniques, and new functionality for data manipulation.

  6. Diffusion bonding aeroengine components

    NASA Astrophysics Data System (ADS)

    Fitzpatrick, G. A.; Broughton, T.

    1988-10-01

    The use of diffusion bonding processes at Rolls-Royce for the manufacture of titanium-alloy aircraft engine components and structures is described. A liquid-phase diffusion bonding process called activated diffusion bonding has been developed for the manufacture of the hollow titanium wide chord fan blade. In addition, solid-state diffusion bonding is being used in the manufacture of hollow vane/blade airfoil constructions mainly in conjunction with superplastic forming and hot forming techniques.

  7. Reduction of the secretory response to Escherichia coli heat-stable enterotoxin by thiol and disulfide compounds. [Mice

    SciTech Connect

    Greenberg, R.N.; Dunn, J.A.; Guerrant, R.L.

    1983-07-01

    We examined the effects of disulfide and thiol compounds on Escherichia coli heat-stable enterotoxin (ST) and cyclic GMP-induced secretion. Both cystamine and cystine (disulfide compounds) reduced the secretory responses to submaximal doses of ST in suckling mice (at 0.5 mumol per mouse) and reduced ST activation of guanylate cyclase (by 33 to 73% at 1 mM). In higher doses, cystamine completely eradicated a maximally effective ST dose as well. In addition, the sulfhydryl (thiol) compounds cysteamine, cysteine, and acetylcysteine strikingly reduced the secretory response and the guanylate cyclase response to ST. Neither the disulfide nor the thiol compounds tested reduced cyclic GMP-induced secretion. These studies suggest that disulfide and thiol compounds both block ST-induced secretion before its activation of guanylate cyclase. Taken with the work of others, these findings suggest that disulfide compounds may alter the oxidation reduction state of a cell or act directly on the guanylate cyclase enzyme, whereas thiol compounds may inactivate ST itself by breaking its disulfide bridges, or it may alter guanylate cyclase activation by ST. Both families of compounds deserve further consideration among potential antisecretory agents for application in the control of ST-induced diarrhea.

  8. Bent Bonds and Multiple Bonds.

    ERIC Educational Resources Information Center

    Robinson, Edward A.; Gillespie, Ronald J.

    1980-01-01

    Considers carbon-carbon multiple bonds in terms of Pauling's bent bond model, which allows direct calculation of double and triple bonds from the length of a CC single bond. Lengths of these multiple bonds are estimated from direct measurements on "bent-bond" models constructed of plastic tubing and standard kits. (CS)