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

DEFF Research Database (Denmark)

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

2016-01-01

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

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

International Nuclear Information System (INIS)

Takeda, Mitsuhiro; Terauchi, Tsutomu; Kainosho, Masatsune

2012-01-01

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

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

Directory of Open Access Journals (Sweden)

Pedro Jacquez

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

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

Science.gov (United States)

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

2018-04-01

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

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

DEFF Research Database (Denmark)

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

1998-01-01

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

3.3 Å structure of Niemann–Pick C1 protein reveals insights into the function of the C-terminal luminal domain in cholesterol transport

Energy Technology Data Exchange (ETDEWEB)

Li, Xiaochun; Lu, Feiran; Trinh, Michael N.; Schmiege, Philip; Seemann, Joachim; Wang, Jiawei; Blobel, Günter

2017-08-07

Niemann–Pick C1 (NPC1) and NPC2 proteins are indispensable for the export of LDL-derived cholesterol from late endosomes. Mutations in these proteins result in Niemann–Pick type C disease, a lysosomal storage disease. Despite recent reports of the NPC1 structure depicting its overall architecture, the function of its C-terminal luminal domain (CTD) remains poorly understood even though 45% of NPC disease-causing mutations are in this domain. Here, we report a crystal structure at 3.3 Å resolution of NPC1* (residues 314–1,278), which—in contrast to previous lower resolution structures—features the entire CTD well resolved. Notably, all eight cysteines of the CTD form four disulfide bonds, one of which (C909–C914) enforces a specific loop that in turn mediates an interaction with a loop of the N-terminal domain (NTD). Importantly, this loop and its interaction with the NTD were not observed in any previous structures due to the lower resolution. Our mutagenesis experiments highlight the physiological relevance of the CTD–NTD interaction, which might function to keep the NTD in the proper orientation for receiving cholesterol from NPC2. Additionally, this structure allows us to more precisely map all of the disease-causing mutations, allowing future molecular insights into the pathogenesis of NPC disease.

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

Science.gov (United States)

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

2017-09-05

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

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

Science.gov (United States)

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

2012-07-13

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

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

Directory of Open Access Journals (Sweden)

M. Graciela Pucciarelli

2017-12-01

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

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

Science.gov (United States)

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

2012-12-18

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

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

Directory of Open Access Journals (Sweden)

Vijay Kumar Ravi

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

Protein disulfide bond generation in Escherichia coli DsbB–DsbA

International Nuclear Information System (INIS)

Inaba, Kenji

2008-01-01

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

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

Science.gov (United States)

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

2017-06-27

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

Analysis of Disulfide Bond Formation

NARCIS (Netherlands)

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

2017-01-01

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

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

Science.gov (United States)

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

2014-12-01

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

Alpha-cyclodextrins reversibly capped with disulfide bonds

Czech Academy of Sciences Publication Activity Database

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

2010-01-01

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

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

Science.gov (United States)

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

2015-10-09

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

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

Science.gov (United States)

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

2016-10-01

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

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

Science.gov (United States)

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

2008-09-01

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

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

Directory of Open Access Journals (Sweden)

Lobstein Julie

2012-05-01

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

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

Science.gov (United States)

Honda, Ryo

2018-02-27

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

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

Science.gov (United States)

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

2015-11-15

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

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

DEFF Research Database (Denmark)

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

2015-01-01

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

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

Directory of Open Access Journals (Sweden)

Lihie Levin

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

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

OpenAIRE

Palanisamy Kalimuthu; Palraj Kalimuthu; S. Abraham John

2007-01-01

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

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

Science.gov (United States)

Yamagami, T; Funatsu, G; Ishiguro, M

2000-06-01

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

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

Directory of Open Access Journals (Sweden)

Chengtuo Niu

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

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

Directory of Open Access Journals (Sweden)

Xiao-Wei Yu

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

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

International Nuclear Information System (INIS)

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

2013-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2013-10-01

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

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

Science.gov (United States)

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

2007-06-01

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

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

International Nuclear Information System (INIS)

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

2012-01-01

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

Akt kinase C-terminal modifications control activation loop dephosphorylation and enhance insulin response.

Science.gov (United States)

Chan, Tung O; Zhang, Jin; Tiegs, Brian C; Blumhof, Brian; Yan, Linda; Keny, Nikhil; Penny, Morgan; Li, Xue; Pascal, John M; Armen, Roger S; Rodeck, Ulrich; Penn, Raymond B

2015-10-01

The Akt protein kinase, also known as protein kinase B, plays key roles in insulin receptor signalling and regulates cell growth, survival and metabolism. Recently, we described a mechanism to enhance Akt phosphorylation that restricts access of cellular phosphatases to the Akt activation loop (Thr(308) in Akt1 or protein kinase B isoform alpha) in an ATP-dependent manner. In the present paper, we describe a distinct mechanism to control Thr(308) dephosphorylation and thus Akt deactivation that depends on intramolecular interactions of Akt C-terminal sequences with its kinase domain. Modifications of amino acids surrounding the Akt1 C-terminal mTORC2 (mammalian target of rapamycin complex 2) phosphorylation site (Ser(473)) increased phosphatase resistance of the phosphorylated activation loop (pThr(308)) and amplified Akt phosphorylation. Furthermore, the phosphatase-resistant Akt was refractory to ceramide-dependent dephosphorylation and amplified insulin-dependent Thr(308) phosphorylation in a regulated fashion. Collectively, these results suggest that the Akt C-terminal hydrophobic groove is a target for the development of agents that enhance Akt phosphorylation by insulin. © 2015 Authors; published by Portland Press Limited.

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

Science.gov (United States)

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

2015-11-25

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

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

DEFF Research Database (Denmark)

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

2015-01-01

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

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

OpenAIRE

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

2015-01-01

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

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

Directory of Open Access Journals (Sweden)

Hatahet Feras

2010-09-01

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

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

OpenAIRE

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

2008-01-01

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

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

Science.gov (United States)

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

2011-12-01

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

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

Science.gov (United States)

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

2015-01-01

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

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

Science.gov (United States)

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

2015-08-28

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

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

DEFF Research Database (Denmark)

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

2001-01-01

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

Structure and function of C-terminal catalytic region of pasteurella multocida toxin

International Nuclear Information System (INIS)

Kitadokoro, Kengo; Kamitami, Shigeki; Horiguchi, Yasuhiko

2008-01-01

Pasteurella multocida toxin (PMT) is one of virulence factors responsible for the pathogenesis in some Pasteurellosis. We determined the crystal structure of the C-terminal region of PMT (C-PMT), which carries an intracellularly active moiety. The overall structure of C-PMT displays three different domains designated C1, C2 and C3. We found in the C3 domain the Cys-His-Asp catalytic triad that is organized only when the Cys is released from a disulfide bond. The steric alignment of the triad corresponded well to that of papain or other enzymes carrying the Cys-His-Asp triad. Our results demonstrate that PMT is an enzymatic toxin carrying the cysteine-protease like catalytic triad, which is organized only under reducing conditions. (author)

Photo-responsive liquid crystalline epoxy networks with exchangeable disulfide bonds

Energy Technology Data Exchange (ETDEWEB)

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

2017-07-27

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

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

DEFF Research Database (Denmark)

Björnberg, Olof; Maeda, Kenji; Svensson, Birte

2012-01-01

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

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

NARCIS (Netherlands)

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

2008-01-01

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

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

Science.gov (United States)

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

2018-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

1991-06-01

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

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

Directory of Open Access Journals (Sweden)

Helena Kellett-Clarke

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

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

International Nuclear Information System (INIS)

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

2012-01-01

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

Characterization of cyclic peptides containing disulfide bonds

OpenAIRE

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

2015-01-01

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

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

Science.gov (United States)

Reiz, Bela; Li, Liang

2010-09-01

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

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

Science.gov (United States)

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

2013-05-01

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

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

Science.gov (United States)

Wang, Wei

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

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

Directory of Open Access Journals (Sweden)

Kajal Gupta

2010-11-01

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

Step-wise addition of disulfide bridge in firefly luciferase controls color shift through a flexible loop: a thermodynamic perspective.

Science.gov (United States)

Nazari, Mahboobeh; Hosseinkhani, Saman; Hassani, Leila

2013-02-01

Multi-color bioluminescence is developed using the introduction of single/double disulfide bridges in firefly luciferase. The bioluminescence reaction, which uses luciferin, Mg(2+)-ATP and molecular oxygen to yield an electronically excited oxyluciferin, is carried out by the luciferase and emits visible light. The bioluminescence color of firefly luciferases is determined by the luciferase sequence and assay conditions. It has been proposed that the stability of a protein may increase through the introduction of a disulfide bridge that decreases the configurational entropy of unfolding. Single and double disulfide bridges are introduced into Photinus pyralis firefly luciferase to make separate mutant enzymes with a single/double bridge (C(81)-A(105)C, L(306)C-L(309)C, P(451)C-V(469)C; C(81)-A(105)C/P(451)C-V(469)C, and A(296)C-A(326)C/P(451)C-V(469)C). By introduction of disulfide bridges using site-directed mutagenesis in Photinus pyralis luciferase the color of emitted light was changed to red or kept in different extents. The bioluminescence color shift occurred with displacement of a critical loop in the luciferase structure without any change in green emitter mutants. Thermodynamic analysis revealed that among mutants, L(306)C-L(309)C shows a remarkable stability against urea denaturation and also a considerable increase in kinetic stability and a clear shift in bioluminescence spectra towards red.

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

Directory of Open Access Journals (Sweden)

Adam A. Campos-Acevedo

2014-12-01

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

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

Science.gov (United States)

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

2014-12-15

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

Quantification of thiols and disulfides

DEFF Research Database (Denmark)

Winther, Jakob R.; Thorpe, Colin

2014-01-01

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

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

International Nuclear Information System (INIS)

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

2008-01-01

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

An Engineered Disulfide Bond Reversibly Traps the IgE-Fc_3-4 in a Closed, Nonreceptor Binding Conformation

Energy Technology Data Exchange (ETDEWEB)

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

2013-08-02

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2013-12-02

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

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

Directory of Open Access Journals (Sweden)

Jihun Lee

2010-12-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

1993-04-01

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

COOH-terminal substitutions in the serpin C1 inhibitor that cause loop overinsertion and subsequent multimerization

NARCIS (Netherlands)

Eldering, E.; Verpy, E.; Roem, D.; Meo, T.; Tosi, M.

1995-01-01

The region COOH-terminal to the reactive center loop is highly conserved in the serine protease inhibitor (serpin) family. We have studied the structural consequences of three substitutions (Val451-->Met, Phe455-->Ser, and Pro476-->Ser) found in this region of C1 inhibitor in patients suffering from

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

Science.gov (United States)

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

1998-12-01

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

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

Science.gov (United States)

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

2015-10-01

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

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

Science.gov (United States)

Shibamoto, Shigeaki; Murata, Tasuku; Yamamoto, Kouhei

2016-09-01

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

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

International Nuclear Information System (INIS)

Matak, Mehdi Youssefi; Moghaddam, Majid Erfani

2009-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2013-06-15

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

The multidrug resistance IncA/C transferable plasmid encodes a novel domain-swapped dimeric protein-disulfide isomerase.

Science.gov (United States)

Premkumar, Lakshmanane; Kurth, Fabian; Neyer, Simon; Schembri, Mark A; Martin, Jennifer L

2014-01-31

The multidrug resistance-encoding IncA/C conjugative plasmids disseminate antibiotic resistance genes among clinically relevant enteric bacteria. A plasmid-encoded disulfide isomerase is associated with conjugation. Sequence analysis of several IncA/C plasmids and IncA/C-related integrative and conjugative elements (ICE) from commensal and pathogenic bacteria identified a conserved DsbC/DsbG homolog (DsbP). The crystal structure of DsbP reveals an N-terminal domain, a linker region, and a C-terminal catalytic domain. A DsbP homodimer is formed through domain swapping of two DsbP N-terminal domains. The catalytic domain incorporates a thioredoxin-fold with characteristic CXXC and cis-Pro motifs. Overall, the structure and redox properties of DsbP diverge from the Escherichia coli DsbC and DsbG disulfide isomerases. Specifically, the V-shaped dimer of DsbP is inverted compared with EcDsbC and EcDsbG. In addition, the redox potential of DsbP (-161 mV) is more reducing than EcDsbC (-130 mV) and EcDsbG (-126 mV). Other catalytic properties of DsbP more closely resemble those of EcDsbG than EcDsbC. These catalytic differences are in part a consequence of the unusual active site motif of DsbP (CAVC); substitution to the EcDsbC-like (CGYC) motif converts the catalytic properties to those of EcDsbC. Structural comparison of the 12 independent subunit structures of DsbP that we determined revealed that conformational changes in the linker region contribute to mobility of the catalytic domain, providing mechanistic insight into DsbP function. In summary, our data reveal that the conserved plasmid-encoded DsbP protein is a bona fide disulfide isomerase and suggest that a dedicated oxidative folding enzyme is important for conjugative plasmid transfer.

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

Energy Technology Data Exchange (ETDEWEB)

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

1979-01-01

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

Multi-terminal memtransistors from polycrystalline monolayer molybdenum disulfide

Science.gov (United States)

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

2018-02-01

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

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

NARCIS (Netherlands)

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

1993-01-01

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

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

Directory of Open Access Journals (Sweden)

Xiaonan Hou

2017-02-01

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

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

Czech Academy of Sciences Publication Activity Database

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

2017-01-01

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

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

Science.gov (United States)

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

2017-10-01

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

How thioredoxin dissociates its mixed disulfide.

Directory of Open Access Journals (Sweden)

Goedele Roos

2009-08-01

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

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

Science.gov (United States)

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

2017-01-22

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

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

Directory of Open Access Journals (Sweden)

Padmesh Anjukandi

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

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

Directory of Open Access Journals (Sweden)

W Wang

2009-01-01

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

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

Science.gov (United States)

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

2017-06-15

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

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

DEFF Research Database (Denmark)

Mysling, Simon; Salbo, Rune; Ploug, Michael

2014-01-01

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

A slow-forming isopeptide bond in the structure of the major pilin SpaD from Corynebacterium diphtheriae has implications for pilus assembly

Energy Technology Data Exchange (ETDEWEB)

Kang, Hae Joo; Paterson, Neil G. [University of Auckland, Private Bag 92019, Auckland 1142 (New Zealand); Kim, Chae Un [Cornell University, Ithaca, NY 14853 (United States); Middleditch, Martin [University of Auckland, Private Bag 92019, Auckland 1142 (New Zealand); Chang, Chungyu; Ton-That, Hung [University of Texas–Houston Medical School, Houston, TX 77030 (United States); Baker, Edward N., E-mail: ted.baker@auckland.ac.nz [University of Auckland, Private Bag 92019, Auckland 1142 (New Zealand)

2014-05-01

Two crystal structures of the major pilin SpaD from C. diphtheriae have been determined at 1.87 and 2.5 Å resolution. The N-terminal domain is found to contain an isopeptide bond that forms slowly over time in the recombinant protein. Given its structural context, this provides insight into the relationship between internal isopeptide-bond formation and pilus assembly. The Gram-positive organism Corynebacterium diphtheriae, the cause of diphtheria in humans, expresses pili on its surface which it uses for adhesion and colonization of its host. These pili are covalent protein polymers composed of three types of pilin subunit that are assembled by specific sortase enzymes. A structural analysis of the major pilin SpaD, which forms the polymeric backbone of one of the three types of pilus expressed by C. diphtheriae, is reported. Mass-spectral and crystallographic analysis shows that SpaD contains three internal Lys–Asn isopeptide bonds. One of these, shown by mass spectrometry to be located in the N-terminal D1 domain of the protein, only forms slowly, implying an energy barrier to bond formation. Two crystal structures, of the full-length three-domain protein at 2.5 Å resolution and of a two-domain (D2-D3) construct at 1.87 Å resolution, show that each of the three Ig-like domains contains a single Lys–Asn isopeptide-bond cross-link, assumed to give mechanical stability as in other such pili. Additional stabilizing features include a disulfide bond in the D3 domain and a calcium-binding loop in D2. The N-terminal D1 domain is more flexible than the others and, by analogy with other major pilins of this type, the slow formation of its isopeptide bond can be attributed to its location adjacent to the lysine used in sortase-mediated polymerization during pilus assembly.

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

Science.gov (United States)

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

2018-05-01

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

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

Science.gov (United States)

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

2018-01-25

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

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

International Nuclear Information System (INIS)

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

2013-01-01

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

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

Science.gov (United States)

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

2016-01-01

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

Structures and related properties of helical, disulfide-stabilized peptides

Energy Technology Data Exchange (ETDEWEB)

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

1993-11-01

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

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

Science.gov (United States)

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

2011-01-01

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

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

Science.gov (United States)

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

2014-04-01

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

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

International Nuclear Information System (INIS)

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

1990-01-01

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

Identification of Thioredoxin Target Disulfides Using Isotope-Coded Affinity Tags

DEFF Research Database (Denmark)

Hägglund, Per; Bunkenborg, Jakob; Maeda, Kenji

2014-01-01

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

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

Science.gov (United States)

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

2018-05-01

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

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

Science.gov (United States)

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

2018-05-30

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

Soft Computing Methods for Disulfide Connectivity Prediction.

Science.gov (United States)

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

2015-01-01

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

Kinetic and Thermodynamic Aspects of Cellular Thiol-Disulfide Redox Regulation

DEFF Research Database (Denmark)

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

2009-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2017-09-01

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

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

Czech Academy of Sciences Publication Activity Database

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

2009-01-01

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

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

Science.gov (United States)

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

2013-12-10

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

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

Science.gov (United States)

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

2010-06-15

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

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

Science.gov (United States)

Mohanram, Harini; Bhattacharjya, Surajit

2014-04-21

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

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

Directory of Open Access Journals (Sweden)

Harini Mohanram

2014-04-01

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

Distal loop flexibility of a regulatory domain modulates dynamics and activity of C-terminal SRC kinase (csk.

Directory of Open Access Journals (Sweden)

Sulyman Barkho

Full Text Available The Src family of tyrosine kinases (SFKs regulate numerous aspects of cell growth and differentiation and are under the principal control of the C-terminal Src Kinase (Csk. Csk and SFKs share a modular design with the kinase domain downstream of the N-terminal SH2 and SH3 domains that regulate catalytic function and membrane localization. While the function of interfacial segments in these multidomain kinases are well-investigated, little is known about how surface sites and long-range, allosteric coupling control protein dynamics and catalytic function. The SH2 domain of Csk is an essential component for the down-regulation of all SFKs. A unique feature of the SH2 domain of Csk is the tight turn in place of the canonical CD loop in a surface site far removed from kinase domain interactions. In this study, we used a combination of experimental and computational methods to probe the importance of this difference by constructing a Csk variant with a longer SH2 CD loop to mimic the flexibility found in homologous kinase SH2 domains. Our results indicate that while the fold and function of the isolated domain and the full-length kinase are not affected by loop elongation, native protein dynamics that are essential for efficient catalysis are perturbed. We also identify key motifs and routes through which the distal SH2 site might influence catalysis at the active site. This study underscores the sensitivity of intramolecular signaling and catalysis to native protein dynamics that arise from modest changes in allosteric regions while providing a potential strategy to alter intrinsic activity and signaling modulation.

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

Czech Academy of Sciences Publication Activity Database

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

2018-01-01

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

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

International Nuclear Information System (INIS)

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

2004-01-01

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

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

Science.gov (United States)

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

1999-04-20

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

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

DEFF Research Database (Denmark)

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

2017-01-01

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

C-H Bond Functionalization via Hydride Transfer: Direct Coupling of Unactivated Alkynes and sp3 C-H Bonds Catalyzed by Platinum Tetraiodide

Science.gov (United States)

Vadola, Paul A.; Sames, Dalibor

2010-01-01

We report a catalytic intramolecular coupling between terminal unactivated alkynes and sp3 C-H bonds via the through-space hydride transfer (HT-cyclization of alkynes). This method enables one-step preparation of complex heterocyclic compounds by α-alkenylation of readily available cyclic ethers and amines. We show that PtI4 is an effective Lewis acid catalyst for the activation of terminal alkynes for the hydride attack and subsequent C-C bond formation. In addition, we have shown that the activity of neutral platinum salts (PtXn) can be modulated by the halide ligands. This modulation in turn allows for fine-tuning of the platinum center reactivity to match the reactivity and stability of selected substrates and products. PMID:19852462

77 FR 29472 - Surety Companies Acceptable on Federal Bonds-Termination: Atlantic Bonding Company, Inc.

Science.gov (United States)

2012-05-17

... be accepted from this company, and bonds that are continuous in nature should not be renewed. The... DEPARTMENT OF THE TREASURY Fiscal Service Surety Companies Acceptable on Federal Bonds--Termination: Atlantic Bonding Company, Inc. AGENCY: Financial Management Service, Fiscal Service, Department...

Site-specific Isopeptide Bridge Tethering of Chimeric gp41 N-terminal Heptad Repeat Helical Trimers for the Treatment of HIV-1 Infection

Science.gov (United States)

Wang, Chao; Li, Xue; Yu, Fei; Lu, Lu; Jiang, Xifeng; Xu, Xiaoyu; Wang, Huixin; Lai, Wenqing; Zhang, Tianhong; Zhang, Zhenqing; Ye, Ling; Jiang, Shibo; Liu, Keliang

2016-01-01

Peptides derived from the N-terminal heptad repeat (NHR) of HIV-1 gp41 can be potent inhibitors against viral entry when presented in a nonaggregating trimeric coiled-coil conformation via the introduction of exogenous trimerization motifs and intermolecular disulfide bonds. We recently discovered that crosslinking isopeptide bridges within the de novo helical trimers added exceptional resistance to unfolding. Herein, we attempted to optimize (CCIZN17)3, a representative disulfide bond-stabilized chimeric NHR-trimer, by incorporating site-specific interhelical isopeptide bonds as the redox-sensitive disulfide surrogate. In this process, we systematically examined the effect of isopeptide bond position and molecular sizes of auxiliary trimeric coiled-coil motif and NHR fragments on the antiviral potency of these NHR-trimers. Pleasingly, (IZ14N24N)3 possessed promising inhibitory activity against HIV-1 infection and markedly increased proteolytic stability relative to its disulfide-tethered counterpart, suggesting good potential for further development as an effective antiviral agent for treatment of HIV-1 infection. PMID:27562370

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

DEFF Research Database (Denmark)

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

2006-01-01

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

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

Directory of Open Access Journals (Sweden)

Jianjun Sun

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

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

Science.gov (United States)

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

2005-01-14

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

Crystal Structure of the Full-Length Feline Immunodeficiency Virus Capsid Protein Shows an N-Terminal β-Hairpin in the Absence of N-Terminal Proline

Directory of Open Access Journals (Sweden)

Christelle Folio

2017-11-01

Full Text Available Feline immunodeficiency virus (FIV is a member of the Retroviridae family. It is the causative agent of an acquired immunodeficiency syndrome (AIDS in cats and wild felines. Its capsid protein (CA drives the assembly of the viral particle, which is a critical step in the viral replication cycle. Here, the first atomic structure of full-length FIV CA to 1.67 Å resolution is determined. The crystallized protein exhibits an original tetrameric assembly, composed of dimers which are stabilized by an intermolecular disulfide bridge induced by the crystallogenesis conditions. The FIV CA displays a standard α-helical CA topology with two domains, separated by a linker shorter than other retroviral CAs. The β-hairpin motif at its amino terminal end, which interacts with nucleotides in HIV-1, is unusually long in FIV CA. Interestingly, this functional β-motif is formed in this construct in the absence of the conserved N-terminal proline. The FIV CA exhibits a cis Arg–Pro bond in the CypA-binding loop, which is absent in known structures of lentiviral CAs. This structure represents the first tri-dimensional structure of a functional, full-length FIV CA.

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

DEFF Research Database (Denmark)

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

2001-01-01

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

C-terminal peptide extension via gas-phase ion/ion reactions

Science.gov (United States)

Peng, Zhou; McLuckey, Scott A.

2015-01-01

The formation of peptide bonds is of great importance from both a biological standpoint and in routine organic synthesis. Recent work from our group demonstrated the synthesis of peptides in the gas-phase via ion/ion reactions with sulfo-NHS reagents, which resulted in conjugation of individual amino acids or small peptides to the N-terminus of an existing ‘anchor’ peptide. Here, we demonstrate a complementary approach resulting in the C-terminal extension of peptides. Individual amino acids or short peptides can be prepared as reagents by incorporating gas phase-labile protecting groups to the reactive C-terminus and then converting the N-terminal amino groups to the active ketenimine reagent. Gas-phase ion/ion reactions between the anionic reagents and doubly protonated “anchor” peptide cations results in extension of the “anchor” peptide with new amide bond formation at the C-terminus. We have demonstrated that ion/ion reactions can be used as a fast, controlled, and efficient means for C-terminal peptide extension in the gas phase. PMID:26640400

Loop-Loop Interactions Regulate KaiA-Stimulated KaiC Phosphorylation in the Cyanobacterial KaiABC Circadian Clock

Energy Technology Data Exchange (ETDEWEB)

Egli, Martin [Vanderbilt Univ., Nashville, TN (United States); Pattanayek, Rekha [Vanderbilt Univ., Nashville, TN (United States); Sheehan, Jonathan H. [Vanderbilt Univ., Nashville, TN (United States); Xu, Yao [Vanderbilt Univ., Nashville, TN (United States); Mori, Tetsuya [Vanderbilt Univ., Nashville, TN (United States); Smith, Jarrod A. [Vanderbilt Univ., Nashville, TN (United States); Johnson, Carl H. [Vanderbilt Univ., Nashville, TN (United States)

2013-01-25

We found that the Synechococcus elongatus KaiA, KaiB, and KaiC proteins in the presence of ATP generate a post-translational oscillator that runs in a temperature-compensated manner with a period of 24 h. KaiA dimer stimulates phosphorylation of KaiC hexamer at two sites per subunit, T432 and S431, and KaiB dimers antagonize KaiA action and induce KaiC subunit exchange. Neither the mechanism of KaiA-stimulated KaiC phosphorylation nor that of KaiB-mediated KaiC dephosphorylation is understood in detail at present. We demonstrate here that the A422V KaiC mutant sheds light on the former mechanism. It was previously reported that A422V is less sensitive to dark pulse-induced phase resetting and has a reduced amplitude of the KaiC phosphorylation rhythm in vivo. A422 maps to a loop (422-loop) that continues toward the phosphorylation sites. By pulling on the C-terminal peptide of KaiC (A-loop), KaiA removes restraints from the adjacent 422-loop whose increased flexibility indirectly promotes kinase activity. We found in the crystal structure that A422V KaiC lacks phosphorylation at S431 and exhibits a subtle, local conformational change relative to wild-type KaiC. Molecular dynamics simulations indicate higher mobility of the 422-loop in the absence of the A-loop and mobility differences in other areas associated with phosphorylation activity between wild-type and mutant KaiCs. Finally, the A-loop–422-loop relay that informs KaiC phosphorylation sites of KaiA dimer binding propagates to loops from neighboring KaiC subunits, thus providing support for a concerted allosteric mechanism of phosphorylation.

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

Science.gov (United States)

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

2017-07-01

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

Ultrahigh and High Resolution Structures and Mutational Analysis of Monomeric Streptococcus pyogenes SpeB Reveal a Functional Role for the Glycine-rich C-terminal Loop

Energy Technology Data Exchange (ETDEWEB)

González-Páez, Gonzalo E.; Wolan, Dennis W. (Scripps)

2012-09-05

Cysteine protease SpeB is secreted from Streptococcus pyogenes and has been studied as a potential virulence factor since its identification almost 70 years ago. Here, we report the crystal structures of apo mature SpeB to 1.06 {angstrom} resolution as well as complexes with the general cysteine protease inhibitor trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane and a novel substrate mimetic peptide inhibitor. These structures uncover conformational changes associated with maturation of SpeB from the inactive zymogen to its active form and identify the residues required for substrate binding. With the use of a newly developed fluorogenic tripeptide substrate to measure SpeB activity, we determined IC{sub 50} values for trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane and our new peptide inhibitor and the effects of mutations within the C-terminal active site loop. The structures and mutational analysis suggest that the conformational movements of the glycine-rich C-terminal loop are important for the recognition and recruitment of biological substrates and release of hydrolyzed products.

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

Directory of Open Access Journals (Sweden)

Tyo Keith EJ

2012-03-01

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

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

Science.gov (United States)

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

2015-04-02

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

Structure of Acostatin, a Dimeric Disintegrin From Southern Copperhead (Agkistrodon Contortrix Contortrix), at 1.7 Angstrom Resolution

Energy Technology Data Exchange (ETDEWEB)

Moiseeva, N.; Bau, R.; Swenson, S.D.; Marklund, F.S.; Jr.; Choe, J.-Y.; Liu, Z.-J.; Allaire, M.

2009-05-26

Disintegrins are a family of small (4-14 kDa) proteins that bind to another class of proteins, integrins. Therefore, as integrin inhibitors, they can be exploited as anticancer and antiplatelet agents. Acostatin, an {alpha}{beta} heterodimeric disintegrin, has been isolated from the venom of Southern copperhead (Agkistrodon contortrix contortrix). The three-dimensional structure of acostatin has been determined by macromolecular crystallography using the molecular-replacement method. The asymmetric unit of the acostatin crystals consists of two heterodimers. The structure has been refined to an R{sub work} and R{sub free} of 18.6% and 21.5%, respectively, using all data in the 20-1.7 {angstrom} resolution range. The structure of all subunits is similar and is well ordered into N-terminal and C-terminal clusters with four intramolecular disulfide bonds. The overall fold consists of short {beta}-sheets, each of which is formed by a pair of antiparallel {beta}-strands connected by {beta}-turns and flexible loops of different lengths. Conformational flexibility is found in the RGD loops and in the C-terminal segment. The interaction of two N-terminal clusters via two intermolecular disulfide bridges anchors the {alpha}{beta}chains of the acostatin dimers. The C-terminal clusters of the heterodimer project in opposite directions and form a larger angle between them in comparison with other dimeric disintegrins. Extensive interactions are observed between two heterodimers, revealing an {alpha}{beta}{beta}{alpha} acostatin tetramer. Further experiments are required to identify whether the {alpha}{beta}{beta}{alpha} acostatin complex plays a functional role in vivo.

Tyrosine B10 triggers a heme propionate hydrogen bonding network loop with glutamine E7 moiety

International Nuclear Information System (INIS)

Ramos-Santana, Brenda J.; López-Garriga, Juan

2012-01-01

Highlights: ► H-bonding network loop by PheB10Tyr mutation is proposed. ► The propionate group H-bonding network restricted the flexibility of the heme. ► The hydrogen bonding interaction modulates the electron density of the iron. ► Propionate H-bonding network loop explains the heme-ligand stabilization. -- Abstract: Propionates, as peripheral groups of the heme active center in hemeproteins have been described to contribute in the modulation of heme reactivity and ligand selection. These electronic characteristics prompted the question of whether the presence of hydrogen bonding networks between propionates and distal amino acids present in the heme ligand moiety can modulate physiological relevant events, like ligand binding association and dissociation activities. Here, the role of these networks was evaluated by NMR spectroscopy using the hemoglobin I PheB10Tyr mutant from Lucina pectinata as model for TyrB10 and GlnE7 hemeproteins. 1 H-NMR results for the rHbICN PheB10Tyr derivative showed chemical shifts of TyrB10 OHη at 31.00 ppm, GlnE7 N ε1 H/N ε2 H at 10.66 ppm/−3.27 ppm, and PheE11 C δ H at 11.75 ppm, indicating the presence of a crowded, collapsed, and constrained distal pocket. Strong dipolar contacts and inter-residues crosspeaks between GlnE7/6-propionate group, GlnE7/TyrB10 and TyrB10/CN suggest that this hydrogen bonding network loop between GlnE7, TyrB10, 6-propionate group, and the heme ligand contribute significantly to the modulation of the heme iron electron density as well as the ligand stabilization mechanism. Therefore, the network loop presented here support the fact that the electron withdrawing character of the hydrogen bonding is controlled by the interaction of the propionates and the nearby electronic environments contributing to the modulation of the heme electron density state. Thus, we hypothesize that in hemeproteins with similar electrostatic environment the flexibility of the heme-6-propionate promotes a hydrogen

Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding

DEFF Research Database (Denmark)

Barington, Line; Rummel, Pia C; Lückmann, Michael

2016-01-01

and aromatic residues in extracellular loop 2 (ECL2) for ligand binding and activation in the chemokine receptor CCR8. We used IP3 accumulation and radioligand binding experiments to determine the impact of receptor mutagenesis on both chemokine and small molecule agonist and antagonist binding and action...... in CCR8. We find that the 7 transmembrane (7TM) receptor conserved disulfide bridge (7TM bridge) linking transmembrane helix (TM)III and ECL2 is crucial for chemokine and small molecule action, whereas the chemokine receptor conserved disulfide bridge between the N terminus and TMVII is needed only...

4-alkyl-L-(Dehydro)proline biosynthesis in actinobacteria involves N-terminal nucleophile-hydrolase activity of γ-glutamyltranspeptidase homolog for C-C bond cleavage

Science.gov (United States)

Zhong, Guannan; Zhao, Qunfei; Zhang, Qinglin; Liu, Wen

2017-07-01

γ-Glutamyltranspeptidases (γ-GTs), ubiquitous in glutathione metabolism for γ-glutamyl transfer/hydrolysis, are N-terminal nucleophile (Ntn)-hydrolase fold proteins that share an autoproteolytic process for self-activation. γ-GT homologues are widely present in Gram-positive actinobacteria where their Ntn-hydrolase activities, however, are not involved in glutathione metabolism. Herein, we demonstrate that the formation of 4-Alkyl-L-(dehydro)proline (ALDP) residues, the non-proteinogenic α-amino acids that serve as vital components of many bioactive metabolites found in actinobacteria, involves unprecedented Ntn-hydrolase activity of γ-GT homologue for C-C bond cleavage. The related enzymes share a key Thr residue, which acts as an internal nucleophile for protein hydrolysis and then as a newly released N-terminal nucleophile for carboxylate side-chain processing likely through the generation of an oxalyl-Thr enzyme intermediate. These findings provide mechanistic insights into the biosynthesis of various ALDP residues/associated natural products, highlight the versatile functions of Ntn-hydrolase fold proteins, and particularly generate interest in thus far less-appreciated γ-GT homologues in actinobacteria.

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

Directory of Open Access Journals (Sweden)

Gregory T. Forcherio

2017-07-01

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

Crystal structure of the starch-binding domain of glucoamylase from Aspergillus niger.

Science.gov (United States)

Suyama, Yousuke; Muraki, Norifumi; Kusunoki, Masami; Miyake, Hideo

2017-10-01

Glucoamylases are widely used commercially to produce glucose syrup from starch. The starch-binding domain (SBD) of glucoamylase from Aspergillus niger is a small globular protein containing a disulfide bond. The structure of A. niger SBD has been determined by NMR, but the conformation surrounding the disulfide bond was unclear. Therefore, X-ray crystal structural analysis was used to attempt to clarify the conformation of this region. The SBD was purified from an Escherichia coli-based expression system and crystallized at 293 K. The initial phase was determined by the molecular-replacement method, and the asymmetric unit of the crystal contained four protomers, two of which were related by a noncrystallographic twofold axis. Finally, the structure was solved at 2.0 Å resolution. The SBD consisted of seven β-strands and eight loops, and the conformation surrounding the disulfide bond was determined from a clear electron-density map. Comparison of X-ray- and NMR-determined structures of the free SBD showed no significant difference in the conformation of each β-strand, but the conformations of the loops containing the disulfide bond and the L5 loop were different. In particular, the difference in the position of the C α atom of Cys509 between the X-ray- and NMR-determined structures was 13.3 Å. In addition, the B factors of the amino-acid residues surrounding the disulfide bond are higher than those of other residues. Therefore, the conformation surrounding the disulfide bond is suggested to be highly flexible.

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

Science.gov (United States)

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

2018-05-01

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

The synthesis of unsymmetric disulfides for use as radio-protectives

International Nuclear Information System (INIS)

Chang, S.H.H.

1988-01-01

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

Covalent Bonding of Pyrrolobenzodiazepines (PBDs) to Terminal Guanine Residues within Duplex and Hairpin DNA Fragments

Science.gov (United States)

Mantaj, Julia; Jackson, Paul J. M.; Karu, Kersti; Rahman, Khondaker M.; Thurston, David E.

2016-01-01

Pyrrolobenzodiazepines (PBDs) are covalent-binding DNA-interactive agents with growing importance as payloads in Antibody Drug Conjugates (ADCs). Until now, PBDs were thought to covalently bond to C2-NH2 groups of guanines in the DNA-minor groove across a three-base-pair recognition sequence. Using HPLC/MS methodology with designed hairpin and duplex oligonucleotides, we have now demonstrated that the PBD Dimer SJG-136 and the C8-conjugated PBD Monomer GWL-78 can covalently bond to a terminal guanine of DNA, with the PBD skeleton spanning only two base pairs. Control experiments with the non-C8-conjugated anthramycin along with molecular dynamics simulations suggest that the C8-substituent of a PBD Monomer, or one-half of a PBD Dimer, may provide stability for the adduct. This observation highlights the importance of PBD C8-substituents, and also suggests that PBDs may bind to terminal guanines within stretches of DNA in cells, thus representing a potentially novel mechanism of action at the end of DNA strand breaks. PMID:27055050

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

Science.gov (United States)

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

2012-10-01

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

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

Science.gov (United States)

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

2014-03-01

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

Functional insight into the C-terminal extension of halolysin SptA from haloarchaeon Natrinema sp. J7.

Directory of Open Access Journals (Sweden)

Zhisheng Xu

Full Text Available Halolysin SptA from haloarchaeon Natrinema sp. J7 consists of a subtilisin-like catalytic domain and a C-terminal extension (CTE containing two cysteine residues. In this report, we have investigated the function of the CTE using recombinant enzymes expressed in Haloferax volcanii WFD11. Deletion of the CTE greatly reduced but did not abolish protease activity, which suggests that the CTE is not essential for enzyme folding. Mutational analysis suggests that residues Cys303 and Cys338 within the CTE form a disulfide bond that make this domain resistant to autocleavage and proteolysis under hypotonic conditions. Characterization of full-length and CTE-truncation enzymes indicates the CTE not only confers extra stability to the enzyme but also assists enzyme activity on protein substrates by facilitating binding at high salinities. Interestingly, homology modeling of the CTE yields a β-jelly roll-like structure similar to those seen in Claudin-binding domain of Clostridium perfringens enterotoxin (clostridial C-CPE and collagen binding domain (CBD, and the CTE also possesses collagen-binding activity, making it a potential candidate as an anchoring unit in drug delivery systems.

Structure of acostatin, a dimeric disintegrin from Southern copperhead (Agkistrodon contortrix contortrix), at 1.7 Å resolution

International Nuclear Information System (INIS)

Moiseeva, Natalia; Bau, Robert; Swenson, Stephen D.; Markland, Francis S. Jr; Choe, Jun-Yong; Liu, Zhi-Jie; Allaire, Marc

2008-01-01

Two acostatin heterodimers interact together to form an αββα tetramer. Disintegrins are a family of small (4–14 kDa) proteins that bind to another class of proteins, integrins. Therefore, as integrin inhibitors, they can be exploited as anticancer and antiplatelet agents. Acostatin, an αβ heterodimeric disintegrin, has been isolated from the venom of Southern copperhead (Agkistrodon contortrix contortrix). The three-dimensional structure of acostatin has been determined by macromolecular crystallography using the molecular-replacement method. The asymmetric unit of the acostatin crystals consists of two heterodimers. The structure has been refined to an R work and R free of 18.6% and 21.5%, respectively, using all data in the 20–1.7 Å resolution range. The structure of all subunits is similar and is well ordered into N-terminal and C-terminal clusters with four intramolecular disulfide bonds. The overall fold consists of short β-sheets, each of which is formed by a pair of antiparallel β-strands connected by β-turns and flexible loops of different lengths. Conformational flexibility is found in the RGD loops and in the C-terminal segment. The interaction of two N-terminal clusters via two intermolecular disulfide bridges anchors the αβ chains of the acostatin dimers. The C-terminal clusters of the heterodimer project in opposite directions and form a larger angle between them in comparison with other dimeric disintegrins. Extensive interactions are observed between two heterodimers, revealing an αββα acostatin tetramer. Further experiments are required to identify whether the αββα acostatin complex plays a functional role in vivo

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

OpenAIRE

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

2014-01-01

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

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

DEFF Research Database (Denmark)

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

1998-01-01

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

Functionalization with C-terminal cysteine enhances transfection efficiency of cell-penetrating peptides through dimer formation

Energy Technology Data Exchange (ETDEWEB)

Amand, Helene L., E-mail: helene.amand@chalmers.se [Chalmers University of Technology, Department of Chemical and Biological Engineering/Physical Chemistry, SE-412 96 Gothenburg (Sweden); Norden, Bengt, E-mail: norden@chalmers.se [Chalmers University of Technology, Department of Chemical and Biological Engineering/Physical Chemistry, SE-412 96 Gothenburg (Sweden); Fant, Kristina, E-mail: kristina.fant@sp.se [Chalmers University of Technology, Department of Chemical and Biological Engineering/Physical Chemistry, SE-412 96 Gothenburg (Sweden)

2012-02-17

Highlights: Black-Right-Pointing-Pointer Reversible CPP dimerisation is a simple yet efficient strategy to improve delivery. Black-Right-Pointing-Pointer Dimer formation enhances peptiplex stability, resulting in increased transfection. Black-Right-Pointing-Pointer By dimerisation, the CPP EB1 even gain endosomal escape properties while lowering cytotoxicity. -- Abstract: Cell-penetrating peptides have the ability to stimulate uptake of macromolecular cargo in mammalian cells in a non-toxic manner and therefore hold promise as efficient and well tolerated gene delivery vectors. Non-covalent peptide-DNA complexes ('peptiplexes') enter cells via endocytosis, but poor peptiplex stability and endosomal entrapment are considered as main barriers to peptide-mediated delivery. We explore a simple, yet highly efficient, strategy to improve the function of peptide-based vectors, by adding one terminal cysteine residue. This allows the peptide to dimerize by disulfide bond formation, increasing its affinity for nucleic acids by the 'chelate effect' and, when the bond is reduced intracellularly, letting the complex dissociate to deliver the nucleic acid. By introducing a single C-terminal cysteine in the classical CPP penetratin and the penetratin analogs PenArg and EB1, we show that this minor modification greatly enhances the transfection capacity for plasmid DNA in HEK293T cells. We conclude that this effect is mainly due to enhanced thermodynamic stability of the peptiplexes as endosome-disruptive chloroquine is still required for transfection and the effect is more pronounced for peptides with lower inherent DNA condensation capacity. Interestingly, for EB1, addition of one cysteine makes the peptide able to mediate transfection in absence of chloroquine, indicating that dimerisation can also improve endosomal escape properties. Further, the cytotoxicity of EB1 peptiplexes is considerably reduced, possibly due to lower concentration of free peptide

Functionalization with C-terminal cysteine enhances transfection efficiency of cell-penetrating peptides through dimer formation

International Nuclear Information System (INIS)

Åmand, Helene L.; Nordén, Bengt; Fant, Kristina

2012-01-01

Highlights: ► Reversible CPP dimerisation is a simple yet efficient strategy to improve delivery. ► Dimer formation enhances peptiplex stability, resulting in increased transfection. ► By dimerisation, the CPP EB1 even gain endosomal escape properties while lowering cytotoxicity. -- Abstract: Cell-penetrating peptides have the ability to stimulate uptake of macromolecular cargo in mammalian cells in a non-toxic manner and therefore hold promise as efficient and well tolerated gene delivery vectors. Non-covalent peptide-DNA complexes (“peptiplexes”) enter cells via endocytosis, but poor peptiplex stability and endosomal entrapment are considered as main barriers to peptide-mediated delivery. We explore a simple, yet highly efficient, strategy to improve the function of peptide-based vectors, by adding one terminal cysteine residue. This allows the peptide to dimerize by disulfide bond formation, increasing its affinity for nucleic acids by the “chelate effect” and, when the bond is reduced intracellularly, letting the complex dissociate to deliver the nucleic acid. By introducing a single C-terminal cysteine in the classical CPP penetratin and the penetratin analogs PenArg and EB1, we show that this minor modification greatly enhances the transfection capacity for plasmid DNA in HEK293T cells. We conclude that this effect is mainly due to enhanced thermodynamic stability of the peptiplexes as endosome-disruptive chloroquine is still required for transfection and the effect is more pronounced for peptides with lower inherent DNA condensation capacity. Interestingly, for EB1, addition of one cysteine makes the peptide able to mediate transfection in absence of chloroquine, indicating that dimerisation can also improve endosomal escape properties. Further, the cytotoxicity of EB1 peptiplexes is considerably reduced, possibly due to lower concentration of free peptide dimer resulting from its stronger binding to DNA.

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

Science.gov (United States)

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

2015-02-01

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

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

International Nuclear Information System (INIS)

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

2006-01-01

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

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

Directory of Open Access Journals (Sweden)

Yen Ten-Yang

2011-02-01

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

77 FR 553 - Surety Companies Acceptable on Federal Bonds: Termination; Western Bonding Company

Science.gov (United States)

2012-01-05

.... Department of the Treasury, Financial Management Service, Financial Accounting and Services Division, Surety..., 2011. Laura Carrico, Director, Financial Accounting and Services Division, Financial Management Service...: Termination; Western Bonding Company AGENCY: Financial Management Service, Fiscal Service, Department of the...

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

DEFF Research Database (Denmark)

Maeda, Kenji; Hägglund, Per; Finnie, Christine

2006-01-01

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

Structural Insight into the Critical Role of the N-Terminal Region in the Catalytic Activity of Dual-Specificity Phosphatase 26.

Directory of Open Access Journals (Sweden)

Eun-Young Won

Full Text Available Human dual-specificity phosphatase 26 (DUSP26 is a novel target for anticancer therapy because its dephosphorylation of the p53 tumor suppressor regulates the apoptosis of cancer cells. DUSP26 inhibition results in neuroblastoma cell cytotoxicity through p53-mediated apoptosis. Despite the previous structural studies of DUSP26 catalytic domain (residues 61-211, DUSP26-C, the high-resolution structure of its catalytically active form has not been resolved. In this study, we determined the crystal structure of a catalytically active form of DUSP26 (residues 39-211, DUSP26-N with an additional N-terminal region at 2.0 Å resolution. Unlike the C-terminal domain-swapped dimeric structure of DUSP26-C, the DUSP26-N (C152S monomer adopts a fold-back conformation of the C-terminal α8-helix and has an additional α1-helix in the N-terminal region. Consistent with the canonically active conformation of its protein tyrosine phosphate-binding loop (PTP loop observed in the structure, the phosphatase assay results demonstrated that DUSP26-N has significantly higher catalytic activity than DUSP26-C. Furthermore, size exclusion chromatography-multiangle laser scattering (SEC-MALS measurements showed that DUSP26-N (C152S exists as a monomer in solution. Notably, the crystal structure of DUSP26-N (C152S revealed that the N-terminal region of DUSP26-N (C152S serves a scaffolding role by positioning the surrounding α7-α8 loop for interaction with the PTP-loop through formation of an extensive hydrogen bond network, which seems to be critical in making the PTP-loop conformation competent for phosphatase activity. Our study provides the first high-resolution structure of a catalytically active form of DUSP26, which will contribute to the structure-based rational design of novel DUSP26-targeting anticancer therapeutics.

Folding and activity of hybrid sequence, disulfide-stabilized peptides

Energy Technology Data Exchange (ETDEWEB)

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

1990-08-01

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

Folding and activity of hybrid sequence, disulfide-stabilized peptides

International Nuclear Information System (INIS)

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

1990-01-01

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

Myotonic Dystrophy Type 1 RNA Crystal Structures Reveal Heterogeneous 1 × 1 Nucleotide UU Internal Loop Conformations

Energy Technology Data Exchange (ETDEWEB)

Kumar, Amit; Park, HaJeung; Fang, Pengfei; Parkesh, Raman; Guo, Min; Nettles, Kendall W.; Disney, Matthew D. (Scripps)

2012-03-27

RNA internal loops often display a variety of conformations in solution. Herein, we visualize conformational heterogeneity in the context of the 5'CUG/3'GUC repeat motif present in the RNA that causes myotonic dystrophy type 1 (DM1). Specifically, two crystal structures of a model DM1 triplet repeating construct, 5'r[{und UU}GGGC(C{und U}G){sub 3}GUCC]{sub 2}, refined to 2.20 and 1.52 {angstrom} resolution are disclosed. Here, differences in the orientation of the 5' dangling UU end between the two structures induce changes in the backbone groove width, which reveals that noncanonical 1 x 1 nucleotide UU internal loops can display an ensemble of pairing conformations. In the 2.20 {angstrom} structure, CUGa, the 5' UU forms a one hydrogen-bonded pair with a 5' UU of a neighboring helix in the unit cell to form a pseudoinfinite helix. The central 1 x 1 nucleotide UU internal loop has no hydrogen bonds, while the terminal 1 x 1 nucleotide UU internal loops each form a one-hydrogen bond pair. In the 1.52 {angstrom} structure, CUGb, the 5' UU dangling end is tucked into the major groove of the duplex. While the canonically paired bases show no change in base pairing, in CUGb the terminal 1 x 1 nucleotide UU internal loops now form two hydrogen-bonded pairs. Thus, the shift in the major groove induced by the 5' UU dangling end alters noncanonical base patterns. Collectively, these structures indicate that 1 x 1 nucleotide UU internal loops in DM1 may sample multiple conformations in vivo. This observation has implications for the recognition of this RNA, and other repeating transcripts, by protein and small molecule ligands.

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

Science.gov (United States)

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

2012-04-17

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

Selective disulfide reduction for labeling and enhancement of Fab antibody fragments

International Nuclear Information System (INIS)

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

2016-01-01

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

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

Science.gov (United States)

Bukowska-Faniband, Ewa; Hederstedt, Lars

2017-07-01

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

Enantioselective functionalization of allylic C-H bonds following a strategy of functionalization and diversification.

Science.gov (United States)

Sharma, Ankit; Hartwig, John F

2013-11-27

We report the enantioselective functionalization of allylic C-H bonds in terminal alkenes by a strategy involving the installation of a temporary functional group at the terminal carbon atom by C-H bond functionalization, followed by the catalytic diversification of this intermediate with a broad scope of reagents. The method consists of a one-pot sequence of palladium-catalyzed allylic C-H bond oxidation under neutral conditions to form linear allyl benzoates, followed by iridium-catalyzed allylic substitution. This overall transformation forms a variety of chiral products containing a new C-N, C-O, C-S, or C-C bond at the allylic position in good yield with a high branched-to-linear selectivity and excellent enantioselectivity (ee ≤97%). The broad scope of the overall process results from separating the oxidation and functionalization steps; by doing so, the scope of nucleophile encompasses those sensitive to direct oxidative functionalization. The high enantioselectivity of the overall process is achieved by developing an allylic oxidation that occurs without acid to form the linear isomer with high selectivity. These allylic functionalization processes are amenable to an iterative sequence leading to (1,n)-functionalized products with catalyst-controlled diastereo- and enantioselectivity. The utility of the method in the synthesis of biologically active molecules has been demonstrated.

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

Science.gov (United States)

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

1996-10-25

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

13C-NMR spectra and bonding situation in ketenimines

International Nuclear Information System (INIS)

Firl, J.; Runge, W.; Hartmann, W.; Utikal, H.P.

1975-01-01

13 C-NMR spectra of a series of substituted ketenimines are reported. The terminal carbon resonances are found at unusual high fields between delta 37 and 78, while the central carbon signals appear around delta 189 - 196. On the basis of these results, the bonding situation in ketenimines has been discussed. (auth.)

Automation of C-terminal sequence analysis of 2D-PAGE separated proteins

Directory of Open Access Journals (Sweden)

P.P. Moerman

2014-06-01

Full Text Available Experimental assignment of the protein termini remains essential to define the functional protein structure. Here, we report on the improvement of a proteomic C-terminal sequence analysis method. The approach aims to discriminate the C-terminal peptide in a CNBr-digest where Met-Xxx peptide bonds are cleaved in internal peptides ending at a homoserine lactone (hsl-derivative. pH-dependent partial opening of the lactone ring results in the formation of doublets for all internal peptides. C-terminal peptides are distinguished as singlet peaks by MALDI-TOF MS and MS/MS is then used for their identification. We present a fully automated protocol established on a robotic liquid-handling station.

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

Energy Technology Data Exchange (ETDEWEB)

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

1985-11-01

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

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

International Nuclear Information System (INIS)

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

1985-01-01

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

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

Science.gov (United States)

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

2016-01-01

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

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

Science.gov (United States)

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

2012-01-01

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

Cobalt-Catalyzed, Aminoquinoline-Directed sp2 C-H Bond Alkenylation by Alkynes**

Science.gov (United States)

Grigorjeva, Liene; Daugulis, Olafs

2014-01-01

We have developed a method for cobalt-catalyzed, aminoquinoline- and picolinamide-directed sp2 C-H bond alkenylation by alkynes. Method shows excellent functional group tolerance and both internal and terminal alkynes are competent substrates for the coupling. The reaction employs Co(OAc)2*4H2O catalyst, Mn(OAc)2 cocatalyst, and oxygen from air as a terminal oxidant. PMID:25060365

Autoactivation of mouse trypsinogens is regulated by chymotrypsin C via cleavage of the autolysis loop.

Science.gov (United States)

Németh, Balázs Csaba; Wartmann, Thomas; Halangk, Walter; Sahin-Tóth, Miklós

2013-08-16

Chymotrypsin C (CTRC) is a proteolytic regulator of trypsinogen autoactivation in humans. CTRC cleavage of the trypsinogen activation peptide stimulates autoactivation, whereas cleavage of the calcium binding loop promotes trypsinogen degradation. Trypsinogen mutations that alter these regulatory cleavages lead to increased intrapancreatic trypsinogen activation and cause hereditary pancreatitis. The aim of this study was to characterize the regulation of autoactivation of mouse trypsinogens by mouse Ctrc. We found that the mouse pancreas expresses four trypsinogen isoforms to high levels, T7, T8, T9, and T20. Only the T7 activation peptide was cleaved by mouse Ctrc, causing negligible stimulation of autoactivation. Surprisingly, mouse Ctrc poorly cleaved the calcium binding loop in all mouse trypsinogens. In contrast, mouse Ctrc readily cleaved the Phe-150-Gly-151 peptide bond in the autolysis loop of T8 and T9 and inhibited autoactivation. Mouse chymotrypsin B also cleaved the same peptide bond but was 7-fold slower. T7 was less sensitive to chymotryptic regulation, which involved slow cleavage of the Leu-149-Ser-150 peptide bond in the autolysis loop. Modeling indicated steric proximity of the autolysis loop and the activation peptide in trypsinogen, suggesting the cleaved autolysis loop may directly interfere with activation. We conclude that autoactivation of mouse trypsinogens is under the control of mouse Ctrc with some notable differences from the human situation. Thus, cleavage of the trypsinogen activation peptide or the calcium binding loop by Ctrc is unimportant. Instead, inhibition of autoactivation via cleavage of the autolysis loop is the dominant mechanism that can mitigate intrapancreatic trypsinogen activation.

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

DEFF Research Database (Denmark)

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

2010-01-01

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

The C-terminus of the B-chain of human insulin-like peptide 5 is critical for cognate RXFP4 receptor activity.

Science.gov (United States)

Patil, Nitin A; Bathgate, Ross A D; Kocan, Martina; Ang, Sheng Yu; Tailhades, Julien; Separovic, Frances; Summers, Roger; Grosse, Johannes; Hughes, Richard A; Wade, John D; Hossain, Mohammed Akhter

2016-04-01

Insulin-like peptide 5 (INSL5) is an orexigenic peptide hormone belonging to the relaxin family of peptides. It is expressed primarily in the L-cells of the colon and has a postulated key role in regulating food intake. Its G protein-coupled receptor, RXFP4, is a potential drug target for treating obesity and anorexia. We studied the effect of modification of the C-terminus of the A and B-chains of human INSL5 on RXFP4 binding and activation. Three variants of human INSL5 were prepared using solid phase peptide synthesis and subsequent sequential regioselective disulfide bond formation. The peptides were synthesized as C-terminal acids (both A- and B-chains with free C-termini, i.e., the native form), amides (both chains as the C-terminal amide) and one analog with the C-terminus of its A-chain as the amide and the C-terminus of the B-chain as the acid. The results showed that C-terminus of the B-chain is more important than that of the A-chain for RXFP4 binding and activity. Amidation of the A-chain C-terminus does not have any effect on the INSL5 activity. The difference in RXFP4 binding and activation between the three peptides is believed to be due to electrostatic interaction of the free carboxylate of INSL5 with a positively charged residue (s), either situated within the INSL5 molecule itself or in the receptor extracellular loops.

Rhodium-Catalyzed C-C Bond Formation via Heteroatom-Directed C-H Bond Activation

Energy Technology Data Exchange (ETDEWEB)

Colby, Denise; Bergman, Robert; Ellman, Jonathan

2010-05-13

Once considered the 'holy grail' of organometallic chemistry, synthetically useful reactions employing C-H bond activation have increasingly been developed and applied to natural product and drug synthesis over the past decade. The ubiquity and relative low cost of hydrocarbons makes C-H bond functionalization an attractive alternative to classical C-C bond forming reactions such as cross-coupling, which require organohalides and organometallic reagents. In addition to providing an atom economical alternative to standard cross - coupling strategies, C-H bond functionalization also reduces the production of toxic by-products, thereby contributing to the growing field of reactions with decreased environmental impact. In the area of C-C bond forming reactions that proceed via a C-H activation mechanism, rhodium catalysts stand out for their functional group tolerance and wide range of synthetic utility. Over the course of the last decade, many Rh-catalyzed methods for heteroatom-directed C-H bond functionalization have been reported and will be the focus of this review. Material appearing in the literature prior to 2001 has been reviewed previously and will only be introduced as background when necessary. The synthesis of complex molecules from relatively simple precursors has long been a goal for many organic chemists. The ability to selectively functionalize a molecule with minimal pre-activation can streamline syntheses and expand the opportunities to explore the utility of complex molecules in areas ranging from the pharmaceutical industry to materials science. Indeed, the issue of selectivity is paramount in the development of all C-H bond functionalization methods. Several groups have developed elegant approaches towards achieving selectivity in molecules that possess many sterically and electronically similar C-H bonds. Many of these approaches are discussed in detail in the accompanying articles in this special issue of Chemical Reviews. One approach

Enhanced stiffness of silk-like fibers by loop formation in the corona leads to stronger gels.

Science.gov (United States)

Rombouts, Wolf H; Domeradzka, Natalia E; Werten, Marc W T; Leermakers, Frans A M; de Vries, Renko J; de Wolf, Frits A; van der Gucht, Jasper

2016-11-01

We study the self-assembly of protein polymers consisting of a silk-like block flanked by two hydrophilic blocks, with a cysteine residue attached to the C-terminal end. The silk blocks self-assemble to form fibers while the hydrophilic blocks form a stabilizing corona. Entanglement of the fibers leads to the formation of hydrogels. Under oxidizing conditions the cysteine residues form disulfide bridges, effectively connecting two corona chains at their ends to form a loop. We find that this leads to a significant increase in the elastic modulus of the gels. Using atomic force microscopy, we show that this stiffening is due to an increase of the persistence length of the fibers. Self-consistent-field calculations indicate a slight decrease of the lateral pressure in the corona upon loop formation. We argue that this small decrease in the repulsive interactions affects the stacking of the silk-like blocks in the core, resulting in a more rigid fiber. © 2016 Wiley Periodicals, Inc.

Resistance of Subtype C HIV-1 Strains to Anti-V3 Loop Antibodies

Directory of Open Access Journals (Sweden)

David Almond

2012-01-01

Full Text Available HIV-1’s subtype C V3 loop consensus sequence exhibits increased resistance to anti-V3 antibody-mediated neutralization as compared to the subtype B consensus sequence. The dynamic 3D structure of the consensus C V3 loop crown, visualized by ab initio folding, suggested that the resistance derives from structural rigidity and non-β-strand secondary protein structure in the N-terminal strand of the β-hairpin of the V3 loop crown, which is where most known anti-V3 loop antibodies bind. The observation of either rigidity or non-β-strand structure in this region correlated with observed resistance to antibody-mediated neutralization in a series of chimeric pseudovirus (psV mutants. The results suggest the presence of an epitope-independent, neutralization-relevant structural difference in the antibody-targeted region of the V3 loop crown between subtype C and subtype B, a difference that we hypothesize may contribute to the divergent pattern of global spread between these subtypes. As antibodies to a variable loop were recently identified as an inverse correlate of risk for HIV infection, the structure-function relationships discussed in this study may have relevance to HIV vaccine research.

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

DEFF Research Database (Denmark)

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

2015-01-01

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

A novel approach for a C-11C bond formation: synthesis of 17α-([11C]prop-1-ynyl)-3-methoxy-3,17β-estradiol

International Nuclear Information System (INIS)

Wuest, F.; Zessin, J.

2002-01-01

A novel method for a 11 C-C bond formation was developed, employing a cross-coupling reaction between a terminal acetylene and [ 11 C]methyl iodide. The method was used for the synthesis of 17α-([ 11 C]prop-1-ynyl)-3-methoxy-3,17β-estadiol. (orig.)

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

International Nuclear Information System (INIS)

Ou Wu; Silver, Jonathan

2006-01-01

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

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

NARCIS (Netherlands)

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

2002-01-01

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

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

Science.gov (United States)

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

2016-11-25

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

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

Science.gov (United States)

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

2018-05-16

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

Metal-organic cooperative catalysis in C-H and C-C bond activation and its concurrent recovery.

Science.gov (United States)

Park, Young Jun; Park, Jung-Woo; Jun, Chul-Ho

2008-02-01

The development of an efficient catalytic activation (cleavage) system for C-H and C-C bonds is an important challenge in organic synthesis, because these bonds comprise a variety of organic molecules such as natural products, petroleum oils, and polymers on the earth. Among many elegant approaches utilizing transition metals to activate C-H and C-C bonds facilely, chelation-assisted protocols based on the coordinating ability of an organic moiety have attracted great attention, though they have often suffered from the need for an intact coordinating group in a substrate. In this Account, we describe our entire efforts to activate C-H or C-C bonds adjacent to carbonyl groups by employing a new concept of metal-organic cooperative catalysis (MOCC), which enables the temporal installation of a 2-aminopyridyl group into common aldehydes or ketones in a catalytic way. Consequently, a series of new catalytic reactions such as alcohol hydroacylation, oxo-ester synthesis, C-C triple bond cleavage, hydrative dimerization of alkynes, and skeletal rearrangements of cyclic ketones was realized through MOCC. In particular, in the quest for an optimized MOCC system composed of a Wilkinson's catalyst (Ph 3P) 3RhCl and an organic catalyst (2-amino-3-picoline), surprising efficiency enhancements could be achieved when benzoic acid and aniline were introduced as promoters for the aldimine formation process. Furthermore, a notable accomplishment of C-C bond activation has been made using 2-amino-3-picoline as a temporary chelating auxiliary in the reactions of unstrained ketones with various terminal olefins and Wilkinson's catalyst. In the case of seven-membered cyclic ketones, an interesting ring contraction to five- or six-membered ones takes place through skeletal rearrangements initiated by the C-C bond activation of MOCC. On the other hand, the fundamental advances of these catalytic systems into recyclable processes could be achieved by immobilizing both metal and organic

75 FR 6791 - Surety Companies Acceptable on Federal Bonds-Termination: Trinity Universal Insurance Company

Science.gov (United States)

2010-02-10

.... However, no new bonds should be accepted from this company, and bonds that are continuous in nature should... DEPARTMENT OF THE TREASURY Fiscal Service Surety Companies Acceptable on Federal Bonds--Termination: Trinity Universal Insurance Company AGENCY: Financial Management Service, Fiscal Service...

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

Energy Technology Data Exchange (ETDEWEB)

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

2014-09-16

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

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

International Nuclear Information System (INIS)

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

2014-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2017-07-01

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

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

International Nuclear Information System (INIS)

Xu, Yurun; Chen, Dajun

2017-01-01

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

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

Science.gov (United States)

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

2013-07-05

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

Cobalt-catalyzed, aminoquinoline-directed C(sp²)-H bond alkenylation by alkynes.

Science.gov (United States)

Grigorjeva, Liene; Daugulis, Olafs

2014-09-15

A method for cobalt-catalyzed, aminoquinoline- and picolinamide-directed C(sp(2))-H bond alkenylation by alkynes was developed. The method shows excellent functional-group tolerance and both internal and terminal alkynes are competent substrates for the coupling. The reaction employs a Co(OAc)2⋅4 H2O catalyst, Mn(OAc)2 co-catalyst, and oxygen (from air) as a terminal oxidant. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Stereochemical determinants of C-terminal specificity in PDZ peptide-binding domains: a novel contribution of the carboxylate-binding loop.

Science.gov (United States)

Amacher, Jeanine F; Cushing, Patrick R; Bahl, Christopher D; Beck, Tobias; Madden, Dean R

2013-02-15

PDZ (PSD-95/Dlg/ZO-1) binding domains often serve as cellular traffic engineers, controlling the localization and activity of a wide variety of binding partners. As a result, they play important roles in both physiological and pathological processes. However, PDZ binding specificities overlap, allowing multiple PDZ proteins to mediate distinct effects on shared binding partners. For example, several PDZ domains bind the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), an epithelial ion channel mutated in CF. Among these binding partners, the CFTR-associated ligand (CAL) facilitates post-maturational degradation of the channel and is thus a potential therapeutic target. Using iterative optimization, we previously developed a selective CAL inhibitor peptide (iCAL36). Here, we investigate the stereochemical basis of iCAL36 specificity. The crystal structure of iCAL36 in complex with the CAL PDZ domain reveals stereochemical interactions distributed along the peptide-binding cleft, despite the apparent degeneracy of the CAL binding motif. A critical selectivity determinant that distinguishes CAL from other CFTR-binding PDZ domains is the accommodation of an isoleucine residue at the C-terminal position (P(0)), a characteristic shared with the Tax-interacting protein-1. Comparison of the structures of these two PDZ domains in complex with ligands containing P(0) Leu or Ile residues reveals two distinct modes of accommodation for β-branched C-terminal side chains. Access to each mode is controlled by distinct residues in the carboxylate-binding loop. These studies provide new insights into the primary sequence determinants of binding motifs, which in turn control the scope and evolution of PDZ interactomes.

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

DEFF Research Database (Denmark)

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

2005-01-01

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

75 FR 36153 - Surety Companies Acceptable on Federal Bonds-Terminations: Victore Insurance Company

Science.gov (United States)

2010-06-24

... from this company, and bonds that are continuous in nature should not be renewed. The Circular may be... DEPARTMENT OF THE TREASURY Fiscal Service Surety Companies Acceptable on Federal Bonds--Terminations: Victore Insurance Company AGENCY: Financial Management Service, Fiscal Service, Department of the...

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

Science.gov (United States)

Fernandez, Julio M.

2008-03-01

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

Solution structure and dynamics of C-terminal regulatory domain of Vibrio vulnificus extracellular metalloprotease

Energy Technology Data Exchange (ETDEWEB)

Yun, Ji-Hye; Kim, Heeyoun [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Park, Jung Eun [Department of Biotechnology, College of Natural Sciences, Chosun University, Gwangju 501-759 (Korea, Republic of); Lee, Jung Sup, E-mail: jsplee@mail.chosun.ac.kr [Department of Biotechnology, College of Natural Sciences, Chosun University, Gwangju 501-759 (Korea, Republic of); Lee, Weontae, E-mail: wlee@spin.yonsei.ac.kr [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of)

2013-01-11

Highlights: Black-Right-Pointing-Pointer We have determined solution structures of vEP C-terminal regulatory domain. Black-Right-Pointing-Pointer vEP C-ter100 has a compact {beta}-barrel structure with eight anti-parallel {beta}-strands. Black-Right-Pointing-Pointer Solution structure of vEP C-ter100 shares its molecular topology with that of the collagen-binding domain of collagenase. Black-Right-Pointing-Pointer Residues in the {beta}3 region of vEP C-ter100 might be important in putative ligand/receptor binding. Black-Right-Pointing-Pointer vEP C-ter100 interacts strongly with iron ion. -- Abstract: An extracellular metalloprotease (vEP) secreted by Vibrio vulnificus ATCC29307 is a 45-kDa proteolytic enzyme that has prothrombin activation and fibrinolytic activities during bacterial infection. The action of vEP could result in clotting that could serve to protect the bacteria from the host defense machinery. Very recently, we showed that the C-terminal propeptide (C-ter100), which is unique to vEP, is involved in regulation of vEP activity. To understand the structural basis of this function of vEP C-ter100, we have determined the solution structure and backbone dynamics using multidimensional nuclear magnetic resonance spectroscopy. The solution structure shows that vEP C-ter100 is composed of eight anti-parallel {beta}-strands with a unique fold that has a compact {beta}-barrel formation which stabilized by hydrophobic and hydrogen bonding networks. Protein dynamics shows that the overall structure, including loops, is very rigid and stabilized. By structural database analysis, we found that vEP C-ter100 shares its topology with that of the collagen-binding domain of collagenase, despite low sequence homology between the two domains. Fluorescence assay reveals that vEP C-ter100 interacts strongly with iron (Fe{sup 3+}). These findings suggest that vEP protease might recruit substrate molecules, such as collagen, by binding at C-ter100 and that vEP participates

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

Directory of Open Access Journals (Sweden)

Juan Martínez-Oliván

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

The Processed Amino-Terminal Fragment of Human TLR7 Acts as a Chaperone To Direct Human TLR7 into Endosomes

Science.gov (United States)

Shepherd, Dawn; Booth, Sarah; Waithe, Dominic; Reis e Sousa, Caetano

2015-01-01

TLR7 mediates innate immune responses to viral RNA in endocytic compartments. Mouse and human (h)TLR7 undergo proteolytic cleavage, resulting in the generation of a C-terminal fragment that accumulates in endosomes and associates with the signaling adaptor MyD88 upon receptor triggering by TLR7 agonists. Although mouse TLR7 is cleaved in endosomes by acidic proteases, hTLR7 processing can occur at neutral pH throughout the secretory pathway through the activity of furin-like proprotein convertases. However, the mechanisms by which cleaved hTLR7 reaches the endosomal compartment remain unclear. In this study, we demonstrate that, after hTLR7 proteolytic processing, the liberated amino (N)-terminal fragment remains bound to the C terminus through disulfide bonds and provides key trafficking information that ensures correct delivery of the complex to endosomal compartments. In the absence of the N-terminal fragment, the C-terminal fragment is redirected to the cell surface, where it is functionally inactive. Our data reveal a novel role for the N terminus of hTLR7 as a molecular chaperone that provides processed hTLR7 with the correct targeting instructions to reach the endosomal compartment, hence ensuring its biological activity and preventing inadvertent cell surface responses to self-RNA. PMID:25917086

Influence of Dimerization of Lipopeptide Laur-Orn-Orn-Cys-NH2 and an N-terminal Peptide of Human Lactoferricin on Biological Activity.

Science.gov (United States)

Kamysz, Elżbieta; Sikorska, Emilia; Dawgul, Małgorzata; Tyszkowski, Rafał; Kamysz, Wojciech

Lactoferrin (LF) is a naturally occurring antimicrobial peptide that is cleaved by pepsin to lactoferricin (LFcin). LFcin has an enhanced antimicrobial activity as compared to that of LF. Recently several hetero- and homodimeric antimicrobial peptides stabilized by a single disulfide bond linking linear polypeptide chains have been discovered. We have demonstrated that the S-S bond heterodimerization of lipopeptide Laur-Orn-Orn-Cys-NH 2 (peptide III) and the synthetic N -terminal peptide of human lactoferricin (peptide I) yields a dimer (peptide V), which is almost as microbiologically active as the more active monomer and at the same time it is much less toxic. Furthermore, it has been found that the S-S bond homodimerization of both peptide I and peptide III did not affect antimicrobial and haemolytic activity of the compounds. The homo- and heterodimerization of peptides I and III resulted in either reduction or loss of antifungal activity. This work suggests that heterodimerization of antimicrobial lipopeptides via intermolecular disulfide bond might be a powerful modification deserving consideration in the design of antimicrobial peptides.

Atomic structure of the sweet-tasting protein thaumatin I at pH 8.0 reveals the large disulfide-rich region in domain II to be sensitive to a pH change

Energy Technology Data Exchange (ETDEWEB)

Masuda, Tetsuya, E-mail: t2masuda@kais.kyoto-u.ac.jp [Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011 (Japan); Department of Natural Resources, Graduate School of Global Environmental Studies, Kyoto University, Gokasho, Uji, Kyoto 611-0011 (Japan); Ohta, Keisuke [Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011 (Japan); Department of Natural Resources, Graduate School of Global Environmental Studies, Kyoto University, Gokasho, Uji, Kyoto 611-0011 (Japan); Mikami, Bunzo [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011 (Japan); Kitabatake, Naofumi [Department of Foods and Human Nutrition, Notre Dame Seishin University, Okayama 700-8516 (Japan); Tani, Fumito [Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011 (Japan); Department of Natural Resources, Graduate School of Global Environmental Studies, Kyoto University, Gokasho, Uji, Kyoto 611-0011 (Japan)

2012-03-02

Highlights: Black-Right-Pointing-Pointer Structure of a recombinant thaumatin at pH 8.0 determined at a resolution of 1.0 A. Black-Right-Pointing-Pointer Substantial fluctuations of a loop in domain II was found in the structure at pH 8.0. Black-Right-Pointing-Pointer B-factors for Lys137, Lys163, and Lys187 were significantly affected by pH change. Black-Right-Pointing-Pointer An increase in mobility might play an important role in the heat-induced aggregation. -- Abstract: Thaumatin, an intensely sweet-tasting plant protein, elicits a sweet taste at 50 nM. Although the sweetness remains when thaumatin is heated at 80 Degree-Sign C for 4 h under acid conditions, it rapidly declines when heating at a pH above 6.5. To clarify the structural difference at high pH, the atomic structure of a recombinant thaumatin I at pH 8.0 was determined at a resolution of 1.0 A. Comparison to the crystal structure of thaumatin at pH 7.3 and 7.0 revealed the root-mean square deviation value of a C{alpha} atom to be substantially greater in the large disulfide-rich region of domain II, especially residues 154-164, suggesting that a loop region in domain II to be affected by solvent conditions. Furthermore, B-factors of Lys137, Lys163, and Lys187 were significantly affected by pH change, suggesting that a striking increase in the mobility of these lysine residues, which could facilitate a reaction with a free sulfhydryl residue produced via the {beta}-elimination of disulfide bonds by heating at a pH above 7.0. The increase in mobility of lysine residues as well as a loop region in domain II might play an important role in the heat-induced aggregation of thaumatin above pH 7.0.

Reactivity of the parent amido complexes of iridium with olefins: C-NH2 bond formation versus C-H activation.

Science.gov (United States)

Mena, Inmaculada; García-Orduña, Pilar; Polo, Víctor; Lahoz, Fernando J; Casado, Miguel A; Oro, Luis A

2017-08-29

Herein we report on the different chemical reactivity displayed by two mononuclear terminal amido compounds depending on the nature of the coordinated diene. Hence, treatment of amido-bridged iridium complexes [{Ir(μ-NH 2 )(tfbb)} 3 ] (1; tfbb = tetrafluorobenzobarrelene) with dppp (dppp = bis(diphenylphosphane)propane) leads to the rupture of the amido bridges forming the mononuclear terminal amido compound [Ir(NH 2 )(dppp)(tfbb)] (3) in the first stage. On changing the reaction conditions, the formation of a C-NH 2 bond between the amido moiety and the coordinated diene is observed and a new dinuclear complex [{Ir(1,2-η 2 -4-κ-C 12 H 8 F 4 N)(dppp)} 2 (μ-dppp)] (4) has been isolated. On the contrary, the diiridium amido-bridged complex [{Ir(μ-NH 2 )(cod)} 2 ] (2; cod = 1,5-cyclooctadiene) in the presence of dppb (dppb = bis(diphenylphosphane)butane) allows the isolation of a mononuclear complex [Ir(1,2,3-η 3 -6-κ-C 8 H 10 )H(dppb)] (5), as a consequence of the extrusion of ammonia. The monitoring of the reaction of 2 with dppb (and dppp) allowed us to detect terminal amido complexes [Ir(NH 2 )(P-P)(cod)] (P-P = dppb (6), dppp (7)) in solution, as confirmed by an X-ray analysis of 7. Complex 7 was observed to evolve into hydrido species 5 at room temperature. DFT studies showed that C-H bond activation occurs through the deprotonation of one methylene fragment of the cod ligand by the highly basic terminal amido moiety instead of C-H oxidative addition to the Ir(i) center.

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

Science.gov (United States)

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

2016-04-26

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

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

Science.gov (United States)

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

2018-07-15

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

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

Directory of Open Access Journals (Sweden)

Minh Tan Nguyen

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

75 FR 33897 - Surety Companies Acceptable on Federal Bonds-Terminations: Commercial Alliance Insurance Company

Science.gov (United States)

2010-06-15

... be accepted from this company, and bonds that are continuous in nature should not be renewed. The... DEPARTMENT OF THE TREASURY Fiscal Service Surety Companies Acceptable on Federal Bonds--Terminations: Commercial Alliance Insurance Company AGENCY: Financial Management Service, Fiscal Service...

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

NARCIS (Netherlands)

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

2006-01-01

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

75 FR 38188 - Surety Companies Acceptable on Federal Bonds-Termination: Stonebridge Casualty Insurance Company

Science.gov (United States)

2010-07-01

... should be accepted from this company, and bonds that are continuous in nature should not be renewed. The... DEPARTMENT OF THE TREASURY Fiscal Service [NAIC 10952] Surety Companies Acceptable on Federal Bonds--Termination: Stonebridge Casualty Insurance Company AGENCY: Financial Management Service, Fiscal...

75 FR 81331 - Surety Companies Acceptable on Federal Bonds: Termination-Penn Millers Insurance Company

Science.gov (United States)

2010-12-27

... should be accepted from this company, and bonds that are continuous in nature should not be renewed. The... DEPARTMENT OF THE TREASURY Fiscal Service Surety Companies Acceptable on Federal Bonds: Termination--Penn Millers Insurance Company AGENCY: Financial Management Service, Fiscal Service, Department...

Probing hydrogen bonds in the antibody-bound HIV-1 gp120 V3 loop by solid state NMR REDOR measurements

Energy Technology Data Exchange (ETDEWEB)

Balbach, John J. [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States); Yang Jun; Weliky, David P. [Michigan State University, Department of Chemistry (United States); Steinbach, Peter J. [National Institutes of Health, Center for Molecular Modeling, Center for Information Technology (United States); Tugarinov, Vitali; Anglister, Jacob [Weizmann Institute of Science, Department of Structural Biology (Israel); Tycko, Robert [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States)

2000-04-15

We describe solid state NMR measurements on frozen solutions of the complex of the 24-residue HIV-1 gp120 V3 loop peptide RP135 with the Fab fragment of the anti-gp120 antibody 0.5{beta}, using rotational echo double resonance (REDOR). In order to probe possible hydrogen bonding between arginine side chains and glycine backbone carbonyls in the region of the conserved Gly-Pro-Gly-Arg (GPGR) motif of the V3 loop, RP135 samples were prepared with {sup 15}N labels at the {eta} nitrogen positions of arginine side chains and {sup 13}C labels at glycine carbonyl positions and {sup 13}C-detected {sup 13}C-{sup 15}N REDOR measurements were performed on peptide/antibody complexes of these labeled samples. Such hydrogen bonding was previously observed in a crystal structure of the V3 loop peptide/antibody complex RP142/59.1 [Ghiara et al. (1994) Science, 264, 82-85], but is shown by the REDOR measurements to be absent in the RP135/0.5{beta} complex. These results confirm the antibody-dependent conformational differences in the GPGR motif suggested by previously reported solid state NMR measurements of {phi} and {psi} backbone dihedral angles in the RP135/0.5{beta} complex. In addition, we describe REDOR measurements on the helical synthetic peptide MB(i+4)EK in frozen solution that establish our ability to detect {sup 13}C-{sup 15}N dipole-dipole couplings in the distance range appropriate to these hydrogen bonding studies. We also report the results of molecular modeling calculations on the central portion RP135, using a combination of the solid state NMR restraints of Weliky et al. [Nat. Struct. Biol., 6, 141-145, 1999] and the liquid state NMR restraints of Tugarinov et al. (Nat. Struct. Biol., 6, 331-335, 1999]. The dynamics calculations demonstrate the mutual compatibility of the two sets of experimental structural restraints and reduce ambiguities in the solid state NMR restraints that result from symmetry and signal-to-noise considerations.

Probing hydrogen bonds in the antibody-bound HIV-1 gp120 V3 loop by solid state NMR REDOR measurements

International Nuclear Information System (INIS)

Balbach, John J.; Yang Jun; Weliky, David P.; Steinbach, Peter J.; Tugarinov, Vitali; Anglister, Jacob; Tycko, Robert

2000-01-01

We describe solid state NMR measurements on frozen solutions of the complex of the 24-residue HIV-1 gp120 V3 loop peptide RP135 with the Fab fragment of the anti-gp120 antibody 0.5β, using rotational echo double resonance (REDOR). In order to probe possible hydrogen bonding between arginine side chains and glycine backbone carbonyls in the region of the conserved Gly-Pro-Gly-Arg (GPGR) motif of the V3 loop, RP135 samples were prepared with 15 N labels at the η nitrogen positions of arginine side chains and 13 C labels at glycine carbonyl positions and 13 C-detected 13 C- 15 N REDOR measurements were performed on peptide/antibody complexes of these labeled samples. Such hydrogen bonding was previously observed in a crystal structure of the V3 loop peptide/antibody complex RP142/59.1 [Ghiara et al. (1994) Science, 264, 82-85], but is shown by the REDOR measurements to be absent in the RP135/0.5β complex. These results confirm the antibody-dependent conformational differences in the GPGR motif suggested by previously reported solid state NMR measurements of φ and Ψ backbone dihedral angles in the RP135/0.5β complex. In addition, we describe REDOR measurements on the helical synthetic peptide MB(i+4)EK in frozen solution that establish our ability to detect 13 C- 15 N dipole-dipole couplings in the distance range appropriate to these hydrogen bonding studies. We also report the results of molecular modeling calculations on the central portion RP135, using a combination of the solid state NMR restraints of Weliky et al. [Nat. Struct. Biol., 6, 141-145, 1999] and the liquid state NMR restraints of Tugarinov et al. (Nat. Struct. Biol., 6, 331-335, 1999]. The dynamics calculations demonstrate the mutual compatibility of the two sets of experimental structural restraints and reduce ambiguities in the solid state NMR restraints that result from symmetry and signal-to-noise considerations

Loop Evolution Observed with AIA and Hi-C

Science.gov (United States)

Mulu-Moore, Fana; Winebarger, Amy R.; Cirtain, Jonathan W.; Kobayashi, Ken; Korreck, Kelly E.; Golub, Leon; Kuzin, Sergei; Walsh, Robert William; DeForest, Craig E.; De Pontieu, Bart;

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

Science.gov (United States)

Tarasava, Katsiaryna; Chesnov, Serge; Freisinger, Eva

2016-05-01

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

Full cyclic coordinate descent: solving the protein loop closure problem in Cα space

Directory of Open Access Journals (Sweden)

Hamelryck Thomas

2005-06-01

Full Text Available Abstract Background Various forms of the so-called loop closure problem are crucial to protein structure prediction methods. Given an N- and a C-terminal end, the problem consists of finding a suitable segment of a certain length that bridges the ends seamlessly. In homology modelling, the problem arises in predicting loop regions. In de novo protein structure prediction, the problem is encountered when implementing local moves for Markov Chain Monte Carlo simulations. Most loop closure algorithms keep the bond angles fixed or semi-fixed, and only vary the dihedral angles. This is appropriate for a full-atom protein backbone, since the bond angles can be considered as fixed, while the (φ, ψ dihedral angles are variable. However, many de novo structure prediction methods use protein models that only consist of Cα atoms, or otherwise do not make use of all backbone atoms. These methods require a method that alters both bond and dihedral angles, since the pseudo bond angle between three consecutive Cα atoms also varies considerably. Results Here we present a method that solves the loop closure problem for Cα only protein models. We developed a variant of Cyclic Coordinate Descent (CCD, an inverse kinematics method from the field of robotics, which was recently applied to the loop closure problem. Since the method alters both bond and dihedral angles, which is equivalent to applying a full rotation matrix, we call our method Full CCD (FCDD. FCCD replaces CCD's vector-based optimization of a rotation around an axis with a singular value decomposition-based optimization of a general rotation matrix. The method is easy to implement and numerically stable. Conclusion We tested the method's performance on sets of random protein Cα segments between 5 and 30 amino acids long, and a number of loops of length 4, 8 and 12. FCCD is fast, has a high success rate and readily generates conformations close to those of real loops. The presence of constraints

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

Energy Technology Data Exchange (ETDEWEB)

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

2002-07-01

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

Interfacial chemical bonding state and band alignment of CaF2/hydrogen-terminated diamond heterojunction

International Nuclear Information System (INIS)

Liu, J. W.; Liao, M. Y.; Cheng, S. H.; Imura, M.; Koide, Y.

2013-01-01

CaF 2 films are deposited on hydrogen-terminated diamond (H-diamond) by a radio-frequency sputter-deposition technique at room temperature. Interfacial chemical bonding state and band alignment of CaF 2 /H-diamond heterojunction are investigated by X-ray photoelectron spectroscopy. It is confirmed that there are only C-Ca bonds at the CaF 2 /H-diamond heterointerface. Valence and conductance band offsets of the CaF 2 /H-diamond heterojunciton are determined to be 3.7 ± 0.2 and 0.3 ± 0.2 eV, respectively. It shows a type I straddling band configuration. The large valence band offset suggests advantage of the CaF 2 /H-diamond heterojunciton for the development of high power and high frequency field effect transistors.

Solution structure of the N-terminal domain of a replication restart primosome factor, PriC, in Escherichia coli

Science.gov (United States)

Aramaki, Takahiko; Abe, Yoshito; Katayama, Tsutomu; Ueda, Tadashi

2013-01-01

In eubacterial organisms, the oriC-independent primosome plays an essential role in replication restart after the dissociation of the replication DNA-protein complex by DNA damage. PriC is a key protein component in the replication restart primosome. Our recent study suggested that PriC is divided into two domains: an N-terminal and a C-terminal domain. In the present study, we determined the solution structure of the N-terminal domain, whose structure and function have remained unknown until now. The revealed structure was composed of three helices and one extended loop. We also observed chemical shift changes in the heteronuclear NMR spectrum and oligomerization in the presence of ssDNA. These abilities may contribute to the PriC-ssDNA complex, which is important for the replication restart primosome. PMID:23868391

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

Directory of Open Access Journals (Sweden)

Christen Y. L. Yuen

2013-10-01

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

Synthesis of Hydroxy-Terminated Dinitropropyl Acrylate Polymers and Improved Characterization of Hydroxy-Terminated Prepolymers

Science.gov (United States)

1983-03-01

CH CH OH P."" 2 2 2 " 2" 2 I2",’C-H CN CN B-2000 Poly-(1,2-butylene)- glycol from DOW Chemical Carbowax Polyethylene glycol from Union Carbide CH...Carbonate GPC Gel Permeation Chromatography HEDS Hydroxyethyl Disulfide HPLC High Pressure Liquid Chromatography NMIM N-methyl imidazole NPGA Neopentyl ...PEG Polyethylene glycol PPG Polypropylene glycol from Wyandotte PY Pyridine R-45M Hydroxy-terminated polybutadeine from ARCO Teracol Poly

Cleavage of sp3 C-O bonds via oxidative addition of C-H bonds.

Science.gov (United States)

Choi, Jongwook; Choliy, Yuriy; Zhang, Xiawei; Emge, Thomas J; Krogh-Jespersen, Karsten; Goldman, Alan S

2009-11-04

(PCP)Ir (PCP = kappa(3)-C(6)H(3)-2,6-[CH(2)P(t-Bu)(2)](2)) is found to undergo oxidative addition of the methyl-oxygen bond of electron-poor methyl aryl ethers, including methoxy-3,5-bis(trifluoromethyl)benzene and methoxypentafluorobenzene, to give the corresponding aryloxide complexes (PCP)Ir(CH(3))(OAr). Although the net reaction is insertion of the Ir center into the C-O bond, density functional theory (DFT) calculations and a significant kinetic isotope effect [k(CH(3))(OAr)/k(CD(3))(OAr) = 4.3(3)] strongly argue against a simple insertion mechanism and in favor of a pathway involving C-H addition and alpha-migration of the OAr group to give a methylene complex followed by hydride-to-methylene migration to give the observed product. Ethoxy aryl ethers, including ethoxybenzene, also undergo C-O bond cleavage by (PCP)Ir, but the net reaction in this case is 1,2-elimination of ArO-H to give (PCP)Ir(H)(OAr) and ethylene. DFT calculations point to a low-barrier pathway for this reaction that proceeds through C-H addition of the ethoxy methyl group followed by beta-aryl oxide elimination and loss of ethylene. Thus, both of these distinct C-O cleavage reactions proceed via initial addition of a C(sp(3))-H bond, despite the fact that such bonds are typically considered inert and are much stronger than C-O bonds.

Platinum-Catalyzed, Terminal-Selective C(sp(3))-H Oxidation of Aliphatic Amines.

Science.gov (United States)

Lee, Melissa; Sanford, Melanie S

2015-10-14

This Communication describes the terminal-selective, Pt-catalyzed C(sp(3))-H oxidation of aliphatic amines without the requirement for directing groups. CuCl2 is employed as a stoichiometric oxidant, and the reactions proceed in high yield at Pt loadings as low as 1 mol%. These transformations are conducted in the presence of sulfuric acid, which reacts with the amine substrates in situ to form ammonium salts. We propose that protonation of the amine serves at least three important roles: (i) it renders the substrates soluble in the aqueous reaction medium; (ii) it limits binding of the amine nitrogen to Pt or Cu; and (iii) it electronically deactivates the C-H bonds proximal to the nitrogen center. We demonstrate that this strategy is effective for the terminal-selective C(sp(3))-H oxidation of a variety of primary, secondary, and tertiary amines.

Functional mechanism of C-terminal tail in the enzymatic role of porcine testicular carbonyl reductase: a combined experiment and molecular dynamics simulation study of the C-terminal tail in the enzymatic role of PTCR.

Directory of Open Access Journals (Sweden)

Minky Son

Full Text Available Porcine testicular carbonyl reductase, PTCR which is one of the short chain dehydrogenases/reductases (SDR superfamily catalyzes the NADPH-dependent reduction of carbonyl compounds including steroids and prostaglandins. Previously we reported C-terminal tail of PTCR was deleted due to a nonsynonymous single nucleotide variation (nsSNV. Here we identified from kinetic studies that the enzymatic properties for 5α-dihydrotestosterone (5α-DHT were different between wild-type and C-terminal-deleted PTCRs. Compared to wild-type PTCR, C-terminal-deleted PTCR has much higher reduction rate. To investigate structural difference between wild-type and C-terminal-deleted PTCRs upon 5α-DHT binding, we performed molecular dynamics simulations for two complexes. Using trajectories, molecular interactions including hydrogen bonding patterns, distance between 5α-DHT and catalytic Tyr193, and interaction energies are analyzed and compared. During the MD simulation time, the dynamic behavior of C-terminal tail in wild-type PTCR is also examined using essential dynamics analysis. The results of our simulations reveal that the binding conformation of 5α-DHT in C-terminal-deleted PTCR is more favorable for reduction reaction in PTCR, which shows strong agreement with kinetic data. These structural findings provide valuable information to understand substrate specificity of PTCR and further kinetic properties of enzymes belonging to the SDR superfamily.

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

Science.gov (United States)

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

2010-07-13

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

77 FR 42554 - Surety Companies Acceptable on Federal Bonds-Termination: Peerless Insurance Company (NAIC #24198)

Science.gov (United States)

2012-07-19

... should be accepted from this company, and bonds that are continuous in nature should not be renewed. The... DEPARTMENT OF THE TREASURY Fiscal Service Surety Companies Acceptable on Federal Bonds--Termination: Peerless Insurance Company (NAIC 24198) AGENCY: Financial Management Service, Fiscal Service...

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

NARCIS (Netherlands)

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

2003-01-01

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

Identity and functions of CxxC-derived motifs.

Science.gov (United States)

Fomenko, Dmitri E; Gladyshev, Vadim N

2003-09-30

Two cysteines separated by two other residues (the CxxC motif) are employed by many redox proteins for formation, isomerization, and reduction of disulfide bonds and for other redox functions. The place of the C-terminal cysteine in this motif may be occupied by serine (the CxxS motif), modifying the functional repertoire of redox proteins. Here we found that the CxxC motif may also give rise to a motif, in which the C-terminal cysteine is replaced with threonine (the CxxT motif). Moreover, in contrast to a view that the N-terminal cysteine in the CxxC motif always serves as a nucleophilic attacking group, this residue could also be replaced with threonine (the TxxC motif), serine (the SxxC motif), or other residues. In each of these CxxC-derived motifs, the presence of a downstream alpha-helix was strongly favored. A search for conserved CxxC-derived motif/helix patterns in four complete genomes representing bacteria, archaea, and eukaryotes identified known redox proteins and suggested possible redox functions for several additional proteins. Catalytic sites in peroxiredoxins were major representatives of the TxxC motif, whereas those in glutathione peroxidases represented the CxxT motif. Structural assessments indicated that threonines in these enzymes could stabilize catalytic thiolates, suggesting revisions to previously proposed catalytic triads. Each of the CxxC-derived motifs was also observed in natural selenium-containing proteins, in which selenocysteine was present in place of a catalytic cysteine.

Effect of oxidative stress on homer scaffolding proteins.

Directory of Open Access Journals (Sweden)

Igor Nepliouev

Full Text Available Homer proteins are a family of multifaceted scaffolding proteins that participate in the organization of signaling complexes at the post-synaptic density and in a variety of tissues including striated muscle. Homer isoforms form multimers via their C-terminal coiled coil domains, which allows for the formation of a polymeric network in combination with other scaffolding proteins. We hypothesized that the ability of Homer isoforms to serve as scaffolds would be influenced by oxidative stress. We have found by standard SDS-PAGE of lysates from adult mouse skeletal muscle exposed to air oxidation that Homer migrates as both a dimer and monomer in the absence of reducing agents and solely as a monomer in the presence of a reducing agent, suggesting that Homer dimers exposed to oxidation could be modified by the presence of an inter-molecular disulfide bond. Analysis of the peptide sequence of Homer 1b revealed the presence of only two cysteine residues located adjacent to the C-terminal coiled-coil domain. HEK 293 cells were transfected with wild-type and cysteine mutant forms of Homer 1b and exposed to oxidative stress by addition of menadione, which resulted in the formation of disulfide bonds except in the double mutant (C246G, C365G. Exposure of myofibers from adult mice to oxidative stress resulted in decreased solubility of endogenous Homer isoforms. This change in solubility was dependent on disulfide bond formation. In vitro binding assays revealed that cross-linking of Homer dimers enhanced the ability of Homer 1b to bind Drebrin, a known interacting partner. Our results show that oxidative stress results in disulfide cross-linking of Homer isoforms and loss of solubility of Homer scaffolds. This suggests that disulfide cross-linking of a Homer polymeric network may contribute to the pathophysiology seen in neurodegenerative diseases and myopathies characterized by oxidative stress.

The catalytic chain of human complement subcomponent C1r. Purification and N-terminal amino acid sequences of the major cyanogen bromide-cleavage fragments.

Science.gov (United States)

Arlaud, G J; Gagnon, J; Porter, R R

1982-01-01

1. The a- and b-chains of reduced and alkylated human complement subcomponent C1r were separated by high-pressure gel-permeation chromatography and isolated in good yield and in pure form. 2. CNBr cleavage of C1r b-chain yielded eight major peptides, which were purified by gel filtration and high-pressure reversed-phase chromatography. As determined from the sum of their amino acid compositions, these peptides accounted for a minimum molecular weight of 28 000, close to the value 29 100 calculated from the whole b-chain. 3. N-Terminal sequence determinations of C1r b-chain and its CNBr-cleavage peptides allowed the identification of about two-thirds of the amino acids of C1r b-chain. From our results, and on the basis of homology with other serine proteinases, an alignment of the eight CNBr-cleavage peptides from C1r b-chain is proposed. 4. The residues forming the 'charge-relay' system of the active site of serine proteinases (His-57, Asp-102 and Ser-195 in the chymotrypsinogen numbering) are found in the corresponding regions of C1r b-chain, and the amino acid sequence around these residues has been determined. 5. The N-terminal sequence of C1r b-chain has been extended to residue 60 and reveals that C1r b-chain lacks the 'histidine loop', a disulphide bond that is present in all other known serine proteinases.

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

Directory of Open Access Journals (Sweden)

Zeynep A Oztug Durer

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

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

Science.gov (United States)

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

2016-09-02

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

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

Science.gov (United States)

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

2016-01-01

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

The specific cleavage of lactone linkage to open-loop in cyclic lipopeptide during negative ESI tandem mass spectrometry: the hydrogen bond interaction effect of 4-ethyl guaiacol.

Directory of Open Access Journals (Sweden)

Mengzhe Guo

Full Text Available Mass spectrometry is a valuable tool for the analysis and identification of chemical compounds, particularly proteins and peptides. Lichenysins G, the major cyclic lipopeptide of lichenysin, and the non-covalent complex of lichenysins G and 4-ethylguaiacol were investigated with negative ion ESI tandem mass spectrometry. The different fragmentation mechanisms for these compounds were investigated. Our study shows the 4-ethylguaiacol hydrogen bond with the carbonyl oxygen of the ester group in the loop of lichenysins G. With the help of this hydrogen bond interaction, the ring structure preferentially opens in lactone linkage rather than O-C bond of the ester-group to produce alcohol and ketene. Isothermal titration 1H-NMR analysis verified the hydrogen bond and determined the proportion of subject and ligand in the non-covalent complex to be 1∶1. Theoretical calculations also suggest that the addition of the ligand can affect the energy of the transition structures (TS during loop opening.

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

Czech Academy of Sciences Publication Activity Database

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

2011-01-01

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

Breakdown voltage at the electric terminals of GCFR-core flow test loop fuel rod simulators in helium and air

International Nuclear Information System (INIS)

Huntley, W.R.; Conley, T.B.

1979-12-01

Tests were performed to determine the ac and dc breakdown voltage at the terminal ends of a fuel rod simulator (FRS) in helium and air atmospheres. The tests were performed at low pressures (1 to 2 atm) and at temperatures from 20 to 350 0 C (68 to 660 0 F). The area of concern was the 0.64-mm (0.025-in.) gap between the coaxial conductor of the FRS and the sheaths of the four internal thermocouples as they exit the FRS. The tests were prformed to ensure a sufficient safety margin during Core Flow Test Loop (CFTL) operations that require potentials up to 350 V ac at the FRS terminals. The primary conclusion from the test results is that the CFTL cannot be operated safely if the terminal ends of the FRSs are surrounded by a helium atmosphere but can be operated safely in air

The human protein disulfide isomerase gene family

Directory of Open Access Journals (Sweden)

Galligan James J

2012-07-01

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

Critical structural and functional roles for the N-terminal insertion sequence in surfactant protein B analogs.

Directory of Open Access Journals (Sweden)

Frans J Walther

2010-01-01

Full Text Available Surfactant protein B (SP-B; 79 residues belongs to the saposin protein superfamily, and plays functional roles in lung surfactant. The disulfide cross-linked, N- and C-terminal domains of SP-B have been theoretically predicted to fold as charged, amphipathic helices, suggesting their participation in surfactant activities. Earlier structural studies with Mini-B, a disulfide-linked construct based on the N- and C-terminal regions of SP-B (i.e., approximately residues 8-25 and 63-78, confirmed that these neighboring domains are helical; moreover, Mini-B retains critical in vitro and in vivo surfactant functions of the native protein. Here, we perform similar analyses on a Super Mini-B construct that has native SP-B residues (1-7 attached to the N-terminus of Mini-B, to test whether the N-terminal sequence is also involved in surfactant activity.FTIR spectra of Mini-B and Super Mini-B in either lipids or lipid-mimics indicated that these peptides share similar conformations, with primary alpha-helix and secondary beta-sheet and loop-turns. Gel electrophoresis demonstrated that Super Mini-B was dimeric in SDS detergent-polyacrylamide, while Mini-B was monomeric. Surface plasmon resonance (SPR, predictive aggregation algorithms, and molecular dynamics (MD and docking simulations further suggested a preliminary model for dimeric Super Mini-B, in which monomers self-associate to form a dimer peptide with a "saposin-like" fold. Similar to native SP-B, both Mini-B and Super Mini-B exhibit in vitro activity with spread films showing near-zero minimum surface tension during cycling using captive bubble surfactometry. In vivo, Super Mini-B demonstrates oxygenation and dynamic compliance that are greater than Mini-B and compare favorably to full-length SP-B.Super Mini-B shows enhanced surfactant activity, probably due to the self-assembly of monomer peptide into dimer Super Mini-B that mimics the functions and putative structure of native SP-B.

Forging C-C Bonds Through Decarbonylation of Aryl Ketones.

Science.gov (United States)

Somerville, Rosie J; Martin, Ruben

2017-06-06

The ability of nickel to cleave strong σ-bonds is again in the spotlight after a recent report that demonstrates the feasibility of using nickel complexes to promote decarbonylation of diaryl ketones. This transformation involves the cleavage of two strong C-C(O) bonds and avoids the use of noble metals, hence reinforcing the potential of decarbonylation as a technique for forging C-C bonds. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

NARCIS (Netherlands)

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

2008-01-01

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

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

NARCIS (Netherlands)

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

2015-01-01

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

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

Science.gov (United States)

Hayashi, Satoko; Tsubomoto, Yutaka; Nakanishi, Waro

2018-02-17

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

Backbone dynamics of the human CC-chemokine eotaxin

Energy Technology Data Exchange (ETDEWEB)

Ye Jiqing; Mayer, Kristen L.; Stone, Martin J. [Indiana University, Department of Chemistry (United States)

1999-10-15

Eotaxin is a CC chemokine with potent chemoattractant activity towards eosinophils. {sup 15}N NMR relaxation data have been used to characterize the backbone dynamics of recombinant human eotaxin. {sup 15}N longitudinal (R{sub 1}) and transverse (R{sub 2}) auto relaxation rates, heteronuclear {sup 1}H-{sup 15}N steady-state NOEs, and transverse cross-relaxation rates ({eta}{sub xy}) were obtained at 30 deg. C for all resolved backbone secondary amide groups using {sup 1} H-detected two-dimensional NMR experiments. Ratios of transverse auto and cross relaxation rates were used to identify NH groups influenced by slow conformational rearrangement. Relaxation data were fit to the extended model free dynamics formalism, yielding parameters describing axially symmetric molecular rotational diffusion and the internal dynamics of each NH group. The molecular rotational correlation time ({tau}{sub m}) is 5.09{+-}0.02 ns, indicating that eotaxin exists predominantly as a monomer under the conditions of the NMR study. The ratio of diffusion rates about unique and perpendicular axes (D{sub parallel}/D{sub perpendicular}) is 0.81{+-}0.02. Residues with large amplitudes of subnanosecond motion are clustered in the N-terminal region (residues 1-19), the C-terminus (residues 68-73) and the loop connecting the first two {beta}-strands (residues 30-37). N-terminal flexibility appears to be conserved throughout the chemokine family and may have implications for the mechanism of chemokine receptor activation. Residues exhibiting significant dynamics on the microsecond-millisecond time scale are located close to the two conserved disulfide bonds, suggesting that these motions may be coupled to disulfide bond isomerization.

The carboxy-terminal tail or the intracellular loop 3 is required for β-arrestin-dependent internalization of a mammalian type II GnRH receptor.

Science.gov (United States)

Madziva, Michael T; Mkhize, Nonhlanhla N; Flanagan, Colleen A; Katz, Arieh A

2015-08-15

The type II GnRH receptor (GnRH-R2) in contrast to mammalian type I GnRH receptor (GnRH-R1) has a cytosolic carboxy-terminal tail. We investigated the role of β-arrestin 1 in GnRH-R2-mediated signalling and mapped the regions in GnRH-R2 required for recruitment of β-arrestin, employing internalization assays. We show that GnRH-R2 activation of ERK is dependent on β-arrestin and protein kinase C. Appending the tail of GnRH-R2 to GnRH-R1 enabled GRK- and β-arrestin-dependent internalization of the chimaeric receptor. Surprisingly, carboxy-terminally truncated GnRH-R2 retained β-arrestin and GRK-dependent internalization, suggesting that β-arrestin interacts with additional elements of GnRH-R2. Mutating serine and threonine or basic residues of intracellular loop 3 did not abolish β-arrestin 1-dependent internalization but a receptor lacking these basic residues and the carboxy-terminus showed no β-arrestin 1-dependent internalization. Our results suggest that basic residues at the amino-terminal end of intracellular loop 3 or the carboxy-terminal tail are required for β-arrestin dependent internalization. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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

Directory of Open Access Journals (Sweden)

Julie K Klint

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

Alkane Activation at Ambient Temperatures: Unusual Selectivities, C-C, C-H Bond Scission versus C-C Bond Coupling

NARCIS (Netherlands)

Trionfetti, C.; Agiral, A.; Gardeniers, Johannes G.E.; Lefferts, Leonardus; Seshan, Kulathuiyer

2008-01-01

Activating bonds: A cold plasma generated by dielectric barrier discharge in a microreactor converts alkanes (C1–C3) at atmospheric pressure. Large amounts of products with higher molecular weight than the starting hydrocarbons are observed showing that C-H activation at lower T favourably leads to

Ab initio study of 3C-SiC/M (M = Ti or Al) nano-hetero interfaces

International Nuclear Information System (INIS)

Tanaka, Shingo; Kohyama, Masanori

2003-01-01

Ab initio pseudopotential calculation of 3C-SiC(1 1 1)/Al nano-hetero interfaces have been performed and interface atom species dependence (IASD) and interface orientation dependence (IOD) of nano-hetero interfaces between 3C-SiC ((1 1 1) or (0 0 1) orientation) and metal (Ti or Al) have been studied systematically. Stable atomic configurations of the 3C-SiC(1 1 1)/Al interfaces are quite different from those of the 3C-SiC(1 1 1)/Ti interfaces. Two terminated, Si-terminated (Si-TERM) and C-terminated (C-TERM), 3C-SiC(1 1 1)/Al interfaces have covalent bonding nature. In 3C-SiC/M (M = Ti or Al) nano-hetero interfaces, the C-terminated interface has relative strong, covalent and ionic C-Ti or C-Al bonds as TiC or SiC while the Si-terminated interface has various type of bonding nature, relative weak Si-Ti or Si-Al bonds from metallic character at the (0 0 1) interface to covalent character at the (1 1 1) interface. Adhesive energy (AE) shows strong IASD and IOD. The AE of the C-terminated interface is larger than that of the Si-terminated one. In the C-terminated interface, the AE of the (1 1 1) interface is smaller than that of the (0 0 1) one while in the Si-terminated interface there exists opposite interrelation. Schottky barrier height (SBH) also shows strong IASD and IOD. The SBH of the C-terminated interface is smaller than that of the Si-terminated one. The C-terminated SiC/Al interfaces have extremely small SBHs. In comparison with some experimental SBH, the present result is reliable as the difference of SBH between the two terminated interfaces and qualitative properties

Structural basis for new pattern of conserved amino acid residues related to chitin-binding in the antifungal peptide from the coconut rhinoceros beetle Oryctes rhinoceros.

Science.gov (United States)

Hemmi, Hikaru; Ishibashi, Jun; Tomie, Tetsuya; Yamakawa, Minoru

2003-06-20

Scarabaecin isolated from hemolymph of the coconut rhinoceros beetle Oryctes rhinoceros is a 36-residue polypeptide that has antifungal activity. The solution structure of scarabaecin has been determined from twodimensional 1H NMR spectroscopic data and hybrid distance geometry-simulated annealing protocol calculation. Based on 492 interproton and 10 hydrogen-bonding distance restraints and 36 dihedral angle restraints, we obtained 20 structures. The average backbone root-mean-square deviation for residues 4-35 is 0.728 +/- 0.217 A from the mean structure. The solution structure consists of a two-stranded antiparallel beta-sheet connected by a type-I beta-turn after a short helical turn. All secondary structures and a conserved disulfide bond are located in the C-terminal half of the peptide, residues 18-36. Overall folding is stabilized by a combination of a disulfide bond, seven hydrogen bonds, and numerous hydrophobic interactions. The structural motif of the C-terminal half shares a significant tertiary structural similarity with chitin-binding domains of plant and invertebrate chitin-binding proteins, even though scarabaecin has no overall sequence similarity to other peptide/polypeptides including chitin-binding proteins. The length of its primary structure, the number of disulfide bonds, and the pattern of conserved functional residues binding to chitin in scarabaecin differ from those of chitin-binding proteins in other invertebrates and plants, suggesting that scarabaecin does not share a common ancestor with them. These results are thought to provide further strong experimental evidence to the hypothesis that chitin-binding proteins of invertebrates and plants are correlated by a convergent evolution process.

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

Energy Technology Data Exchange (ETDEWEB)

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

2013-08-01

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

Scandium Terminal Imido Chemistry.

Science.gov (United States)

Lu, Erli; Chu, Jiaxiang; Chen, Yaofeng

2018-02-20

of two p-d π bonds between the scandium ion and the nitrogen atom of the imido ligand and showed that the dianionic [NR] 2- imido ligand acts as a 2σ,4π electron donor. Subsequent studies of the scandium terminal imides revealed highly versatile and intriguing reactivity of the Sc═N bond. This included cycloaddition toward various unsaturated bonds, C-H/Si-H/B-H bond activations and catalytic hydrosilylation, dehydrofluorination of fluoro-substituted benzenes/alkanes, CO 2 and H 2 activations, activation of elemental selenium, coordination with other transition metal halides, etc. Since our initial success in 2010, and with contributions from us and across the community, this young, vibrant research field has rapidly flourished into one of the most active frontiers of rare-earth metal chemistry. The prospect of extending Ln═N chemistry to other rare-earth metals and/or different metal oxidation states, as well as exploiting their stoichiometric and catalytic reactivities, continues to attract research effort. Herein we present an account of our investigations into scandium terminal imido chemistry as a timely summary, in the hope that our studies will be of interest to this readership.

Disruption of a hydrogen bond network in human versus spider monkey cytochrome c affects heme crevice stability.

Science.gov (United States)

Goldes, Matthew E; Jeakins-Cooley, Margaret E; McClelland, Levi J; Mou, Tung-Chung; Bowler, Bruce E

2016-05-01

The hypothesis that the recent rapid evolution of primate cytochromes c, which primarily involves residues in the least stable Ω-loop (Ω-loop C, residues 40-57), stabilizes the heme crevice of cytochrome c relative to other mammals, is tested. To accomplish this goal, we have compared the properties of human and spider monkey cytochrome c and a set of four variants produced in the process of converting human cytochrome c into spider monkey cytochrome c. The global stability of all variants has been measured by guanidine hydrochloride denaturation. The stability of the heme crevice has been assessed with the alkaline conformational transition. Structural insight into the effects of the five amino acid substitutions needed to convert human cytochrome c into spider monkey cytochrome c is provided by a 1.15Å resolution structure of spider monkey cytochrome c. The global stability for all variants is near 9.0kcal/mol at 25°C and pH7, which is higher than that observed for other mammalian cytochromes c. The heme crevice stability is more sensitive to the substitutions required to produce spider monkey cytochrome c with decreases of up to 0.5 units in the apparent pKa of the alkaline conformational transition relative to human cytochrome c. The structure of spider monkey cytochrome c indicates that the Y46F substitution destabilizes the heme crevice by disrupting an extensive hydrogen bond network that connects three surface loops including Ω-loop D (residues 70-85), which contains the Met80 heme ligand. Copyright © 2015 Elsevier Inc. All rights reserved.

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

DEFF Research Database (Denmark)

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

2012-01-01

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

Platinum-Catalyzed Terminal-Selective C(sp3)–H Oxidation of Aliphatic Amines

Science.gov (United States)

Lee, Melissa; Sanford, Melanie S.

2016-01-01

This paper describes the terminal-selective Pt-catalyzed C(sp3)–H oxidation of aliphatic amines without the requirement for directing groups. CuCl2 is employed as a stoichiometric oxidant, and the reactions proceed in high yield at Pt loadings as low as 1 mol %. These transformations are conducted in the presence of sulfuric acid, which reacts with the amine substrates in situ to form ammonium salts. We propose that protonation of the amine serves at least three important roles: (i) it renders the substrates soluble in the aqueous reaction medium; (ii) it limits binding of the amine nitrogen to Pt or Cu; and (ii) it electronically deactivates the C–H bonds proximal to the nitrogen center. We demonstrate that this strategy is effective for the terminal-selective C(sp3)–H oxidation of a variety of primary, secondary and tertiary amines. PMID:26439251

Stability assessment on a library scale: a rapid method for the evaluation of the commutability and insertion of residues in C-terminal loops of the CH3 domains of IgG1-Fc.

Science.gov (United States)

Hasenhindl, Christoph; Traxlmayr, Michael W; Wozniak-Knopp, Gordana; Jones, Phil C; Stadlmayr, Gerhard; Rüker, Florian; Obinger, Christian

2013-10-01

Antigen-binding Fc fragments (Fcab) are generated by engineering the C-terminal loop regions in the CH3 domain of human immunoglobulin G class 1-crystallizable fragment (IgG1-Fc). For an optimum library design with high percentage of well-folded clones for efficient binder selection, information about the correlation between primary structure and stability is needed. Here, we present a rapid method that allows determination of the overall stability of whole libraries of IgG1-Fc on the surface of yeast by flow cytometry. Libraries of IgG1-Fc mutants with distinct regions in AB-, CD- and EF-loops of the CH3 domains randomized or carrying therein insertions of five additional residues were constructed, incubated at increasing temperatures and probed for residual binding of generic Fc ligands. Calculated temperatures of half-maximal irreversible denaturation of the libraries gave a clear hierarchy of tolerance to randomization of distinct loop positions. Experimental data were evaluated by a computational approach and are discussed with respect to the structure of IgG1-Fc and variation in sequence and length of these loops in homologous Fc proteins. Generally, the described method allows for quick assessment of the effects of randomization of distinct regions on the foldability and stability of a yeast-displayed protein library.

Epimerization-free C-terminal peptide activation, elongation and cyclization

NARCIS (Netherlands)

Popović, S.

2015-01-01

C-terminal peptide activation and cyclization reactions are generally accompanied with epimerization (partial loss of C‐terminal stereointegrity). Therefore, the focus of this thesis was to develop epimerization-free methods for C-terminal peptide activation to enable C-terminal peptide elongation

Designing new catalytic C-C and C-N bond formations promoted by organoactinides

International Nuclear Information System (INIS)

Eisen, M.S.; Straub, T.; Haskel, A.

1998-01-01

Organoactinides of the type Cp 2 * AcMe 2 (Cp * =C 5 Me 5 ; Ac=Th; U) are active catalytic precursors for the oligomerization of terminal alkynes HC≡CR (R=alkyl, aryl, SiMe 3 ). The regioselectivity and the extent of oligomerization depend strongly on the alkyne substituent R, whereas the catalytic reactivity is similar for both organoactinides. Reaction with tert-butylacetylene yields regioselectively the E-2,4-disubstituted 1-buten-3-yne dimer whereas trimethylsilylacetylene is regioselective trimerized to the E,E-1,4,6-tris(trimethylsilyl)-1,3-hexa diene-5-yne, with small amounts (3-5%) of the corresponding E-2,4-disubstituted 1-buten-3-yne dimer. Oligomerization with less bulky alkyl and aryl substituted alkynes produces a mixture of higher oligomers with no regioselectivity. Using the Cp 2 * ThMe 2 catalyst, we have recently developed a strategic method to control the extent and in some cases the regioselectivity of the catalyzed oligomerization of nonbulky terminal alkynes to dimers and/or trimers. The metallocene catalytic precursors ensure the selective synthesis of small oligomers by the addition of specific amines. Catalytic ''tailoring'' to dimer and trimers can be achieved by using small or bulky amines, respectively. Kinetic and mechanistic data for the controlling experiments argue that the turnover-limiting step involves the acetylide actinide complex formation with the rapid insertion of the alkyne and protonolysis by the amine. The analog Cp 2 * UMe 2 in the presence of primary amines induce the selective C-N bond formation, producing enamines which are tautomerized to the corresponding imines. (orig.)

Ionic interaction of myosin loop 2 with residues located beyond the N-terminal part of actin probed by chemical cross-linking.

Science.gov (United States)

Pliszka, Barbara; Martin, Brian M; Karczewska, Emilia

2008-02-01

To probe ionic contacts of skeletal muscle myosin with negatively charged residues located beyond the N-terminal part of actin, myosin subfragment 1 (S1) and actin split by ECP32 protease (ECP-actin) were cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). We have found that unmodified S1 can be cross-linked not only to the N-terminal part, but also to the C-terminal 36 kDa fragment of ECP-actin. Subsequent experiments performed on S1 cleaved by elastase or trypsin indicate that the cross-linking site in S1 is located within loop 2. This site is composed of Lys-636 and Lys-637 and can interact with negatively charged residues of the 36 kDa actin fragment, most probably with Glu-99 and Glu-100. Cross-links are formed both in the absence and presence of MgATP.P(i) analog, although the addition of nucleotide decreases the efficiency of the cross-linking reaction.

A new crystal form of human tear lipocalin reveals high flexibility in the loop region and induced fit in the ligand cavity

International Nuclear Information System (INIS)

Breustedt, Daniel A.; Chatwell, Lorenz; Skerra, Arne

2009-01-01

The crystal structure of tear lipocalin determined in space group P2 1 revealed large structural deviations from the previously solved X-ray structure in space group C2, especially in the loop region and adjoining parts of the β-barrel which give rise to the ligand-binding site. These findings illustrate a novel mechanism for promiscuity in ligand recognition by the lipocalin protein family. Tear lipocalin (TLC) with the bound artificial ligand 1,4-butanediol has been crystallized in space group P2 1 with four protein molecules in the asymmetric unit and its X-ray structure has been solved at 2.6 Å resolution. TLC is a member of the lipocalin family that binds ligands with diverse chemical structures, such as fatty acids, phospholipids and cholesterol as well as microbial siderophores and the antibiotic rifampin. Previous X-ray structural analysis of apo TLC crystallized in space group C2 revealed a rather large bifurcated ligand pocket and a partially disordered loop region at the entrace to the cavity. Analysis of the P2 1 crystal form uncovered major conformational changes (i) in β-strands B, C and D, (ii) in loops 1, 2 and 4 at the open end of the β-barrel and (iii) in the extended C-terminal segment, which is attached to the β-barrel via a disulfide bridge. The structural comparison indicates high conformational plasticity of the loop region as well as of deeper parts of the ligand pocket, thus allowing adaptation to ligands that differ vastly in size and shape. This illustrates a mechanism for promiscuity in ligand recognition which may also be relevant for some other physiologically important members of the lipocalin protein family

Boron-Based Catalysts for C-C Bond-Formation Reactions.

Science.gov (United States)

Rao, Bin; Kinjo, Rei

2018-05-02

Because the construction of the C-C bond is one of the most significant reactions in organic chemistry, the development of an efficient strategy has attracted much attention throughout the synthetic community. Among various protocols to form C-C bonds, organoboron compounds are not just limited to stoichiometric reagents, but have also made great achievements as catalysts because of the easy modification of the electronic and steric impacts on the boron center. This review presents recent developments of boron-based catalysts applied in the field of C-C bond-formation reactions, which are classified into four kinds on the basis of the type of boron catalyst: 1) highly Lewis acidic borane, B(C 6 F 5 ) 3 ; 2) organoboron acids, RB(OH) 2 , and their ester derivatives; 3) borenium ions, (R 2 BL)X; and 4) other miscellaneous kinds. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Resolution of Disulfide Heterogeneity in Nogo Receptor 1 Fusion Proteins by Molecular Engineering

Energy Technology Data Exchange (ETDEWEB)

P Weinreb; D Wen; F Qian; C Wildes; E Garber; L Walus; M Jung; J Wang; J Relton; et al.

2011-12-31

NgRI (Nogo-66 receptor) is part of a signalling complex that inhibits axon regeneration in the central nervous system. Truncated soluble versions of NgRI have been used successfully to promote axon regeneration in animal models of spinal-cord injury, raising interest in this protein as a potential therapeutic target. The LRR (leucine-rich repeat) regions in NgRI are flanked by N- and C-terminal disulfide-containing 'cap' domains (LRRNT and LRRCT respectively). In the present work we show that, although functionally active, the NgRI(310)-Fc fusion protein contains mislinked and heterogeneous disulfide patterns in the LRRCT domain, and we report the generation of a series of variant molecules specifically designed to prevent this heterogeneity. Using these variants we explored the effects of modifying the NgRI truncation site or the spacing between the NgRI and Fc domains, or replacing cysteines within the NgRI or IgG hinge regions. One variant, which incorporates replacements of Cys{sup 266} and Cys{sup 309} with alanine residues, completely eliminated disulfide scrambling while maintaining functional in vitro and in vivo efficacy. This modified NgRI-Fc molecule represents a significantly improved candidate for further pharmaceutical development, and may serve as a useful model for the optimization of other IgG fusion proteins made from LRR proteins.

Insights into PG-binding, conformational change, and dimerization of the OmpA C-terminal domains from Salmonella enterica serovar Typhimurium and Borrelia burgdorferi: Characterization of OmpA C-Terminal Domain

Energy Technology Data Exchange (ETDEWEB)

Tan, Kemin [Center for Structural Genomics of Infectious Diseases, University of Chicago, 5735 South Ellis Avenue Chicago Illinois 60637; Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Structural Biology Center, Biosciences, Argonne National Laboratory, Argonne Illinois 60439; Deatherage Kaiser, Brooke L. [National Security Directorate, Pacific Northwest National Laboratory, Richland Washington 99352; Wu, Ruiying [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Cuff, Marianne [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Fan, Yao [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Bigelow, Lance [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Jedrzejczak, Robert P. [Center for Structural Genomics of Infectious Diseases, University of Chicago, 5735 South Ellis Avenue Chicago Illinois 60637; Adkins, Joshua N. [Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland Washington 99352; Cort, John R. [Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland Washington 99352; Babnigg, Gyorgy [Center for Structural Genomics of Infectious Diseases, University of Chicago, 5735 South Ellis Avenue Chicago Illinois 60637; Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Joachimiak, Andrzej [Center for Structural Genomics of Infectious Diseases, University of Chicago, 5735 South Ellis Avenue Chicago Illinois 60637; Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Structural Biology Center, Biosciences, Argonne National Laboratory, Argonne Illinois 60439

2017-06-19

S. Typhimurium can induce both humoral and cell-mediated responses when establishing itself in the host. These responses are primarily stimulated against the lipopolysaccharide and major outer membrane (OM) proteins of the bacterium. OmpA is one of these major OM proteins. It comprises a N-terminal eight-stranded -barrel membrane domain and a C-terminal so-called OmpA C-terminal domain (OmpACTD). The OmpACTD and its homologs are believed to bind to peptidoglycan (PG) within the periplasm, maintaining bacterial osmotic homeostasis and modulating the permeability and integrity of the outer membrane. Here we present the structures of two forms of the OmpACTD of S. Typhimurium (STOmpACTD) and one structure of the less-studied OmpACTD of Borrelia burgdorferi (BbOmpACTD). In the open form of STOmpACTD, an aspartic acid residue from a long 2-3 loop points into the binding pocket, suggesting that an anion group such as a carboxylate group from PG is favored at the binding site. In the closed form of STOmpACTD and in the structure of BbOmpACTD, a sulfate group from the crystallization buffer is tightly bound at the equivalent site. The differences between the closed and open forms of STOmpACTD, suggest a large conformational change that includes an extension of 3 helix by ordering a part of 2-3 loop. We suggest that the sulfate anion observed in these structures mimics the carboxylate group of PG when bound to STOmpACTD. In addition, the binding of PG or a ligand mimic may enhance dimerization of STOmpACTD, or possibly that of full length STOmpA.

Independence of protein kinase C-delta activity from activation loop phosphorylation: structural basis and altered functions in cells.

Science.gov (United States)

Liu, Yin; Belkina, Natalya V; Graham, Caroline; Shaw, Stephen

2006-04-28

Activation loop phosphorylation plays critical regulatory roles for many kinases. Unlike other protein kinase Cs (PKC), PKC-delta does not require phosphorylation of its activation loop (Thr-507) for in vitro activity. We investigated the structural basis for this unusual capacity and its relevance to PKC-delta function in intact cells. Mutational analysis demonstrated that activity without Thr-507 phosphorylation depends on 20 residues N-terminal to the kinase domain and a pair of phenylalanines (Phe-500/Phe-527) unique to PKC-delta in/near the activation loop. Molecular modeling demonstrated that these elements stabilize the activation loop by forming a hydrophobic chain of interactions from the C-lobe to activation loop to N-terminal (helical) extension. In cells PKC-delta mediates both apoptosis and transcription regulation. We found that the T507A mutant of the PKC-delta kinase domain resembled the corresponding wild type in mediating apoptosis in transfected HEK293T cells. But the T507A mutant was completely defective in AP-1 and NF-kappaB reporter assays. A novel assay in which the kinase domain of PKC-delta and its substrate (a fusion protein of PKC substrate peptide with green fluorescent protein) were co-targeted to lipid rafts revealed a major substrate-selective defect of the T507A mutant in phosphorylating the substrate in cells. In vitro analysis showed strong product inhibition on the T507A mutant with particular substrates whose characteristics suggest it contributes to the substrate selective defect of the PKC-delta T507A mutant in cells. Thus, activation loop phosphorylation of PKC-delta may regulate its function in cells in a novel way.

Coronal Loop Evolution Observed with AIA and Hi-C

Science.gov (United States)

Mulu-Moore, Fana; Winebarger, A.; Cirtain, J.; Kobayashi, K.; Korreck, K.; Golub, L.; Kuzin. S.; Walsh, R.; DeForest, C.; DePontieu, B.;

C-Terminally modified peptides via cleavage of the HMBA linker by O-, N- or S-nucleophiles

DEFF Research Database (Denmark)

Hansen, Jonas; Diness, Frederik; Meldal, Morten Peter

2016-01-01

A large variety of C-terminally modified peptides was obtained by nucleophilic cleavage of the ester bond in solid phase linked peptide esters of 4-hydroxymethyl benzamide (HMBA). The developed methods provided peptides, C-terminally functionalized as esters, amides and thioesters, with high purity...... directly from the resin in a single reaction step. A comprehensive screening of the reaction conditions and scope for nucleophilic cleavage of peptides from the HMBA linker was performed....

Study of a photo-induced lysozyme-riboflavin bond

Energy Technology Data Exchange (ETDEWEB)

Ferrer, I; Silva, E

1985-01-01

Irradiation of lysozyme in the presence of riboflavin results in the formation of a lysozyme-riboflavin adduct. Reduction and carboxymethylation of the four disulfide bonds as well as the chemical modification of the Tyr residues and the photochemical alteration of the His residue in lysozyme, do not affect the formation of the photo-induced lysozyme-riboflavin bond. When the lysozyme-riboflavin adduct was subjected to mild acid hydrolysis and ion exchange chromatography, the retention of a compound containing /sup 14/C-riboflavin was observed. Free /sup 14/C-riboflavin, on the contrary is not retained by the column. The photo-oxidation of free Trp in the presence of /sup 14/C-riboflavin, gave a compound which bound to the ion exchange resin like the above-mentioned derivative. The photo-oxidation of the Trp residues in lysozyme and in peptides obtained from lysozyme showed very high quantum yields, and these values were directly related to the incorporation of /sup 14/C-riboflavin in these samples.

Study of a photo-induced lysozyme-riboflavin bond

International Nuclear Information System (INIS)

Ferrer, I.; Silva, E.

1985-01-01

Irradiation of lysozyme in the presence of riboflavin results in the formation of a lysozyme-riboflavin adduct. Reduction and carboxymethylation of the four disulfide bonds as well as the chemical modification of the Tyr residues and the photochemical alteration of the His residue in lysozyme, do not affect the formation of the photo-induced lysozyme-riboflavin bond. When the lysozyme-riboflavin adduct was subjected to mild acid hydrolysis and ion exchange chromatography, the retention of a compound containing 14 C-riboflavin was observed. Free 14 C-ribboflavin, on the contrary is not retained by the column. The photo-oxidation of free Trp in the presence of 14 C-riboflavin, gave a compound which bound to the ion exchange resin like the above-mentioned derivative. The photo-oxidation of the Trp residues in lysozyme and in peptides obtained from lysozyme showed very high quantum yields, and these values were directly related to the incorporation of 14 C-riboflavin in these samples. (orig.)

13C NMR spectra and bonding situation of the B-C bond in alkynylboranes

International Nuclear Information System (INIS)

Yamamoto, Yoshinori; Moritani, Ichiro

1975-01-01

13 C NMR spectra of boron substituted alkynes reveal that the β-carbon is deshielded by ca. 21 ppm by a B(O-n-C 4 H 9 ) 2 group. This clearly indicates the presence of a B-C π-bonding in alkynylboranes. (auth.)

Closed-loop thrust and pressure profile throttling of a nitrous oxide/hydroxyl-terminated polybutadiene hybrid rocket motor

Science.gov (United States)

Peterson, Zachary W.

Hybrid motors that employ non-toxic, non-explosive components with a liquid oxidizer and a solid hydrocarbon fuel grain have inherently safe operating characteristics. The inherent safety of hybrid rocket motors offers the potential to greatly reduce overall operating costs. Another key advantage of hybrid rocket motors is the potential for in-flight shutdown, restart, and throttle by controlling the pressure drop between the oxidizer tank and the injector. This research designed, developed, and ground tested a closed-loop throttle controller for a hybrid rocket motor using nitrous oxide and hydroxyl-terminated polybutadiene as propellants. The research simultaneously developed closed-loop throttle algorithms and lab scale motor hardware to evaluate the fidelity of the throttle simulations and algorithms. Initial open-loop motor tests were performed to better classify system parameters and to validate motor performance values. Deep-throttle open-loop tests evaluated limits of stable thrust that can be achieved on the test hardware. Open-loop tests demonstrated the ability to throttle the motor to less than 10% of maximum thrust with little reduction in effective specific impulse and acoustical stability. Following the open-loop development, closed-loop, hardware-in-the-loop tests were performed. The closed-loop controller successfully tracked prescribed step and ramp command profiles with a high degree of fidelity. Steady-state accuracy was greatly improved over uncontrolled thrust.

A General Catalyst for Site-Selective C(sp(3))-H Bond Amination of Activated Secondary over Tertiary Alkyl C(sp(3))-H Bonds.

Science.gov (United States)

Scamp, Ryan J; Jirak, James G; Dolan, Nicholas S; Guzei, Ilia A; Schomaker, Jennifer M

2016-06-17

The discovery of transition metal complexes capable of promoting general, catalyst-controlled and selective carbon-hydrogen (C-H) bond amination of activated secondary C-H bonds over tertiary alkyl C(sp(3))-H bonds is challenging, as substrate control often dominates when reactive nitrene intermediates are involved. In this letter, we report the design of a new silver complex, [(Py5Me2)AgOTf]2, that displays general and good-to-excellent selectivity for nitrene insertion into propargylic, benzylic, and allylic C-H bonds over tertiary alkyl C(sp(3))-H bonds.

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

Science.gov (United States)

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

2016-01-01

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

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

DEFF Research Database (Denmark)

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

2016-01-01

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

Si/C and H coadsorption at 4H-SiC{0001} surfaces

Energy Technology Data Exchange (ETDEWEB)

Wachowicz, E., E-mail: elwira@ifd.uni.wroc.pl [Institute of Experimental Physics, University of Wrocław, Plac M. Borna 9, PL-50-204 Wrocław (Poland); Interdisciplinary Centre for Mathematical and Computational Modelling, University of Warsaw, Pawińskiego 5a, PL-02-106 Warsaw (Poland)

2016-06-15

Highlights: • Si on C-terminated and C on Si-terminated surface adsorb in the H{sub 3} hollow site. • The preferred adsorption site is in contrary to the stacking order of bulk crystal. • The presence of hydrogen increases the adsorption energy of Si/C. • Hydrogen weakens the bonds between the adsorbed Si or C and the surface. • Carbon adsorbs on top of the surface carbon on the C-terminated surface. • With both C and H on Si-terminated surface the surface state vanishes. - Abstract: Density functional theory (DFT) study of adsorption of 0.25 monolayer of either Si or C on 4H-SiC{0001} surfaces is presented. The adsorption in high-symmetry sites on both Si- and C-terminated surfaces was examined and the influence of the preadsorbed 0.25 ML of hydrogen on the Si/C adsorption was considered. It was found out that for Si on C-terminated surface and C on Si-terminated the most favourable is threefolded adsorption site on both clean and H-precovered surface. This is contrary to the bulk crystal stacking order which would require adsorption on top of the topmost surface atom. In those cases, the presence of hydrogen weakens the bonding of the adsorbate. Carbon on the C-terminated surface, only binds on-top of the surface atom. The C−C bond-length is almost the same for the clean surface and for one with H and equals to ∼1.33 Å which is shorter by ∼0.2 than in diamond. The analysis of the electronic structure changes under adsorption is also presented.

Atomistic simulations of the formation of <c>-component dislocation loops in α-zirconium

Energy Technology Data Exchange (ETDEWEB)

Dai, Cong, E-mail: dai.cong@queensu.ca; Balogh, Levente; Yao, Zhongwen; Daymond, Mark R., E-mail: mark.daymond@queensu.ca

2016-09-15

The formation of <c>-component dislocation loops in α-Zr is believed to be responsible for the breakaway irradiation growth experimentally observed under high irradiation fluences. However, while <c>-loop growth is well described by existing models, the atomic mechanisms responsible for the nucleation of <c>-component dislocation loops are still not clear. In the present work, both interstitial and vacancy -type dislocation loops are initially equilibrated at different temperatures. Cascades simulations in the vicinity of the -type loops are then performed by selecting an atom as the primary knock-on atoms (PKAs) with different kinetic energies, using molecular dynamics simulations. No <c>-component dislocation loop was formed in cascades simulations with a 10 keV PKA, but <c>-component interstitial loops were observed after the interaction between discontinuous 50 keV PKAs and pre-existing -type interstitial loops. The comparisons of cascades simulations in volumes having pre-existing -type interstitial and vacancy loops suggest that the reaction between the PKAs and -type interstitial loops is responsible for the formation of <c>-component interstitial loops.

Thermodynamics of single polyethylene and polybutylene glycols with hydrogen-bonding ends: A transition from looped to open conformations

Science.gov (United States)

Lee, Eunsang; Paul, Wolfgang

2018-02-01

A variety of linear polymer precursors with hydrogen bonding motifs at both ends enable us to design supramolecular polymer systems with tailored macroscopic properties including self-healing. In this study, we investigate thermodynamic properties of single polyethylene and polybutylene glycols with hydrogen bonding motifs. In this context, we first build a coarse-grained model of building blocks of the supramolecular polymer system based on all-atom molecular structures. The density of states of the single precursor is obtained using the stochastic approximation Monte Carlo method. Constructing canonical partition functions from the density of states, we find the transition from looped to open conformations at transition temperatures which are non-monotonously changing with an increasing degree of polymerization due to the competition between chain stiffness and loop-forming entropy penalty. In the complete range of chain length under investigation, a coexistence of the looped and open morphologies at the transition temperature is shown regardless of whether the transition is first-order-like or continuous. Polyethylene and polybutylene glycols show similar behavior in all the thermodynamic properties but the transition temperature of the more flexible polybutylene glycol is shown to change more gradually.

Disulphide bond formation in food protein aggregation and gelation

NARCIS (Netherlands)

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

2005-01-01

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

Structural Characterization of the Loop at the Alpha-Subunit C-Terminus of the Mixed Lineage Leukemia Protein Activating Protease Taspase1.

Directory of Open Access Journals (Sweden)

Johannes van den Boom

Full Text Available Type 2 asparaginases, a subfamily of N-terminal nucleophile (Ntn hydrolases, are activated by limited proteolysis. This activation yields a heterodimer and a loop region at the C-terminus of the α-subunit is released. Since this region is unresolved in all type 2 asparaginase crystal structures but is close to the active site residues, we explored this loop region in six members of the type 2 asparaginase family using homology modeling. As the loop model for the childhood cancer-relevant protease Taspase1 differed from the other members, Taspase1 activation as well as the conformation and dynamics of the 56 amino acids loop were investigated by CD and NMR spectroscopy. We propose a helix-turn-helix motif, which can be exploited as novel anticancer target to inhibit Taspase1 proteolytic activity.

The functional significance of the autolysis loop in protein C and activated protein C.

Science.gov (United States)

Yang, Likui; Manithody, Chandrashekhara; Rezaie, Alireza R

2005-07-01

The autolysis loop of activated protein C (APC) is five residues longer than the autolysis loop of other vitamin K-dependent coagulation proteases. To investigate the role of this loop in the zymogenic and anticoagulant properties of the molecule, a protein C mutant was constructed in which the autolysis loop of the protein was replaced with the corresponding loop of factor X. The protein C mutant was activated by thrombin with approximately 5-fold higher rate in the presence of Ca2+. Both kinetics and direct binding studies revealed that the Ca2+ affinity of the mutant has been impaired approximately 3-fold. The result of a factor Va degradation assay revealed that the anticoagulant function of the mutant has been improved 4-5-fold in the absence but not in the presence of protein S. The improvement was due to a better recognition of both the P1-Arg506 and P1-Arg306 cleavage sites by the mutant protease. However, the plasma half-life of the mutant was markedly shortened due to faster inactivation by plasma serpins. These results suggest that the autolysis loop of protein C is critical for the Ca(2+)-dependence of activation by thrombin. Moreover, a longer autolysis loop in APC is not optimal for interaction with factor Va in the absence of protein S, but it contributes to the lack of serpin reactivity and longer half-life of the protease in plasma.

Role of the ERC motif in the proximal part of the second intracellular loop and the C-terminal domain of the human prostaglandin F2alpha receptor (hFP-R) in G-protein coupling control.

Science.gov (United States)

Pathe-Neuschäfer-Rube, Andrea; Neuschäfer-Rube, Frank; Püschel, Gerhard P

2005-05-15

The human FP-R (F2alpha prostaglandin receptor) is a Gq-coupled heptahelical ectoreceptor, which is of significant medical interest, since it is a potential target for the treatment of glaucoma and preterm labour. On agonist exposure, it mediates an increase in intracellular inositol phosphate formation. Little is known about the structures that govern the agonist-dependent receptor activation. In other prostanoid receptors, the C-terminal domain has been inferred in the control of agonist-dependent receptor activation. A DRY motif at the beginning of the second intracellular loop is highly conserved throughout the G-protein-coupled receptor family and appears to be crucial for controlling agonist-dependent receptor activation. It is replaced by an ERC motif in the FP-R and no evidence for the relevance of this motif in ligand-dependent activation of prostanoid receptors has been provided so far. The aim of the present study was to elucidate the potential role of the C-terminal domain and the ERC motif in agonist-controlled intracellular signalling in FP-R mutants generated by site-directed mutagenesis. It was found that substitution of the acidic Glu(132) in the ERC motif by a threonine residue led to full constitutive activation, whereas truncation of the receptor's C-terminal domain led to partial constitutive activation of all three intracellular signal pathways that had previously been shown to be activated by the FP-R, i.e. inositol trisphosphate formation, focal adhesion kinase activation and T-cell factor signalling. Inositol trisphosphate formation and focal adhesion kinase phosphorylation were further enhanced by ligand binding in cells expressing the truncation mutant but not the E132T (Glu132-->Thr) mutant. Thus C-terminal truncation appeared to result in a receptor with partial constitutive activation, whereas substitution of Glu132 by threonine apparently resulted in a receptor with full constitutive activity.

First-Principles Study of the Polar TiC/Ti Interface

Institute of Scientific and Technical Information of China (English)

Limin LIU; Shaoqing WANG; Hengqiang YE

2003-01-01

The interface structure, work of adhesion, and bonding character of the polar TiC/Ti interface have been examined by the first-principles density functional plane-wave pseudopotential calculations. Both Ti- and C-terminated interfaces including six different interface structures were calculated, which present quite different features. For the Ti-terminated interface, the interfacial Ti-Ti bond has a strong metallic and weak covalent character; while for the C-terminated interface, the interfacial bond is a strong polar covalent interaction between the Ti-3d and C-2p orbital.The work of adhesion of C-terminated interface is nearly 9 J/m2 stronger than that of the Ti-terminated. It is found that each termination has relatively large work of adhesion, which is consistent with other polar interfaces.

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

Science.gov (United States)

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

2016-01-01

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

C-terminal low-complexity sequence repeats of Mycobacterium smegmatis Ku modulate DNA binding.

Science.gov (United States)

Kushwaha, Ambuj K; Grove, Anne

2013-01-24

Ku protein is an integral component of the NHEJ (non-homologous end-joining) pathway of DSB (double-strand break) repair. Both eukaryotic and prokaryotic Ku homologues have been characterized and shown to bind DNA ends. A unique feature of Mycobacterium smegmatis Ku is its basic C-terminal tail that contains several lysine-rich low-complexity PAKKA repeats that are absent from homologues encoded by obligate parasitic mycobacteria. Such PAKKA repeats are also characteristic of mycobacterial Hlp (histone-like protein) for which they have been shown to confer the ability to appose DNA ends. Unexpectedly, removal of the lysine-rich extension enhances DNA-binding affinity, but an interaction between DNA and the PAKKA repeats is indicated by the observation that only full-length Ku forms multiple complexes with a short stem-loop-containing DNA previously designed to accommodate only one Ku dimer. The C-terminal extension promotes DNA end-joining by T4 DNA ligase, suggesting that the PAKKA repeats also contribute to efficient end-joining. We suggest that low-complexity lysine-rich sequences have evolved repeatedly to modulate the function of unrelated DNA-binding proteins.

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

International Nuclear Information System (INIS)

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

2010-01-01

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

Structure of bacteriophage T4 fibritin: a segmented coiled coil and the role of the C-terminal domain.

Science.gov (United States)

Tao, Y; Strelkov, S V; Mesyanzhinov, V V; Rossmann, M G

1997-06-15

Oligomeric coiled-coil motifs are found in numerous protein structures; among them is fibritin, a structural protein of bacteriophage T4, which belongs to a class of chaperones that catalyze a specific phage-assembly process. Fibritin promotes the assembly of the long tail fibers and their subsequent attachment to the tail baseplate; it is also a sensing device that controls the retraction of the long tail fibers in adverse environments and, thus, prevents infection. The structure of fibritin had been predicted from sequence and biochemical analyses to be mainly a triple-helical coiled coil. The determination of its structure at atomic resolution was expected to give insights into the assembly process and biological function of fibritin, and the properties of modified coiled-coil structures in general. The three-dimensional structure of fibritin E, a deletion mutant of wild-type fibritin, was determined to 2.2 A resolution by X-ray crystallography. Three identical subunits of 119 amino acid residues form a trimeric parallel coiled-coil domain and a small globular C-terminal domain about a crystallographic threefold axis. The coiled-coil domain is divided into three segments that are separated by insertion loops. The C-terminal domain, which consists of 30 residues from each subunit, contains a beta-propeller-like structure with a hydrophobic interior. The residues within the C-terminal domain make extensive hydrophobic and some polar intersubunit interactions. This is consistent with the C-terminal domain being important for the correct assembly of fibritin, as shown earlier by mutational studies. Tight interactions between the C-terminal residues of adjacent subunits counteract the latent instability that is suggested by the structural properties of the coiled-coil segments. Trimerization is likely to begin with the formation of the C-terminal domain which subsequently initiates the assembly of the coiled coil. The interplay between the stabilizing effect of the C-terminal

Effect of strain on bond-specific reaction kinetics during the oxidation of H-terminated (111) Si

International Nuclear Information System (INIS)

Gokce, Bilal; Aspnes, David E.; Gundogdu, Kenan

2011-01-01

Although strain is used in semiconductor technology for manipulating optical, electronic, and chemical properties of semiconductors, the understanding of the microscopic phenomena that are affected or influenced by strain is still incomplete. Second-harmonic generation data obtained during the air oxidation of H-terminated (111) Si reveal the effect of compressive strain on this chemical reaction. Even small amounts of strain manipulate the reaction kinetics of surface bonds significantly, with tensile strain enhancing oxidation and compressive strain retarding it. This dramatic change suggests a strain-driven charge transfer mechanism between Si-H up bonds and Si-Si back bonds in the outer layer of Si atoms.

Development of a tetrameric streptavidin mutein with reversible biotin binding capability: engineering a mobile loop as an exit door for biotin.

Directory of Open Access Journals (Sweden)

Valerie J O'Sullivan

Full Text Available A novel form of tetrameric streptavidin has been engineered to have reversible biotin binding capability. In wild-type streptavidin, loop(3-4 functions as a lid for the entry and exit of biotin. When biotin is bound, interactions between biotin and key residues in loop(3-4 keep this lid in the closed state. In the engineered mutein, a second biotin exit door is created by changing the amino acid sequence of loop(7-8. This door is mobile even in the presence of the bound biotin and can facilitate the release of biotin from the mutein. Since loop(7-8 is involved in subunit interactions, alteration of this loop in the engineered mutein results in an 11° rotation between the two dimers in reference to wild-type streptavidin. The tetrameric state of the engineered mutein is stabilized by a H127C mutation, which leads to the formation of inter-subunit disulfide bonds. The biotin binding kinetic parameters (k(off of 4.28×10(-4 s(-1 and K(d of 1.9×10(-8 M make this engineered mutein a superb affinity agent for the purification of biotinylated biomolecules. Affinity matrices can be regenerated using gentle procedures, and regenerated matrices can be reused at least ten times without any observable reduction in binding capacity. With the combination of both the engineered mutein and wild-type streptavidin, biotinylated biomolecules can easily be affinity purified to high purity and immobilized to desirable platforms without any leakage concerns. Other potential biotechnological applications, such as development of an automated high-throughput protein purification system, are feasible.

Development of a Tetrameric Streptavidin Mutein with Reversible Biotin Binding Capability: Engineering a Mobile Loop as an Exit Door for Biotin

Science.gov (United States)

O'Sullivan, Valerie J.; Barrette-Ng, Isabelle; Hommema, Eric; Hermanson, Greg T.; Schofield, Mark; Wu, Sau-Ching; Honetschlaeger, Claudia; Ng, Kenneth K.-S.; Wong, Sui-Lam

2012-01-01

A novel form of tetrameric streptavidin has been engineered to have reversible biotin binding capability. In wild-type streptavidin, loop3–4 functions as a lid for the entry and exit of biotin. When biotin is bound, interactions between biotin and key residues in loop3–4 keep this lid in the closed state. In the engineered mutein, a second biotin exit door is created by changing the amino acid sequence of loop7–8. This door is mobile even in the presence of the bound biotin and can facilitate the release of biotin from the mutein. Since loop7–8 is involved in subunit interactions, alteration of this loop in the engineered mutein results in an 11° rotation between the two dimers in reference to wild-type streptavidin. The tetrameric state of the engineered mutein is stabilized by a H127C mutation, which leads to the formation of inter-subunit disulfide bonds. The biotin binding kinetic parameters (koff of 4.28×10−4 s−1 and Kd of 1.9×10−8 M) make this engineered mutein a superb affinity agent for the purification of biotinylated biomolecules. Affinity matrices can be regenerated using gentle procedures, and regenerated matrices can be reused at least ten times without any observable reduction in binding capacity. With the combination of both the engineered mutein and wild-type streptavidin, biotinylated biomolecules can easily be affinity purified to high purity and immobilized to desirable platforms without any leakage concerns. Other potential biotechnological applications, such as development of an automated high-throughput protein purification system, are feasible. PMID:22536357

Controlling formation of single-molecule junctions by electrochemical reduction of diazonium terminal groups.

Science.gov (United States)

Hines, Thomas; Díez-Pérez, Ismael; Nakamura, Hisao; Shimazaki, Tomomi; Asai, Yoshihiro; Tao, Nongjian

2013-03-06

We report controlling the formation of single-molecule junctions by means of electrochemically reducing two axialdiazonium terminal groups on a molecule, thereby producing direct Au-C covalent bonds in situ between the molecule and gold electrodes. We report a yield enhancement in molecular junction formation as the electrochemical potential of both junction electrodes approach the reduction potential of the diazonium terminal groups. Step length analysis shows that the molecular junction is significantly more stable, and can be pulled over a longer distance than a comparable junction created with amine anchoring bonds. The stability of the junction is explained by the calculated lower binding energy associated with the direct Au-C bond compared with the Au-N bond.

Novel function of glutathione transferase in rat liver mitochondrial membrane: Role for cytochrome c release from mitochondria

International Nuclear Information System (INIS)

Lee, Kang Kwang; Shimoji, Manami; Hossain, Quazi Sohel; Sunakawa, Hajime; Aniya, Yoko

2008-01-01

Microsomal glutathione transferase (MGST1) is activated by oxidative stress. Although MGST1 is found in mitochondrial membranes (mtMGST1), there is no information about the oxidative activation of mtMGST1. In the present study, we aimed to determine whether mtMGST1 also undergoes activation and about its function. When rats were treated with galactosamine/lipopolysaccharide (GalN/LPS), mtMGST1 activity was significantly increased, and the increased activity was reduced by the disulfide reducing agent dithiothreitol. In mitochondria from GalN/LPS-treated rats, disulfide-linked mtMGST1 dimer and mixed protein glutathione disulfides (glutathionylation) were detected. In addition, cytochrome c release from mitochondria isolated from GalN/LPS-treated rats was observed, and the release was inhibited by anti-MGST1 antibodies. Incubation of mitochondria from control rats with diamide and diamide plus GSH in vitro resulted in dimer- and mixed disulfide bond-mediated activation of mtMGST1, respectively. The activation of mtMGST1 by diamide plus GSH caused cytochrome c release from the mitochondria, and the release was prevented by treatment with anti-MGST1 antibodies. In addition, diamide plus GSH treatment caused mitochondrial swelling accompanied by cytochrome c release, which was inhibited by cyclosporin A (CsA) and bongkrekic acid (BKA), inhibitors of the mitochondrial permeability transition (MPT) pore. Furthermore, mtMGST1 activity was also inhibited by CsA and BKA. These results indicate that mtMGST1 is activated through mixed disulfide bond formation that contributes to cytochrome c release from mitochondria through the MPT pore

Dipole and Coulomb forces in electron capture dissociation and electron transfer dissociation mass spectroscopy.

Science.gov (United States)

Świerszcz, Iwona; Skurski, Piotr; Simons, Jack

2012-02-23

Ab initio electronic structure calculations were performed on a doubly charged polypeptide model H(+)-Lys(Ala)(19)-CO-CH(NH(2))-CH(2)-SS-CH(2)-(NH(2))CH-CO-(Ala)(19)-Lys-H(+) consisting of a C-terminal protonated Lys followed by a 19-Ala α-helix with a 20th Ala-like unit whose side chain is linked by a disulfide bond to a corresponding Ala-like unit connected to a second 19-Ala α-helix terminated by a second C-terminal-protonated Lys. The Coulomb potentials arising from the two charged Lys residues and dipole potentials arising from the two oppositely directed 72 D dipoles of the α-helices act to stabilize the SS bond's σ* orbital. The Coulomb potentials provide stabilization of 1 eV, while the two large dipoles generate an additional 4 eV. Such stabilization allows the SS σ* orbital to attach an electron and thereby generate disulfide bond cleavage products. Although calculations are performed only on SS bond cleavage, discussion of N-C(α) bond cleavage caused by electron attachment to amide π* orbitals is also presented. The magnitudes of the stabilization energies as well as the fact that they arise from Coulomb and dipole potentials are supported by results on a small model system consisting of a H(3)C-SS-CH(3) molecule with positive and negative fractional point charges to its left and right designed to represent (i) two positive charges ca. 32 Å distant (i.e., the two charged Lys sites of the peptide model) and (ii) two 72 D dipoles (i.e., the two α-helices). Earlier workers suggested that internal dipole forces in polypeptides could act to guide incoming free electrons (i.e., in electron capture dissociation (ECD)) toward the positive end of the dipole and thus affect the branching ratios for cleaving various bonds. Those workers argued that, because of the huge mass difference between an anion donor and a free electron, internal dipole forces would have a far smaller influence over the trajectory of a donor (i.e., in electron transfer dissociation

RNA/DNA Hybrid Interactome Identifies DXH9 as a Molecular Player in Transcriptional Termination and R-Loop-Associated DNA Damage.

Science.gov (United States)

Cristini, Agnese; Groh, Matthias; Kristiansen, Maiken S; Gromak, Natalia

2018-05-08

R-loops comprise an RNA/DNA hybrid and displaced single-stranded DNA. They play important biological roles and are implicated in pathology. Even so, proteins recognizing these structures are largely undefined. Using affinity purification with the S9.6 antibody coupled to mass spectrometry, we defined the RNA/DNA hybrid interactome in HeLa cells. This consists of known R-loop-associated factors SRSF1, FACT, and Top1, and yet uncharacterized interactors, including helicases, RNA processing, DNA repair, and chromatin factors. We validate specific examples of these interactors and characterize their involvement in R-loop biology. A top candidate DHX9 helicase promotes R-loop suppression and transcriptional termination. DHX9 interacts with PARP1, and both proteins prevent R-loop-associated DNA damage. DHX9 and other interactome helicases are overexpressed in cancer, linking R-loop-mediated DNA damage and disease. Our RNA/DNA hybrid interactome provides a powerful resource to study R-loop biology in health and disease. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

A C-terminal segment of the V{sub 1}R vasopressin receptor is unstructured in the crystal structure of its chimera with the maltose-binding protein

Energy Technology Data Exchange (ETDEWEB)

Adikesavan, Nallini Vijayarangan; Mahmood, Syed Saad; Stanley, Nithianantham; Xu, Zhen; Wu, Nan [Department of Biochemistry, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4935 (United States); Thibonnier, Marc [Department of Medicine, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4935 (United States); Shoham, Menachem, E-mail: mxs10@case.edu [Department of Biochemistry, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4935 (United States)

2005-04-01

The 1.8 Å crystal structure of an MBP-fusion protein with the C-terminal cytoplasmic segment of the V1 vasopressin receptor reveals that the receptor segment is unstructured. The V{sub 1} vascular vasopressin receptor (V{sub 1}R) is a G-protein-coupled receptor (GPCR) involved in the regulation of body-fluid osmolality, blood volume and blood pressure. Signal transduction is mediated by the third intracellular loop of this seven-transmembrane protein as well as by the C-terminal cytoplasmic segment. A chimera of the maltose-binding protein (MBP) and the C-terminal segment of V{sub 1}R has been cloned, expressed, purified and crystallized. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 51.10, b = 66.56, c = 115.72 Å, β = 95.99°. The 1.8 Å crystal structure reveals the conformation of MBP and part of the linker region of this chimera, with the C-terminal segment being unstructured. This may reflect a conformational plasticity in the C-terminal segment that may be necessary for proper function of V{sub 1}R.

C-C bond formation in the intramolecular Diels-Alder reaction of triene amides.

Science.gov (United States)

Benallou, Abdelilah; El Alaoui El Abdallaoui, Habib; Garmes, Hocine

2018-02-01

The mechanism nature of the intramolecular Diels-Alder reaction has been performed; and thus, the changes of C-C bond forming/breaking along IRC are characterized in this study. Conceptual DFT analyses of the most favorable adduct fused/exo shows that the flux electronic will take place from diene to dienophile moiety. Moreover, ELF topological analysis based on the electron density predicts that C-C bond is formed by the coupling of two pseudoradical centers generated at the most significant atoms of the molecules. However, C2 vs C3, also C1 and C4 interaction comes mainly from the global electron density transfer which takes place along the reaction. Two- stage one-step is the proposed mechanism of this reaction, the first stage aims for the formation of C2-C3 σ bond while the second stage aims for C1-C4 σ bond formation. Interestingly, the observed asynchronicity of this IMDA reaction due principally to the asymmetric reorganization of electron density at the most attractive centers.

A bonding study of c-C5H8 adsorption on Pt(111)

International Nuclear Information System (INIS)

Simonetti, S.; Jasen, P.; Gonzalez, E.; Juan, A.; Brizuela, G.

2006-01-01

The chemisorption of cyclopentane (c-C 5 H 8 ) on Pt(111) has been studied using a qualitative band-structure calculations in the framework of tight-binding implementation with the YAeHMOP package. We modeled the metal surface by a two-dimensional slab of finite thickness with an overlayer of c-C 5 H 8 , in a (3x3) di-σ geometry. The c-C 5 H 8 molecule is attached to the surface with its C?C atoms bonded mainly with two Pt atoms while the opposite CH 2 bends towards the surface. The Pt?Pt bonds in the underlying surface and the C?C bonds of c-C 5 H 8 are weakened upon the chemisorption. A noticeable Pt-H and Pt-C interactions has been observed. We found that of Pt 5d z 2 band plays an important role in the bonding between c-C 5 H 8 and the surface, as do the Pt 6s and 6p z bands. The HOMO-LUMO bands of c-C 5 H 8 are very dispersed, indicative of a strong interaction with the metal surface

Dimerization of the docking/adaptor protein HEF1 via a carboxy-terminal helix-loop-helix domain.

Science.gov (United States)

Law, S F; Zhang, Y Z; Fashena, S J; Toby, G; Estojak, J; Golemis, E A

1999-10-10

HEF1, p130(Cas), and Efs define a family of multidomain docking proteins which plays a central coordinating role for tyrosine-kinase-based signaling related to cell adhesion. HEF1 function has been specifically implicated in signaling pathways important for cell adhesion and differentiation in lymphoid and epithelial cells. While the SH3 domains and SH2-binding site domains (substrate domains) of HEF1 family proteins are well characterized and binding partners known, to date the highly conserved carboxy-terminal domains of the three proteins have lacked functional definition. In this study, we have determined that the carboxy-terminal domain of HEF1 contains a divergent helix-loop-helix (HLH) motif. This motif mediates HEF1 homodimerization and HEF1 heterodimerization with a recognition specificity similar to that of the transcriptional regulatory HLH proteins Id2, E12, and E47. We had previously demonstrated that the HEF1 carboxy-terminus expressed as a separate domain in yeast reprograms cell division patterns, inducing constitutive pseudohyphal growth. Here we show that pseudohyphal induction by HEF1 requires an intact HLH, further supporting the idea that this motif has an effector activity for HEF1, and implying that HEF1 pseudohyphal activity derives in part from interactions with yeast helix-loop-helix proteins. These combined results provide initial insight into the mode of function of the HEF1 carboxy-terminal domain and suggest that the HEF1 protein may interact with cellular proteins which control differentiation. Copyright 1999 Academic Press.

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

Directory of Open Access Journals (Sweden)

Vamsi K Kodali

2011-03-01

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

Formation of stable Si–O–C submonolayers on hydrogen-terminated silicon(111 under low-temperature conditions

Directory of Open Access Journals (Sweden)

Yit Lung Khung

2015-01-01

Full Text Available In this letter, we report results of a hydrosilylation carried out on bifunctional molecules by using two different approaches, namely through thermal treatment and photochemical treatment through UV irradiation. Previously, our group also demonstrated that in a mixed alkyne/alcohol solution, surface coupling is biased towards the formation of Si–O–C linkages instead of Si–C linkages, thus indirectly supporting the kinetic model of hydrogen abstraction from the Si–H surface (Khung, Y. L. et al. Chem. – Eur. J. 2014, 20, 15151–15158. To further examine the probability of this kinetic model we compare the results from reactions with bifunctional alkynes carried out under thermal treatment (<130 °C and under UV irradiation, respectively. X-ray photoelectron spectroscopy and contact angle measurements showed that under thermal conditions, the Si–H surface predominately reacts to form Si–O–C bonds from ethynylbenzyl alcohol solution while the UV photochemical route ensures that the alcohol-based alkyne may also form Si–C bonds, thus producing a monolayer of mixed linkages. The results suggested the importance of surface radicals as well as the type of terminal group as being essential towards directing the nature of surface linkage.

Studies on the selectivity of the reaction of (CO){sub 5}W=C(aryl)H with enynes: transfer of the carbene ligand to the C=C Bond versus insertion of the C triple bond C into the W=C Bond

Energy Technology Data Exchange (ETDEWEB)

Fischer, H.; Volkland, H.P.; Stumpf, R.

1996-10-01

The strongly electrophilic monophenylcarbene complex [(CO){sub 5}W=C(Ph)H] (2a) reacts with the enynes H-C triple bond C-R(R=-C(Me)=CH{sub 2})(3), -C{sub 6}H{sub 4}-CH=CH{sub 2}-p (5) and subsequently with PMe{sub 3} to form the C{sub a}lpha-PMe{sub 3} adducts of the vinylidene complexes [(CO){sub 5}W-{l_brace}C(PMe{sub 3})=CH-C{sub 3}H{sub 3}(Me)Ph{r_brace}] (4) and [(CO){sub 5}W {l_brace}C(PMe{sub 3})=CH-C{sub 6}H{sub 4}-C{sub 3}H{sub 4}Ph{r_brace}] (6). The reaction very likely proceeds by transfer of the carbene ligand to the C=C bond of the enyne to form a cyclopropyl-substituted alkyne complex which is in equilibrium with its vinylidene isomer.

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

Science.gov (United States)

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

2017-09-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2010-01-29

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

PTEN C-Terminal Deletion Causes Genomic Instability and Tumor Development

Directory of Open Access Journals (Sweden)

Zhuo Sun

2014-03-01

Full Text Available Tumor suppressor PTEN controls genomic stability and inhibits tumorigenesis. The N-terminal phosphatase domain of PTEN antagonizes the PI3K/AKT pathway, but its C-terminal function is less defined. Here, we describe a knockin mouse model of a nonsense mutation that results in the deletion of the entire Pten C-terminal region, referred to as PtenΔC. Mice heterozygous for PtenΔC develop multiple spontaneous tumors, including cancers and B cell lymphoma. Heterozygous deletion of the Pten C-terminal domain also causes genomic instability and common fragile site rearrangement. We found that Pten C-terminal disruption induces p53 and its downstream targets. Simultaneous depletion of p53 promotes metastasis without influencing the initiation of tumors, suggesting that p53 mainly suppresses tumor progression. Our data highlight the essential role of the PTEN C terminus in the maintenance of genomic stability and suppression of tumorigenesis.

Properties of catalase-peroxidase lacking its C-terminal domain

International Nuclear Information System (INIS)

Baker, Ruletha D.; Cook, Carma O.; Goodwin, Douglas C.

2004-01-01

Catalase-peroxidases have a two-domain structure. The N-terminal domain contains the bifunctional active site, but the function of the C-terminal domain is unknown. We produced catalase-peroxidase containing only its N-terminal domain (KatG Nterm ). Removal of the C-terminal domain did not result in unexpected changes in secondary structure as evaluated by CD, but KatG Nterm had neither catalase nor peroxidase activity. Partial recovery of both activities was achieved by incubating KatG Nterm with the separately expressed and isolated KatG C-terminal domain. Spectroscopic measurements revealed a shift in heme environment from a mixture of high-spin species (wtKatG) to exclusively hexacoordinate, low-spin (KatG Nterm ). Moreover, a >1000-fold lower k on for CN - binding was observed for KatG Nterm . EPR spectra for KatG Nterm and the results of site-specific substitution of active site histidines suggested that the distal histidine was the sixth ligand. Thus, one important role for the C-terminal domain may be to support the architecture of the active site, preventing heme ligation by this catalytically essential residue

Ligand-Controlled Chemoselective C(acyl)–O Bond vs C(aryl)–C Bond Activation of Aromatic Esters in Nickel Catalyzed C(sp2)–C(sp3) Cross-Couplings

KAUST Repository

Chatupheeraphat, Adisak

2018-02-20

A ligand-controlled and site-selective nickel catalyzed Suzuki-Miyaura cross-coupling reaction with aromatic esters and alkyl organoboron reagents as coupling partners was developed. This methodology provides a facile route for C(sp2)-C(sp3) bond formation in a straightforward fashion by successful suppression of the undesired β-hydride elimination process. By simply switching the phosphorus ligand, the ester substrates are converted into the alkylated arenes and ketone products, respectively. The utility of this newly developed protocol was demonstrated by its wide substrate scope, broad functional group tolerance and application in the synthesis of key intermediates for the synthesis of bioactive compounds. DFT studies on the oxidative addition step helped rationalizing this intriguing reaction chemoselectivity: whereas nickel complexes with bidentate ligands favor the C(aryl)-C bond cleavage in the oxidative addition step leading to the alkylated product via a decarbonylative process, nickel complexes with monodentate phosphorus ligands favor activation of the C(acyl)-O bond, which later generates the ketone product.

Analysis of the link between the redox state and enzymatic activity of the HtrA (DegP protein from Escherichia coli.

Directory of Open Access Journals (Sweden)

Tomasz Koper

Full Text Available Bacterial HtrAs are proteases engaged in extracytoplasmic activities during stressful conditions and pathogenesis. A model prokaryotic HtrA (HtrA/DegP from Escherichia coli requires activation to cleave its substrates efficiently. In the inactive state of the enzyme, one of the regulatory loops, termed LA, forms inhibitory contacts in the area of the active center. Reduction of the disulfide bond located in the middle of LA stimulates HtrA activity in vivo suggesting that this S-S bond may play a regulatory role, although the mechanism of this stimulation is not known. Here, we show that HtrA lacking an S-S bridge cleaved a model peptide substrate more efficiently and exhibited a higher affinity for a protein substrate. An LA loop lacking the disulfide was more exposed to the solvent; hence, at least some of the interactions involving this loop must have been disturbed. The protein without S-S bonds demonstrated lower thermal stability and was more easily converted to a dodecameric active oligomeric form. Thus, the lack of the disulfide within LA affected the stability and the overall structure of the HtrA molecule. In this study, we have also demonstrated that in vitro human thioredoxin 1 is able to reduce HtrA; thus, reduction of HtrA can be performed enzymatically.

Vaccatides: Antifungal Glutamine-Rich Hevein-Like Peptides from Vaccaria hispanica

Directory of Open Access Journals (Sweden)

Ka H. Wong

2017-06-01

Full Text Available Hevein and hevein-like peptides are disulfide-constrained chitin-binding cysteine-rich peptides. They are divided into three subfamilies, 6C-, 8C-, and 10C-hevein-like peptides, based on the number of cysteine residues. In addition, hevein-like peptides can exist in two forms, short and long. The long C-terminal form found in hevein and 10C-hevein-like peptides contain a C-terminal protein cargo. In contrast, the short form without a protein cargo is found in all three subfamilies. Here, we report the discovery and characterization of two novel glutamine-rich and protein cargo-free 8C-hevein-like peptides, vaccatides vH1 and vH2, from Vaccaria hispanica of the Caryophyllaceae family. Proteomic analyses showed that the vaccatides are 40–41 amino acids in length and contain a chitin-binding domain. NMR determination revealed that vaccatide vH2 displays a highly compact structure with a N-terminal cystine knot and an addition C-terminal disulfide bond. Stability studies showed that this compact structure renders vaccatide vH2 resistant to thermal, chemical and proteolytic degradation. The chitin-binding vH2 was shown to inhibit the mycelium growth of four phyto-pathogenic fungal strains with IC50 values in the micromolar range. Our findings show that vaccatides represent a new family of 8C-hevein-like peptides, which are protein cargo-free and glutamine-rich, characteristics that differentiate them from the prototypic hevein and the 10C-hevein-like peptides. In summary, this study enriches the existing library of hevein-like peptides and provides insight into their molecular diversity in sequence, structure and biosynthesis. Additionally, their highly disulfide-constrained structure could be used as a scaffold for developing metabolically and orally active peptidyl therapeutics.

Metal-free oxidative olefination of primary amines with benzylic C-H bonds through direct deamination and C-H bond activation.

Science.gov (United States)

Gong, Liang; Xing, Li-Juan; Xu, Tong; Zhu, Xue-Ping; Zhou, Wen; Kang, Ning; Wang, Bin

2014-09-14

An oxidative olefination reaction between aliphatic primary amines and benzylic sp(3) C-H bonds has been achieved using N-bromosuccinimide as catalyst and tert-butyl hydroperoxide as oxidant. The olefination proceeds under mild metal-free conditions through direct deamination and benzylic C-H bond activation, and provides easy access to biologically active 2-styrylquinolines with (E)-configuration.

Direct approaches to nitriles via highly efficient nitrogenation strategy through C-H or C-C bond cleavage.

Science.gov (United States)

Wang, Teng; Jiao, Ning

2014-04-15

Because of the importance of nitrogen-containing compounds in chemistry and biology, organic chemists have long focused on the development of novel methodologies for their synthesis. For example, nitrogen-containing compounds show up within functional materials, as top-selling drugs, and as bioactive molecules. To synthesize these compounds in a green and sustainable way, researchers have focused on the direct functionalization of hydrocarbons via C-H or C-C bond cleavage. Although researchers have made significant progress in the direct functionalization of simple hydrocarbons, direct C-N bond formation via C-H or C-C bond cleavage remains challenging, in part because of the unstable character of some N-nucleophiles under oxidative conditions. The nitriles are versatile building blocks and precursors in organic synthesis. Recently, chemists have achieved the direct C-H cyanation with toxic cyanide salts in the presence of stoichiometric metal oxidants. In this Account, we describe recent progress made by our group in nitrile synthesis. C-H or C-C bond cleavage is a key process in our strategy, and azides or DMF serve as the nitrogen source. In these reactions, we successfully realized direct nitrile synthesis using a variety of hydrocarbon groups as nitrile precursors, including methyl, alkenyl, and alkynyl groups. We could carry out C(sp(3))-H functionalization on benzylic, allylic, and propargylic C-H bonds to produce diverse valuable synthetic nitriles. Mild oxidation of C═C double-bonds and C≡C triple-bonds also produced nitriles. The incorporation of nitrogen within the carbon skeleton typically involved the participation of azide reagents. Although some mechanistic details remain unclear, studies of these nitrogenation reactions implicate the involvement of a cation or radical intermediate, and an oxidative rearrangement of azide intermediate produced the nitrile. We also explored environmentally friendly oxidants, such as molecular oxygen, to make our

Enhanced radiosyntheses of [¹¹C]raclopride and [¹¹C]DASB using ethanolic loop chemistry.

Science.gov (United States)

Shao, Xia; Schnau, Paul L; Fawaz, Maria V; Scott, Peter J H

2013-01-01

To improve the synthesis and quality control of carbon-11 labeled radiopharmaceuticals, we report the fully automated loop syntheses of [¹¹C]raclopride and [¹¹C]DASB using ethanol as the only organic solvent for synthesis module cleaning, carbon-11 methylation, HPLC purification, and reformulation. Ethanolic loop chemistry is fully automated using a GE TRACERLab FX(C-Pro) synthesis module, and is readily adaptable to any other carbon-11 synthesis apparatus. Precursors (1 mg) were dissolved in ethanol (100 μL) and loaded into the HPLC loop. [¹¹C]MeOTf was passed through the HPLC loop and then the labeled products were purified by semi-preparative HPLC and reformulated into ethanolic saline. Both [¹¹C]raclopride (3.7% RCY; >95% RCP; SA=20831 Ci/mmol; n=64) and [¹¹C]DASB, both with (3.0% RCY; >95% RCP; SA=15152Ci/mmol; n=9) and without (3.0% RCY; >95% RCP; SA=10931 Ci/mmol; n=3) sodium ascorbate, have been successfully prepared using the described methodology. Doses are suitable for human use and the described methods are now employed for routine clinical production of both radiopharmaceuticals at the University of Michigan. Ethanolic loop chemistry is a powerful technique for preparing [¹¹C]raclopride and [¹¹C]DASB, and we are in the process of adapting it for other carbon-11 radiopharmaceuticals prepared in our laboratories ([¹¹C]PMP, [¹¹C]PBR28 etc.). Copyright © 2013 Elsevier Inc. All rights reserved.

ALERGENUL ARA H3, GLOBULINA DE REZERVĂ 11S DIN SEMINŢELE DE ARAHIDE 1. PROTEOLIZA LIMITATĂ CU PAPAINĂ

Directory of Open Access Journals (Sweden)

Ala CHERDIVARĂ

2017-07-01

Full Text Available Proteoliza globulinei de rezervă 11S din seminţele de arahide, Ara h3, cu papaină începe cu cuscindarea secvenței C-terminale extinse a α-catenelor, care cuprinde regiunea α-helixurilor. Clivarea ulterioară a buclei dintre β-strendurile E' şi F' din partea centrală a β-barrelui din α-catene duce la formarea fragmentelor, legate cu β-catenele intacte prin legătură disulfidică, precum şi reţinute în molecula globulinei 11S parţial hidrolizată prin interacţiuni necovalente. Conform scenariului proteolizei descrise, acţiunea iniţială a papainei distruge regiunea C-terminală a α-catenelor, în care sunt prezenţi trei din cei patru determinanţi antigenici (epitopii IgE, identificaţi în globulina 11S din arahide. Astfel, gradul de alergenicitate a Ara h3 poate fi substanțial redus prin proteoliza sa limitată cu papaină.ALLERGEN ARA H3, STORAGE 11S GLOBULIN FROM PEANUT SEEDS 1. PAPAIN LIMITED PROTEOLYSISPapain proteolysis of the storage 11S globulin Ara h3 from peanut seeds starts from the detachment of an extended a-chain C-terminal sequence covering the region of a-helices. Further cleavage of a loop between b-strands E’ and F’ inside the central part of a-chain b-barrel occurs generating fragments connected with intact b-chains via a disulfide bond and those retained inside the molecule of partially hydrolyzed 11S globulin due to non-covalent interactions. In accordance with the described proteolysis scenario, the C-terminal a-chain region containing three of the four antigen determinants (IgE epitopes identified in the peanut 11S globulin is destroyed during the initial papain action. Therefore, the level of Ara h3 allergenicity can be decreased via papain limited proteolysis.

Social evolution. Oxytocin-gaze positive loop and the coevolution of human-dog bonds.

Science.gov (United States)

Nagasawa, Miho; Mitsui, Shouhei; En, Shiori; Ohtani, Nobuyo; Ohta, Mitsuaki; Sakuma, Yasuo; Onaka, Tatsushi; Mogi, Kazutaka; Kikusui, Takefumi

2015-04-17

Human-like modes of communication, including mutual gaze, in dogs may have been acquired during domestication with humans. We show that gazing behavior from dogs, but not wolves, increased urinary oxytocin concentrations in owners, which consequently facilitated owners' affiliation and increased oxytocin concentration in dogs. Further, nasally administered oxytocin increased gazing behavior in dogs, which in turn increased urinary oxytocin concentrations in owners. These findings support the existence of an interspecies oxytocin-mediated positive loop facilitated and modulated by gazing, which may have supported the coevolution of human-dog bonding by engaging common modes of communicating social attachment. Copyright © 2015, American Association for the Advancement of Science.

Ab initio characterization of coupling strength for all types of dangling-bond pairs on the hydrogen-terminated Si(100)-2 × 1 surface

Science.gov (United States)

Shaterzadeh-Yazdi, Zahra; Sanders, Barry C.; DiLabio, Gino A.

2018-04-01

Recent work has suggested that coupled silicon dangling bonds sharing an excess electron may serve as building blocks for quantum-cellular-automata cells and quantum computing schemes when constructed on hydrogen-terminated silicon surfaces. In this work, we employ ab initio density-functional theory to examine the details associated with the coupling between two dangling bonds sharing one excess electron and arranged in various configurations on models of phosphorous-doped hydrogen-terminated silicon (100) surfaces. Our results show that the coupling strength depends strongly on the relative orientation of the dangling bonds on the surface and on the separation between them. The orientation of dangling bonds is determined by the anisotropy of the silicon (100) surface, so this feature of the surface is a significant contributing factor to variations in the strength of coupling between dangling bonds. The results demonstrate that simple models for approximating tunneling, such as the Wentzel-Kramer-Brillouin method, which do not incorporate the details of surface structure, are incapable of providing reasonable estimates of tunneling rates between dangling bonds. The results provide guidance to efforts related to the development of dangling-bond based computing elements.

Synthesis and Anti-HIV-1 Evaluation of New Sonogashira-Modified Emivirine (MKC-442) Analogues

DEFF Research Database (Denmark)

Danel, Krzystof; Jørgensen, Per Trolle; La Colla, Paolo

2009-01-01

The MKC-442 analogue 6-(3,5-dimethylbenzyl)-5-ethyluracil substituted with a (propargyloxo)methyl group at N(1) has previously been found highly active against HIV-1. The C C bond in the substituent at N(1) is here utilized in a series of chemical reactions in order to develop new agents with hig......The MKC-442 analogue 6-(3,5-dimethylbenzyl)-5-ethyluracil substituted with a (propargyloxo)methyl group at N(1) has previously been found highly active against HIV-1. The C C bond in the substituent at N(1) is here utilized in a series of chemical reactions in order to develop new agents...... with higher activity against HIV-1-resistant mutants. The syntheses involved Pd-catalyzed C,C-coupling reactions, addition of disulfides, and click chemistry on the terminal C C bond as well as addition of bromine to the so formed internal C C bonds. Sonogashira coupling were performed with silyl...

Structural Basis for Target Protein Regcognition by Thiredoxin

DEFF Research Database (Denmark)

Maeda, Kenji

2007-01-01

Ser) and a mutant of an in vitro substrate alpha-amylase/subtilisin inhibitor (BASI) (Cys144Ser), as a reaction intermediate-mimic of Trx-catalyzed disulfide reduction. The resultant structure showed a sequence of BASI residues along a conserved hydrophobic groove constituted of three loop segments...... of Trx-fold proteins glutaredoxin and glutathione transferase. This study suggests that the features of main chain conformation as well as charge property around disulfide bonds in protein substrates are important factors for interaction with Trx. Moreover, this study describes a detailed structural......Thioredoxin (Trx) is an ubiquitous protein disulfide reductase that possesses two redox active cysteines in the conserved active site sequence motif, Trp-CysN-Gly/Pro-Pro-CysC situated in the so called Trx-fold. The lack of insight into the protein substrate recognition mechanism of Trx has to date...

Thermal shock resistances of a bonding material of C/C composite and copper

International Nuclear Information System (INIS)

Kurumada, Akira; Oku, Tatsuo; Kawamata, Kiyohiro; Motojima, Osamu; Noda, Nobuaki; McEnaney, B.

1997-01-01

The purpose of this study is to contribute to the development and the safety design of plasma facing components for fusion reactor devices. We evaluated the thermal shock resistance and the thermal shock fracture toughness of a bonding material which was jointed a carbon-fiber-reinforced carbon composite (C/C composite) to oxygen-free copper. We also examined the microstructures of the bonding layers using a scanning electron microscope before and after thermal shock tests. The bonding material did not fracture during thermal shock tests. However, thermal cracks and delamination cracks were observed in the bonding layers. (author)

C-terminal domains of bacterial proteases: structure, function and the biotechnological applications.

Science.gov (United States)

Huang, J; Wu, C; Liu, D; Yang, X; Wu, R; Zhang, J; Ma, C; He, H

2017-01-01

C-terminal domains widely exist in the C-terminal region of multidomain proteases. As a β-sandwich domain in multidomain protease, the C-terminal domain plays an important role in proteolysis including regulation of the secretory process, anchoring and swelling the substrate molecule, presenting as an inhibitor for the preprotease and adapting the protein structural flexibility and stability. In this review, the diversity, structural characteristics and biological function of C-terminal protease domains are described. Furthermore, the application prospects of C-terminal domains, including polycystic kidney disease, prepeptidase C-terminal and collagen-binding domain, in the area of medicine and biological artificial materials are also discussed. © 2016 The Society for Applied Microbiology.

Role of the DELSEED Loop in Torque Transmission of F1-ATPase

Science.gov (United States)

Tanigawara, Mizue; Tabata, Kazuhito V.; Ito, Yuko; Ito, Jotaro; Watanabe, Rikiya; Ueno, Hiroshi; Ikeguchi, Mitsunori; Noji, Hiroyuki

2012-01-01

F1-ATPase is an ATP-driven rotary motor that generates torque at the interface between the catalytic β-subunits and the rotor γ-subunit. The β-subunit inwardly rotates the C-terminal domain upon nucleotide binding/dissociation; hence, the region of the C-terminal domain that is in direct contact with γ—termed the DELSEED loop—is thought to play a critical role in torque transmission. We substituted all the DELSEED loop residues with alanine to diminish specific DELSEED loop-γ interactions and with glycine to disrupt the loop structure. All the mutants rotated unidirectionally with kinetic parameters comparable to those of the wild-type F1, suggesting that the specific interactions between DELSEED loop and γ is not involved in cooperative interplays between the catalytic β-subunits. Glycine substitution mutants generated half the torque of the wild-type F1, whereas the alanine mutant generated comparable torque. Fluctuation analyses of the glycine/alanine mutants revealed that the γ-subunit was less tightly held in the α3β3-stator ring of the glycine mutant than in the wild-type F1 and the alanine mutant. Molecular dynamics simulation showed that the DELSEED loop was disordered by the glycine substitution, whereas it formed an α-helix in the alanine mutant. Our results emphasize the importance of loop rigidity for efficient torque transmissions. PMID:23009846

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

Directory of Open Access Journals (Sweden)

Julien Becker

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

Heparan sulfate regulates fibrillin-1 N- and C-terminal interactions

DEFF Research Database (Denmark)

Cain, Stuart A; Baldwin, Andrew K; Mahalingam, Yashithra

2008-01-01

Fibrillin-1 N- and C-terminal heparin binding sites have been characterized. An unprocessed monomeric N-terminal fragment (PF1) induced a very high heparin binding response, indicating heparin-mediated multimerization. Using PF1 deletion and short fragments, a heparin binding site was localized w......-terminal interactions with heparin/heparan sulfate directly influence cell behavior, whereas C-terminal interactions with heparin/heparan sulfate regulate elastin deposition. These data highlight how heparin/heparan sulfate controls fibrillin-1 interactions....

Thermal Stability of RNA Phage Virus-Like Particles Displaying Foreign Peptides

Directory of Open Access Journals (Sweden)

Peabody David S

2011-05-01

Full Text Available Abstract Background To be useful for genetic display of foreign peptides a viral coat protein must tolerate peptide insertions without major disruption of subunit folding and capsid assembly. The folding of the coat protein of RNA phage MS2 does not normally tolerate insertions in its AB-loop, but an engineered single-chain dimer readily accepts them as long as they are restricted to one of its two halves. Results Here we characterize the effects of peptide insertions on the thermal stabilities of MS2 virus-like particles (VLPs displaying a variety of different peptides in one AB-loop of the coat protein single-chain dimer. These particles typically denature at temperatures around 5-10°C lower than unmodified VLPs. Even so, they are generally stable up to about 50°C. VLPs of the related RNA phage PP7 are cross-linked with intersubunit disulfide bonds and are therefore significantly more stable. An AB-loop insertion also reduces the stability of PP7 VLPs, but they only begin to denature above about 70°C. Conclusions VLPs assembled from MS2 single-chain dimer coat proteins with peptide insertions in one of their AB-loops are somewhat less stable than the wild-type particle, but still resist heating up to about 50°C. Because they possess disulfide cross-links, PP7-derived VLPs provide an alternate platform with even higher stability.

Distribution of sizes of erased loops for loop-erased random walks

OpenAIRE

Dhar, Deepak; Dhar, Abhishek

1997-01-01

We study the distribution of sizes of erased loops for loop-erased random walks on regular and fractal lattices. We show that for arbitrary graphs the probability $P(l)$ of generating a loop of perimeter $l$ is expressible in terms of the probability $P_{st}(l)$ of forming a loop of perimeter $l$ when a bond is added to a random spanning tree on the same graph by the simple relation $P(l)=P_{st}(l)/l$. On $d$-dimensional hypercubical lattices, $P(l)$ varies as $l^{-\\sigma}$ for large $l$, whe...

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

International Nuclear Information System (INIS)

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

2011-01-01

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

Molecularly Tuning the Radicaloid N-H···O═C Hydrogen Bond.

Science.gov (United States)

Lu, Norman; Chung, Wei-Cheng; Ley, Rebecca M; Lin, Kwan-Yu; Francisco, Joseph S; Negishi, Ei-Ichi

2016-03-03

Substituent effects on the open shell N-H···O═C hydrogen-bond has never been reported. This study examines how 12 functional groups composed of electron donating groups (EDG), halogen atoms and electron withdrawing groups (EWG) affect the N-H···O═C hydrogen-bond properties in a six-membered cyclic model system of O═C(Y)-CH═C(X)N-H. It is found that group effects on this open shell H-bonding system are significant and have predictive trends when X = H and Y is varied. When Y is an EDG, the N-H···O═C hydrogen-bond is strengthened; and when Y is an EWG, the bond is weakened; whereas the variation in electronic properties of X group do not exhibit a significant impact upon the hydrogen bond strength. The structural impact of the stronger N-H···O═C hydrogen-bond are (1) shorter H and O distance, r(H···O) and (2) a longer N-H bond length, r(NH). The stronger N-H···O═C hydrogen-bond also acts to pull the H and O in toward one another which has an effect on the bond angles. Our findings show that there is a linear relationship between hydrogen-bond angle and N-H···O═C hydrogen-bond energy in this unusual H-bonding system. In addition, there is a linear correlation of the r(H···O) and the hydrogen bond energy. A short r(H···O) distance corresponds to a large hydrogen bond energy when Y is varied. The observed trends and findings have been validated using three different methods (UB3LYP, M06-2X, and UMP2) with two different basis sets.

Multiple C-H Bond Activations and Ring-Opening C-S Bond Cleavage of Thiophene by Dirhenium Carbonyl Complexes.

Science.gov (United States)

Adams, Richard D; Dhull, Poonam; Tedder, Jonathan D

2018-06-14

The reaction of Re 2 (CO) 8 (μ-C 6 H 5 )(μ-H) (1) with thiophene in CH 2 Cl 2 at 40 °C yielded the new compound Re 2 (CO) 8 (μ-η 2 -SC 4 H 3 )(μ-H) (2), which contains a bridging σ-π-coordinated thienyl ligand formed by the activation of the C-H bond at the 2 position of the thiophene. Compound 2 exhibits dynamical activity on the NMR time scale involving rearrangements of the bridging thienyl ligand. The reaction of compound 2 with a second 1 equiv of 1 at 45 °C yielded the doubly metalated product [Re 2 (CO) 8 (μ-H)] 2 (μ-η 2 -2,3-μ-η 2 -4,5-C 4 H 2 S) (3), formed by the activation of the C-H bond at the 5 position of the thienyl ligand in 2. Heating 3 in a hexane solvent to reflux transformed it into the ring-opened compound Re(CO) 4 [μ-η 5 -η 2 -SCC(H)C(H)C(H)][Re(CO) 3 ][Re 2 (CO) 8 (μ-H)] (4) by the loss of one CO ligand. Compound 4 contains a doubly metalated 1-thiapentadienyl ligand formed by the cleavage of one of the C-S bonds. When heated to reflux (125 °C) in an octane solvent in the presence of H 2 O, the new compound Re(CO) 4 [η 5 -μ-η 2 -SC(H)C(H)C(H)C(H)]Re(CO) 3 (5) was obtained by cleavage of the Re 2 (CO) 8 (μ-H) group from 4 with formation of the known coproduct [Re(CO) 3 (μ 3 -OH)] 4 . All new products were characterized by single-crystal X-ray diffraction analyses.

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

Czech Academy of Sciences Publication Activity Database

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

2014-01-01

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

DETECTING NANOFLARE HEATING EVENTS IN SUBARCSECOND INTER-MOSS LOOPS USING Hi-C

International Nuclear Information System (INIS)

Winebarger, Amy R.; Moore, Ronald; Cirtain, Jonathan; Walsh, Robert W.; De Pontieu, Bart; Title, Alan; Hansteen, Viggo; Golub, Leon; Korreck, Kelly; Weber, Mark; Kobayashi, Ken; DeForest, Craig; Kuzin, Sergey

2013-01-01

The High-resolution Coronal Imager (Hi-C) flew aboard a NASA sounding rocket on 2012 July 11 and captured roughly 345 s of high-spatial and temporal resolution images of the solar corona in a narrowband 193 Å channel. In this paper, we analyze a set of rapidly evolving loops that appear in an inter-moss region. We select six loops that both appear in and fade out of the Hi-C images during the short flight. From the Hi-C data, we determine the size and lifetimes of the loops and characterize whether these loops appear simultaneously along their length or first appear at one footpoint before appearing at the other. Using co-aligned, co-temporal data from multiple channels of the Atmospheric Imaging Assembly on the Solar Dynamics Observatory, we determine the temperature and density of the loops. We find the loops consist of cool (∼10 5 K), dense (∼10 10 cm –3 ) plasma. Their required thermal energy and their observed evolution suggest they result from impulsive heating similar in magnitude to nanoflares. Comparisons with advanced numerical simulations indicate that such dense, cold and short-lived loops are a natural consequence of impulsive magnetic energy release by reconnection of braided magnetic field at low heights in the solar atmosphere.

UV Resonance Raman Elucidation of the Terminal and Internal Peptide Bond Conformations of Crystalline and Solution Oligoglycines.

Science.gov (United States)

Bykov, Sergei V; Asher, Sanford A

2010-11-30

Spectroscopic investigations of macromolecules generally attempt to interpret the measured spectra in terms of the summed contributions of the different molecular fragments. This is the basis of the local mode approximation in vibrational spectroscopy. In the case of resonance Raman spectroscopy independent contributions of molecular fragments require both a local mode-like behavior and the uncoupled electronic transitions. Here we show that the deep UV resonance Raman spectra of aqueous solution phase oligoglycines show independent peptide bond molecular fragment contributions indicating that peptide bonds electronic transitions and vibrational modes are uncoupled. We utilize this result to separately determine the conformational distributions of the internal and penultimate peptide bonds of oligoglycines. Our data indicate that in aqueous solution the oligoglycine terminal residues populate conformations similar to those found in crystals (3(1)-helices and β-strands), but with a broader distribution, while the internal peptide bond conformations are centered around the 3(1)-helix Ramachandran angles.

Triamidoamine-uranium(IV)-stabilized terminal parent phosphide and phosphinidene complexes

Energy Technology Data Exchange (ETDEWEB)

Gardner, Benedict M.; McMaster, Jonathan; Lewis, William; Blake, Alexander J.; Liddle, Stephen T. [School of Chemistry, University of Nottingham (United Kingdom); Balazs, Gabor; Scheer, Manfred [Institut of Inorganic Chemistry, University of Regensburg (Germany); Tuna, Floriana; McInnes, Eric J.L. [School of Chemistry and Photon Science Institute, University of Manchester (United Kingdom)

2014-04-22

Reaction of [U(Tren{sup TIPS})(THF)][BPh{sub 4}] (1; Tren{sup TIPS}=N{CH_2CH_2NSi(iPr)_3}{sub 3}) with NaPH{sub 2} afforded the novel f-block terminal parent phosphide complex [U(Tren {sup TIPS})(PH{sub 2})] (2; U-P=2.883(2) Aa). Treatment of 2 with one equivalent of KCH{sub 2}C{sub 6}H{sub 5} and two equivalents of benzo-15-crown-5 ether (B15C5) afforded the unprecedented metal-stabilized terminal parent phosphinidene complex [U(Tren{sup TIPS})(PH)][K(B15C5){sub 2}] (4; U=P=2.613(2) Aa). DFT calculations reveal a polarized-covalent U=P bond with a Mayer bond order of 1.92. (copyright 2014 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

The first N-terminal unprotected (Gly-Aib)n peptide: H-Gly-Aib-Gly-Aib-OtBu.

Science.gov (United States)

Gessmann, Renate; Brückner, Hans; Petratos, Kyriacos

2015-12-01

Glycine (Gly) is incorporated in roughly half of all known peptaibiotic (nonribosomally biosynthesized antibiotic peptides of fungal origin) sequences and is the residue with the greatest conformational flexibility. The conformational space of Aib (α-aminoisobutyric acid) is severely restricted by the second methyl group attached to the Cα atom. Most of the crystal structures containing Aib are N-terminal protected. Deprotection of the N- or C-terminus of peptides may alter the hydrogen-bonding scheme and/or the structure and may facilitate crystallization. The structure reported here for glycyl-α-aminoisobutyrylglycyl-α-aminoisobutyric acid tert-butyl ester, C16H30N4O5, describes the first N-terminal-unprotected (Gly-Aib)n peptide. The achiral peptide could form an intramolecular hydrogen bond between the C=O group of Gly1 and the N-H group of Aib4. This hydrogen bond is found in all tetrapeptides and N-terminal-protected tripeptides containing Aib, apart from one exception. In the present work, this hydrogen bond is not observed (N...O = 5.88 Å). Instead, every molecule is hydrogen bonded to six other symmetry-related molecules with a total of eight hydrogen bonds per molecule. The backbone conformation starts in the right-handed helical region (and the left-handed helical region for the inverted molecule) and reverses the screw sense in the last two residues.

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

Science.gov (United States)

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

2018-02-14

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

Selective C(sp2)-C(sp) bond cleavage: the nitrogenation of alkynes to amides.

Science.gov (United States)

Qin, Chong; Feng, Peng; Ou, Yang; Shen, Tao; Wang, Teng; Jiao, Ning

2013-07-22

Breakthrough: A novel catalyzed direct highly selective C(sp2)-C(sp) bond functionalization of alkynes to amides has been developed. Nitrogenation is achieved by the highly selective C(sp2)-C(sp) bond cleavage of aryl-substituted alkynes. The oxidant-free and mild conditions and wide substrate scope make this method very practical. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of Runella slithyformis HD-Pnk, a bifunctional DNA/RNA end-healing enzyme composed of an N-terminal 2',3' -phosphoesterase HD domain and a C-terminal 5' -OH polynucleotide kinase domain.

Science.gov (United States)

Munir, Annum; Shuman, Stewart

2016-11-28

5' and 3' end healing are key steps in nucleic acid break repair in which 5' -OH ends are phosphorylated by a polynucleotide kinase and 3' -PO 4 or 2',3' -cyclic-PO 4 ends are hydrolyzed by a phosphoesterase to generate the 5' -PO 4 and 3' -OH termini required for sealing by classic polynucleotide ligases. End healing and sealing enzymes are present in diverse bacterial taxa, often organized as modular units within a single multifunctional polypeptide or as subunits of a repair complex. Here we identify and characterize Runella slithyformis HD-Pnk as a novel bifunctional end-healing enzyme composed of an N-terminal 2',3' -phosphoesterase HD domain and a C-terminal 5' -OH polynucleotide kinase P-loop domain. HD-Pnk phosphorylates 5' -OH polynucleotides (9-mers or longer) in the presence of magnesium and any NTP donor. HD-Pnk dephosphorylates RNA 2',3' -cyclic phosphate, RNA 3' -phosphate, RNA 2' -phosphate, and DNA 3' -phosphate ends in the presence of a transition metal cofactor, which can be nickel, copper or cobalt. HD-Pnkp homologs are present in genera from eleven bacterial phyla and are often encoded in an operon with a putative ATP-dependent polynucleotide ligase. The present study provides insights to the diversity of nucleic acid repair strategies via the characterization of Runella slithyformis HD-Pnkp as the exemplar of a novel clade of dual 5' and 3' end-healing enzymes that phosphorylate 5' -OH termini and dephosphorylate 2',3' -cyclic-PO 4 , 3' -PO 4 , and 2' -PO 4 ends. The distinctive feature of HD-Pnk is its domain composition: a fusion of an N-terminal HD phosphohydrolase module to a C-terminal P-loop polynucleotide kinase module. Homologs of Runella HD-Pnk with the same domain composition, domain order, and similar polypeptide size are distributed widely among genera from eleven bacterial phyla. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Rhodium(III)-Catalyzed Amidation of Unactivated C(sp(3) )-H Bonds.

Science.gov (United States)

Wang, He; Tang, Guodong; Li, Xingwei

2015-10-26

Nitrogenation by direct functionalization of C-H bonds represents an important strategy for constructing C-N bonds. Rhodium(III)-catalyzed direct amidation of unactivated C(sp(3) )-H bonds is rare, especially under mild reaction conditions. Herein, a broad scope of C(sp(3) )-H bonds are amidated under rhodium catalysis in high efficiency using 3-substituted 1,4,2-dioxazol-5-ones as the amide source. The protocol broadens the scope of rhodium(III)-catalyzed C(sp(3) )-H activation chemistry, and is applicable to the late-stage functionalization of natural products. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

DEFF Research Database (Denmark)

Ferré, Henrik; Ruffet, Emmanuel; Blicher, Thomas

2003-01-01

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

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

DEFF Research Database (Denmark)

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

2003-01-01

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

The human selenoprotein VCP-interacting membrane protein (VIMP) is non-globular and harbors a reductase function in an intrinsically disordered region

DEFF Research Database (Denmark)

Christensen, Lea Cecilie; Jensen, Njal Winther; Lages Lino Vala, Andrea

2012-01-01

that Cys-188 in cVIMP-Cys forms a disulfide bond with Cys-174, consistent with the presence of a Cys174-Sec188 selenosulfide bond in the native sequence. For the disulfide bond in cVIMP-Cys we determined the reduction potential to -200 mV, and showed it to be a good substrate of thioredoxin. Based...

On the Michael addition of water to C = C bonds

NARCIS (Netherlands)

Chen, B.

2015-01-01

?-Hydroxy carbonyl compounds are an important class of compounds often found as a common structural motif in natural products. Although the molecules themselves look rather simple, their synthesis can be challenging. Water addition to conjugated C = C bonds opens up a straightforward route for the

Internal mobility of reactive-site-hydrolyzed recombinant Cucurbita maxima trypsin inhibitor-V characterized by NMR spectroscopy: evidence for differential stabilization of newly formed C- and N-termini.

Science.gov (United States)

Liu, J; Prakash, O; Huang, Y; Wen, L; Wen, J J; Huang, J K; Krishnamoorthi, R

1996-09-24

The solution structure and internal dynamics of the reactive-site (Lys44-Asp45 peptide bond) hydrolyzed form of recombinant Cucurbita maxima trypsin inhibitor-V (rCMTI-V*) were characterized by the application of two-dimensional 1H-15N NMR methods to the uniformly 15N-labeled protein. The 1H-15N chemical shift correlation spectra of rCMTI-V* were assigned, and the chemical shift data were compared with those available for rCMTI-V [Liu, J., Prakash, O., Cai, M., Gong, Y., Huang, Y., Wen, L., Wen, J. J., Huang, J.-K., & Krishnamoorthi, R. (1996) Biochemistry 35, 1516-1524] and CMTI-V* [Cai, M., Gong, Y., Prakash, O., & Krishnamoorthi, R. (1995) Biochemistry 34, 12087-12094] for which three-dimensional solution structures have been determined. It was deduced that the solution structure of rCMTI-V* was almost the same as that of CMTI-V*. 15N spin-lattice and spin-spin relaxation rate constants (R1 and R2, respectively) and ¿1H¿-15N steady-state heteronuclear Overhauser effects were measured for the peptide NH units and arginine and tryptophan N epsilon H groups in rCMTI-V*, and the model-free parameters [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559, 4559-4570] were computed. Most of the backbone of rCMTI-V* is found to be highly constrained (S2 = 0.85), including the N-terminal residues 3-6 (S2 = 0.77). Residues 39-44, forming the C-terminal fragment of the binding loop, exhibit increased mobility (S2 = 0.51); however, the N-terminal segment (residues 46-48) retains rigidity as in the intact form (S2 = 0.83). The S2 values, 0.78 and 0.59, respectively, of Arg50 and Arg52 side chain NHs provide evidence not only for the conservation of the Arg hydrogen-bonds with the binding loop segments but also for the difference in strength between them. This is consistent with the earlier observation made from a study of rCMTI-V at two different pHs and its R50 and R52 mutants [Cai, M., Huang, Y., Prakash, O., Wen, L., Dunkelbarger, S. P., Huang, J.-K., Liu, J

Copper-catalyzed transformation of ketones to amides via C(CO)-C(alkyl) bond cleavage directed by picolinamide.

Science.gov (United States)

Ma, Haojie; Zhou, Xiaoqiang; Zhan, Zhenzhen; Wei, Daidong; Shi, Chong; Liu, Xingxing; Huang, Guosheng

2017-09-13

Copper catalyzed chemoselective cleavage of the C(CO)-C(alkyl) bond leading to C-N bond formation with chelation assistance of N-containing directing groups is described. Inexpensive Cu(ii)-acetate serves as a convenient catalyst for this transformation. This method highlights the emerging strategy to transform unactivated alkyl ketones into amides in organic synthesis and provides a new strategy for C-C bond cleavage.

Characterization and photoconductivity study of well-defined C60 terminated poly(tert-butyl acrylate-b-styrene)

International Nuclear Information System (INIS)

Yang Dong; Li Lang; Wang Changchun

2004-01-01

Atom transfer radical polymerization (ATRP) catalyzed by N,N',N',N'',N''-pentamethyldiethylenetriamine (PMDETA)/CuBr was performed to synthesize poly (tert-butyl acrylate) (PtBA-Br) with predesigned molecular weight and narrow polydispersity. Using the PtBA-Br as macroinitiator, the PtBA-block-PS copolymer was prepared through ATRP. After terminated bromine atom was converted to end-functional azide group (P(tBA-b-St)-N 3 ), C 60 end-capped PtBA-PS block copolymers (P(tBA-b-St)-C 60 ) were synthesized by reacting C 60 with P(tBA-b-St)-N 3 . GPC characterizations showed that C 60 was chemically bonded to the end of P(tBA-b-St) chain and the products were mono-substituted. FT-IR, UV-Vis measurements confirmed that hydrolysis of P(tBA-b-St)-C 60 resulted in amphiphilic C 60 end-capped PAA-PS block copolymers. In additional, the photoconductivity of P(tBA-b-St)-C 60 was measured

Density functional theory study on water-gas-shift reaction over molybdenum disulfide

DEFF Research Database (Denmark)

Shi, X. R.; Wang, Shengguang; Hu, J.

2009-01-01

. The pathway for water-gas-shift reaction on both terminations has been carefully studied where the most favorable reaction path precedes the redox mechanism, namely the reaction takes place as follows: CO + H2O --> CO + OH + H --> CO + O + 2H --> CO2 + H-2. The most likely reaction candidates for the formate......Density functional theory calculations have been carried out to investigate the adsorption of reaction intermediates appearing during water-gas-shift reaction at the sulfur covered MoS2 (1 0 0)surfaces, Mo-termination with 37.5% S coverage and S-termination with 50% S coverage using periodic slabs...... species HCOO formation is the surface CO2 reaction with H as a side reaction of CO2 desorption on S-termination with 50% S coverage. The formed HCOO species will react further with adsorbed hydrogen yielding H2COO followed by breaking its C-O bond to form the surface CH2O and O species....

Internal motion time scales of a small, highly stable and disulfide-rich protein: A 15N, 13C NMR and molecular dynamics study

International Nuclear Information System (INIS)

Guenneugues, Marc; Gilquin, Bernard; Wolff, Nicolas; Menez, Andre; Zinn-Justin, Sophie

1999-01-01

Motions of the backbone CαHα and threonine CβHβ bonds of toxin α were investigated using natural abundance 13C NMR and molecular dynamics. Measurement of the 13C longitudinal and transverse relaxation rates employed ACCORDION techniques together with coherence selection by pulsed field gradients and sensitivity enhancement through the use of preservation of equivalent pathway, thus allowing a considerable reduction of the required spectrometer time. 13C R1, R2, 1H → 13C NOE were obtained, as well as the variations of R1ρ(90 deg.) as a function of the rf field strength. These data were compared to those recorded by 1H and 15N NMR on a labelled sample of the toxin [Guenneugues et al. (1997) Biochemistry, 36, 16097-16108]. Both sets of data showed that picosecond to nanosecond time scale motions are well correlated to the secondary structure of the protein. This was further reinforced by the analysis of a 1 ns molecular dynamics simulation in water. Several CαHα and threonine CβHβ experimentally exhibit fast motions with a correlation time longer than 500 ps, that cannot be sampled along the simulation. In addition, the backbone exhibits motions on the microsecond to millisecond time scale on more than half of its length. Thus, toxin α, a highly stable protein (Tm=75 deg. C at acidic pH) containing 61 amino acids and 4 disulfides, shows important internal motions on time scales ranging from 0.1-0.5 ps, to 10-100 ps, 1 ns, and about 30 μs to 10 ms

26 CFR 11.412(c)-11 - Election with respect to bonds.

Science.gov (United States)

2010-04-01

... amount payable at maturity (or, in the case of a bond which is callable prior to maturity, the earliest... 26 Internal Revenue 14 2010-04-01 2010-04-01 false Election with respect to bonds. 11.412(c)-11... OF 1974 § 11.412(c)-11 Election with respect to bonds. (a) In general. Section 412(c)(2)(B) provides...

DETECTING NANOFLARE HEATING EVENTS IN SUBARCSECOND INTER-MOSS LOOPS USING Hi-C

Energy Technology Data Exchange (ETDEWEB)

Winebarger, Amy R.; Moore, Ronald; Cirtain, Jonathan [NASA Marshall Space Flight Center, ZP 13, Huntsville, AL 35812 (United States); Walsh, Robert W. [University of Central Lancashire, Preston, Lancashire PR1 2HE (United Kingdom); De Pontieu, Bart; Title, Alan [Lockheed Martin Solar and Astrophysics Lab, 3251 Hanover St., Org. A0215, Bldg. 252, Palo Alto, CA 94304 (United States); Hansteen, Viggo [Institute of Theoretical Astrophysics, University of Oslo, P.O. Box 1029 Blindern, N-0315 Oslo (Norway); Golub, Leon; Korreck, Kelly; Weber, Mark [Harvard-Smithsonian Center for Astrophysics, 60 Garden St., Cambridge, MA 02138 (United States); Kobayashi, Ken [Center for Space Plasma and Aeronomic Research, University of Alabama in Huntsville, 320 Sparkman Dr, Huntsville, AL 35805 (United States); DeForest, Craig [Southwest Research Institute, 1050 Walnut Street, Suite 300, Boulder, CO 80302 (United States); Kuzin, Sergey, E-mail: amy.r.winebarger@nasa.gov [P.N. Lebedev Physical institute of the Russian Academy of Sciences, Leninskii prospekt 53 119991, Moscow (Russian Federation)

2013-07-01

The High-resolution Coronal Imager (Hi-C) flew aboard a NASA sounding rocket on 2012 July 11 and captured roughly 345 s of high-spatial and temporal resolution images of the solar corona in a narrowband 193 A channel. In this paper, we analyze a set of rapidly evolving loops that appear in an inter-moss region. We select six loops that both appear in and fade out of the Hi-C images during the short flight. From the Hi-C data, we determine the size and lifetimes of the loops and characterize whether these loops appear simultaneously along their length or first appear at one footpoint before appearing at the other. Using co-aligned, co-temporal data from multiple channels of the Atmospheric Imaging Assembly on the Solar Dynamics Observatory, we determine the temperature and density of the loops. We find the loops consist of cool ({approx}10{sup 5} K), dense ({approx}10{sup 10} cm{sup -3}) plasma. Their required thermal energy and their observed evolution suggest they result from impulsive heating similar in magnitude to nanoflares. Comparisons with advanced numerical simulations indicate that such dense, cold and short-lived loops are a natural consequence of impulsive magnetic energy release by reconnection of braided magnetic field at low heights in the solar atmosphere.

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

Science.gov (United States)

Shokri, Ehsan; Hosseini, Morteza; Davari, Mehdi D.; Ganjali, Mohammad R.; Peppelenbosch, Maikel P.; Rezaee, Farhad

2017-04-01

A modified non-cross-linking gold-nanoparticles (Au-NPs) aggregation strategy has been developed for the label free colorimetric detection of DNAs/RNAs based on self-assembling target species in the presence of thiolated probes. Two complementary thiol- modified probes, each of which specifically binds at one half of the target introduced SH groups at both ends of dsDNA. Continuous disulfide bond formation at 3‧ and 5‧ terminals of targets leads to the self-assembly of dsDNAs into the sulfur- rich and flexible products with different lengths. These products have a high affinity for the surface of Au-NPs and efficiently protect the surface from salt induced aggregation. To evaluate the assay efficacy, a small part of the citrus tristeza virus (CTV) genome was targeted, leading to a detection limit of about 5 × 10-9 mol.L-1 over a linear ranged from 20 × 10-9 to 10 × 10-7 mol.L-1. This approach also exhibits good reproducibility and recovery levels in the presence of plant total RNA or human plasma total circulating RNA extracts. Self-assembled targets can be then sensitively distinguished from non-assembled or mismatched targets after gel electrophoresis. The disulfide reaction method and integrating self-assembled DNAs/RNAs targets with bare AuNPs as a sensitive indicator provide us a powerful and simple visual detection tool for a wide range of applications.

N-Terminal Domains in Two-Domain Proteins Are Biased to Be Shorter and Predicted to Fold Faster Than Their C-Terminal Counterparts

Directory of Open Access Journals (Sweden)

Etai Jacob

2013-04-01

Full Text Available Computational analysis of proteomes in all kingdoms of life reveals a strong tendency for N-terminal domains in two-domain proteins to have shorter sequences than their neighboring C-terminal domains. Given that folding rates are affected by chain length, we asked whether the tendency for N-terminal domains to be shorter than their neighboring C-terminal domains reflects selection for faster-folding N-terminal domains. Calculations of absolute contact order, another predictor of folding rate, provide additional evidence that N-terminal domains tend to fold faster than their neighboring C-terminal domains. A possible explanation for this bias, which is more pronounced in prokaryotes than in eukaryotes, is that faster folding of N-terminal domains reduces the risk for protein aggregation during folding by preventing formation of nonnative interdomain interactions. This explanation is supported by our finding that two-domain proteins with a shorter N-terminal domain are much more abundant than those with a shorter C-terminal domain.

Thiol-Disulfide Exchange between Glutaredoxin and Glutathione

DEFF Research Database (Denmark)

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

2010-01-01

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

Multiple ways to make disulfides

DEFF Research Database (Denmark)

Bulleid, Neil J; Ellgaard, Lars

2011-01-01

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

Crystal structure of type I ryanodine receptor amino-terminal [beta]-trefoil domain reveals a disease-associated mutation 'hot spot' loop

Energy Technology Data Exchange (ETDEWEB)

Amador, Fernando J.; Liu, Shuang; Ishiyama, Noboru; Plevin, Michael J.; Wilson, Aaron; MacLennan, David H.; Ikura, Mitsuhiko; (Toronto)

2009-12-01

Muscle contraction and relaxation is regulated by transient elevations of myoplasmic Ca{sup 2+}. Ca{sup 2+} is released from stores in the lumen of the sarco(endo)plasmic reticulum (SER) to initiate formation of the Ca{sup 2+} transient by activation of a class of Ca{sup 2+} release channels referred to as ryanodine receptors (RyRs) and is pumped back into the SER lumen by Ca{sup 2+}-ATPases (SERCAs) to terminate the Ca{sup 2+} transient. Mutations in the type 1 ryanodine receptor gene, RYR1, are associated with 2 skeletal muscle disorders, malignant hyperthermia (MH), and central core disease (CCD). The evaluation of proposed mechanisms by which RyR1 mutations cause MH and CCD is hindered by the lack of high-resolution structural information. Here, we report the crystal structure of the N-terminal 210 residues of RyR1 (RyR{sub NTD}) at 2.5 {angstrom}. The RyR{sub NTD} structure is similar to that of the suppressor domain of type 1 inositol 1,4,5-trisphosphate receptor (IP3Rsup), but lacks most of the long helix-turn-helix segment of the 'arm' domain in IP3Rsup. The N-terminal {beta}-trefoil fold, found in both RyR and IP{sub 3}R, is likely to play a critical role in regulatory mechanisms in this channel family. A disease-associated mutation 'hot spot' loop was identified between strands 8 and 9 in a highly basic region of RyR1. Biophysical studies showed that 3 MH-associated mutations (C36R, R164C, and R178C) do not adversely affect the global stability or fold of RyRNTD, supporting previously described mechanisms whereby mutations perturb protein-protein interactions.

Ligand-Controlled Chemoselective C(acyl)–O Bond vs C(aryl)–C Bond Activation of Aromatic Esters in Nickel Catalyzed C(sp2)–C(sp3) Cross-Couplings

KAUST Repository

Chatupheeraphat, Adisak; Liao, Hsuan-Hung; Srimontree, Watchara; Guo, Lin; Minenkov, Yury; Poater, Albert; Cavallo, Luigi; Rueping, Magnus

2018-01-01

step helped rationalizing this intriguing reaction chemoselectivity: whereas nickel complexes with bidentate ligands favor the C(aryl)-C bond cleavage in the oxidative addition step leading to the alkylated product via a decarbonylative process, nickel

Gold-Catalyzed Formal C-C Bond Insertion Reaction of 2-Aryl-2-diazoesters with 1,3-Diketones.

Science.gov (United States)

Ren, Yuan-Yuan; Chen, Mo; Li, Ke; Zhu, Shou-Fei

2018-06-29

The transition-metal-catalyzed formal C-C bond insertion reaction of diazo compounds with monocarbonyl compounds is well established, but the related reaction of 1,3-diketones instead gives C-H bond insertion products. Herein, we report a protocol for a gold-catalyzed formal C-C bond insertion reaction of 2-aryl-2-diazoesters with 1,3-diketones, which provides efficient access to polycarbonyl compounds with an all-carbon quaternary center. The aryl ester moiety plays a crucial role in the unusual chemoselectivity, and the addition of a Brønsted acid to the reaction mixture improves the yield of the C-C bond insertion product. A reaction mechanism involving cyclopropanation of a gold carbenoid with an enolate and ring-opening of the resulting donor-acceptor-type cyclopropane intermediate is proposed. This mechanism differs from that of the traditional Lewis-acid-catalyzed C-C bond insertion reaction of diazo compounds with monocarbonyl compounds, which involves a rearrangement of a zwitterion intermediate as a key step. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Alteration of the mode of antibacterial action of a defensin by the amino-terminal loop substitution

Energy Technology Data Exchange (ETDEWEB)

Gao, Bin [Group of Animal Innate Immunity, State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District, 100101 Beijing (China); Zhu, Shunyi, E-mail: Zhusy@ioz.ac.cn [Group of Animal Innate Immunity, State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District, 100101 Beijing (China)

2012-10-05

Highlights: Black-Right-Pointing-Pointer Al-M is an engineered fungal defensin with the n-loop of an insect defensin. Black-Right-Pointing-Pointer Al-M adopts a native defensin-like structure with high antibacterial potency. Black-Right-Pointing-Pointer Al-M kills bacteria through a membrane disruptive mechanism. Black-Right-Pointing-Pointer This work sheds light on the functional evolution of CS{alpha}{beta}-type defensins. -- Abstract: Ancient invertebrate-type and classical insect-type defensins (AITDs and CITDs) are two groups of evolutionarily related antimicrobial peptides (AMPs) that adopt a conserved cysteine-stabilized {alpha}-helical and {beta}-sheet (CS{alpha}{beta}) fold with a different amino-terminal loop (n-loop) size and diverse modes of antibacterial action. Although they both are identified as inhibitors of cell wall biosynthesis, only CITDs evolved membrane disruptive ability by peptide oligomerization to form pores. To understand how this occurred, we modified micasin, a fungus-derived AITDs with a non-membrane disruptive mechanism, by substituting its n-loop with that of an insect-derived CITDs. After air oxidization, the synthetic hybrid defensin (termed Al-M) was structurally identified by circular dichroism (CD) and functionally evaluated by antibacterial and membrane permeability assays and electronic microscopic observation. Results showed that Al-M folded into a native-like defensin structure, as determined by its CD spectrum that is similar to that of micasin. Al-M was highly efficacious against the Gram-positive bacterium Bacillus megaterium with a lethal concentration of 1.76 {mu}M. As expected, in contrast to micasin, Al-M killed the bacteria through a membrane disruptive mechanism of action. The alteration in modes of action supports a key role of the n-loop extension in assembling functional surface of CITDs for membrane disruption. Our work provides mechanical evidence for evolutionary relationship between AITDs and CITDs.

Alteration of the mode of antibacterial action of a defensin by the amino-terminal loop substitution

International Nuclear Information System (INIS)

Gao, Bin; Zhu, Shunyi

2012-01-01

Highlights: ► Al-M is an engineered fungal defensin with the n-loop of an insect defensin. ► Al-M adopts a native defensin-like structure with high antibacterial potency. ► Al-M kills bacteria through a membrane disruptive mechanism. ► This work sheds light on the functional evolution of CSαβ-type defensins. -- Abstract: Ancient invertebrate-type and classical insect-type defensins (AITDs and CITDs) are two groups of evolutionarily related antimicrobial peptides (AMPs) that adopt a conserved cysteine-stabilized α-helical and β-sheet (CSαβ) fold with a different amino-terminal loop (n-loop) size and diverse modes of antibacterial action. Although they both are identified as inhibitors of cell wall biosynthesis, only CITDs evolved membrane disruptive ability by peptide oligomerization to form pores. To understand how this occurred, we modified micasin, a fungus-derived AITDs with a non-membrane disruptive mechanism, by substituting its n-loop with that of an insect-derived CITDs. After air oxidization, the synthetic hybrid defensin (termed Al-M) was structurally identified by circular dichroism (CD) and functionally evaluated by antibacterial and membrane permeability assays and electronic microscopic observation. Results showed that Al-M folded into a native-like defensin structure, as determined by its CD spectrum that is similar to that of micasin. Al-M was highly efficacious against the Gram-positive bacterium Bacillus megaterium with a lethal concentration of 1.76 μM. As expected, in contrast to micasin, Al-M killed the bacteria through a membrane disruptive mechanism of action. The alteration in modes of action supports a key role of the n-loop extension in assembling functional surface of CITDs for membrane disruption. Our work provides mechanical evidence for evolutionary relationship between AITDs and CITDs.

Reduction of bonding resistance of two-terminal III-V/Si tandem solar cells fabricated using smart-stack technology

Science.gov (United States)

Baba, Masaaki; Makita, Kikuo; Mizuno, Hidenori; Takato, Hidetaka; Sugaya, Takeyoshi; Yamada, Noboru

2017-12-01

This paper describes a method that remarkably reduces the bonding resistance of mechanically stacked two-terminal GaAs/Si and InGaP/Si tandem solar cells, where the top and bottom cells are bonded using a Pd nanoparticle array. A transparent conductive oxide (TCO) layer, which partially covers the surface of the Si bottom cell below the electrodes of the III-V top cell, significantly enhances the fill factor (FF) and cell conversion efficiency. The partial TCO layer reduces the bonding resistance and thus, increases the FF and efficiency of InGaP/Si by factors of 1.20 and 1.11, respectively. Eventually, the efficiency exceeds 15%. Minimizing the optical losses at the bonding interfaces of the TCO layer is important in the fabrication of high-efficiency solar cells. To help facilitate this, the optical losses in the tandem solar cells are thoroughly characterized through optical simulations and experimental verifications.

Conformational and receptor-binding properties of the insect neuropeptide proctolin and its analogues

Science.gov (United States)

Odell, Barbara; Hammond, Stephen J.; Osborne, Richard; Goosey, Michael W.

1996-04-01

Proctolin (Arg-Tyr-Leu-Pro-Thr) was the first insect neuropeptide to be chemically characterised. It plays an essential role in insect neurophysiology and is involved in muscular contraction and neuromodulation. Elements of secondary structure in solution have been studied by comparing data obtained from NMR and molecular dynamics simulations. Different secondary structural requirements are associated with agonist and antagonist activities. A favoured conformation of proctolin has an inverse γ-turn, comprising an intramolecular hydrogen bond near the C-terminal end between Thr NH and Leu CO. Antagonists have a more compact structure resembling a `paperclip' loop, containing an intramolecular hydrogen bond between Tyr NH and Pro CO, possibly stabilised by a salt bridge between the N- and C-terminal groups. A cyclic analogue retains antagonist activity and resembles a β-bulge loop, also comprising intramolecular hydrogen bonds between Tyr NH and Pro CO and Thr CO. These models may offer feasible starting points for designing novel compounds with proctolinergic activity.

MTH1745, a protein disulfide isomerase-like protein from thermophilic archaea, Methanothermobacter thermoautotrophicum involving in stress response.

Science.gov (United States)

Ding, Xia; Lv, Zhen-Mei; Zhao, Yang; Min, Hang; Yang, Wei-Jun

2008-01-01

MTH1745 is a putative protein disulfide isomerase characterized with 151 amino acid residues and a CPAC active-site from the anaerobic archaea Methanothermobacter thermoautotrophicum. The potential functions of MTH1745 are not clear. In the present study, we show a crucial role of MTH1745 in protecting cells against stress which may be related to its functions as a disulfide isomerase and its chaperone properties. Using real-time polymerase chain reaction analyses, the level of MTH1745 messenger RNA (mRNA) in the thermophilic archaea M. thermoautotrophicum was found to be stress-induced in that it was significantly higher under low (50 degrees C) and high (70 degrees C) growth temperatures than under the optimal growth temperature for the organism (65 degrees C). Additionally, the expression of MTH1745 mRNA was up-regulated by cold shock (4 degrees C). Furthermore, the survival of MTH1745 expressing Escherichia coli cells was markedly higher than that of control cells in response to heat shock (51.0 degrees C). These results indicated that MTH1745 plays an important role in the resistance of stress. By assay of enzyme activities in vitro, MTH1745 also exhibited a chaperone function by promoting the functional folding of citrate synthase after thermodenaturation. On the other hand, MTH1745 was also shown to function as a disulfide isomerase on the refolding of denatured and reduced ribonuclease A. On the basis of its single thioredoxin domain, function as a disulfide isomerase, and its chaperone activity, we suggest that MTH1745 may be an ancient protein disulfide isomerase. These studies may provide clues to the understanding of the function of protein disulfide isomerase in archaea.

Friedel-Crafts reaction of benzyl fluorides: selective activation of C-F bonds as enabled by hydrogen bonding.

Science.gov (United States)

Champagne, Pier Alexandre; Benhassine, Yasmine; Desroches, Justine; Paquin, Jean-François

2014-12-08

A Friedel-Crafts benzylation of arenes with benzyl fluorides has been developed. The reaction produces 1,1-diaryl alkanes in good yield under mild conditions without the need for a transition metal or a strong Lewis acid. A mechanism involving activation of the C-F bond through hydrogen bonding is proposed. This mode of activation enables the selective reaction of benzylic C-F bonds in the presence of other benzylic leaving groups. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Energy and Rate Determinations to Activate the C-C σ-BOND of Acetone by Gaseous NI^+

Science.gov (United States)

Castleberry, Vanessa A.; Dee, S. Jason; Villarroel, Otsmar J.; Laboren, Ivanna E.; Frey, Sarah E.; Bellert, Darrin J.

2009-06-01

A unique application of a custom fabricated photodissociation spectrometer permits the determination of thermodynamic properties (activation energies), reaction rates, and mechanistic details of bare metal cation mediated C-C σ-bond activation in the gas phase. Specifically, the products and rates resulting from the unimolecular decomposition of the Ni^+Acetone (Ni^+Ac) adduct are monitored after absorption of a known amount of energy. The three dissociative products which are observed in high yield are Ni^+, Ni^+CO, and CH3CO^+. The latter two fragment ions result from the activation of a C-C σ-bond. It was found that minimally 14 000 cm^{-1} of energy must be deposited into the adduct ion to induce C-C bond breakage. Preliminary results for the Ni^+ activation of the C-C σ-bond of acetone indicate that there are (at least) two low energy reaction coordinates leading to C-C bond breakage. The lower energy pathway emerges from the doublet ground state with an upper limit to the activation energy of 14 000 cm^{-1} and reaction rate ≈0.14 molecules/μs. The higher energy path is assumed to be along the quartet reaction coordinate with a minimum activation energy of 18 800 cm^{-1} (relative to the ground state) and a slightly slower reaction rate.

Renormalization of loop functions for all loops

International Nuclear Information System (INIS)

Brandt, R.A.; Neri, F.; Sato, M.

1981-01-01

It is shown that the vacuum expectation values W(C 1 ,xxx, C/sub n/) of products of the traces of the path-ordered phase factors P exp[igcontour-integral/sub C/iA/sub μ/(x)dx/sup μ/] are multiplicatively renormalizable in all orders of perturbation theory. Here A/sub μ/(x) are the vector gauge field matrices in the non-Abelian gauge theory with gauge group U(N) or SU(N), and C/sub i/ are loops (closed paths). When the loops are smooth (i.e., differentiable) and simple (i.e., non-self-intersecting), it has been shown that the generally divergent loop functions W become finite functions W when expressed in terms of the renormalized coupling constant and multiplied by the factors e/sup -K/L(C/sub i/), where K is linearly divergent and L(C/sub i/) is the length of C/sub i/. It is proved here that the loop functions remain multiplicatively renormalizable even if the curves have any finite number of cusps (points of nondifferentiability) or cross points (points of self-intersection). If C/sub γ/ is a loop which is smooth and simple except for a single cusp of angle γ, then W/sub R/(C/sub γ/) = Z(γ)W(C/sub γ/) is finite for a suitable renormalization factor Z(γ) which depends on γ but on no other characteristic of C/sub γ/. This statement is made precise by introducing a regularization, or via a loop-integrand subtraction scheme specified by a normalization condition W/sub R/(C-bar/sub γ/) = 1 for an arbitrary but fixed loop C-bar/sub γ/. Next, if C/sub β/ is a loop which is smooth and simple except for a cross point of angles β, then W(C/sub β/) must be renormalized together with the loop functions of associated sets S/sup i//sub β/ = ]C/sup i/ 1 ,xxx, C/sup i//sub p/i] (i = 2,xxx,I) of loops C/sup i//sub q/ which coincide with certain parts of C/sub β/equivalentC 1 1 . Then W/sub R/(S/sup i//sub β/) = Z/sup i/j(β)W(S/sup j//sub β/) is finite for a suitable matrix Z/sup i/j

Insights into PG-binding, conformational change, and dimerization of the OmpA C-terminal domains from Salmonella enterica serovar Typhimurium and Borrelia burgdorferi: Characterization of OmpA C-Terminal Domain

Energy Technology Data Exchange (ETDEWEB)

Tan, Kemin [Center for Structural Genomics of Infectious Diseases, University of Chicago, 5735 South Ellis Avenue Chicago Illinois 60637; Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Structural Biology Center, Biosciences, Argonne National Laboratory, Argonne Illinois 60439; Deatherage Kaiser, Brooke L. [National Security Directorate, Pacific Northwest National Laboratory, Richland Washington 99352; Wu, Ruiying [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Cuff, Marianne [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Fan, Yao [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Bigelow, Lance [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Jedrzejczak, Robert P. [Center for Structural Genomics of Infectious Diseases, University of Chicago, 5735 South Ellis Avenue Chicago Illinois 60637; Adkins, Joshua N. [Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland Washington 99352; Cort, John R. [Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland Washington 99352; Babnigg, Gyorgy [Center for Structural Genomics of Infectious Diseases, University of Chicago, 5735 South Ellis Avenue Chicago Illinois 60637; Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Joachimiak, Andrzej [Center for Structural Genomics of Infectious Diseases, University of Chicago, 5735 South Ellis Avenue Chicago Illinois 60637; Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Structural Biology Center, Biosciences, Argonne National Laboratory, Argonne Illinois 60439

2017-06-19

S. Typhimurium can induce both humoral and cell-mediated responses when establishing itself in the host. These responses are primarily stimulated against the lipopolysaccharide and major outer membrane (OM) proteins. OmpA is one of these major OM proteins. It comprises a N-terminal eight-stranded b-barrel trans membrane domain and a C-terminal domain (OmpACTD). The OmpACTD and its homologs are believed to bind to peptidoglycan (PG) within the periplasm, maintaining bacterial osmotic homeostasis and modulating the permeability and integrity of the OM. Here we present the first crystal structures of the OmpACTD from two pathogens: S. Typhimurium (STOmpACTD) in open and closed forms and causative agent of Lyme Disease Borrelia burgdorferi (BbOmpACTD), in closed form. In the open form of STOmpACTD, an aspartic acid residue from a long b2-a3 loop points into the binding pocket, suggesting that an anion group such as a carboxylate group from PG is favored at the binding site. In the closed form of STOmpACTD and in the structure of BbOmpACTD, a sulfate group from the crystallization buffer is tightly bound at the binding site. The differences between the closed and open forms of STOmpACTD, suggest a large conformational change that includes an extension of a3 helix by ordering a part of b2-a3 loop. We propose that the sulfate anion observed in these structures mimics the carboxylate group of PG when bound to STOmpACTD suggesting PG-anchoring mechanism. In addition, the binding of PG or a ligand mimic may enhance dimerization of STOmpACTD, or possibly that of full length STOmpA.

Structure of a eukaryotic voltage-gated sodium channel at near-atomic resolution.

Science.gov (United States)

Shen, Huaizong; Zhou, Qiang; Pan, Xiaojing; Li, Zhangqiang; Wu, Jianping; Yan, Nieng

2017-03-03

Voltage-gated sodium (Na v ) channels are responsible for the initiation and propagation of action potentials. They are associated with a variety of channelopathies and are targeted by multiple pharmaceutical drugs and natural toxins. Here, we report the cryogenic electron microscopy structure of a putative Na v channel from American cockroach (designated Na v PaS) at 3.8 angstrom resolution. The voltage-sensing domains (VSDs) of the four repeats exhibit distinct conformations. The entrance to the asymmetric selectivity filter vestibule is guarded by heavily glycosylated and disulfide bond-stabilized extracellular loops. On the cytoplasmic side, a conserved amino-terminal domain is placed below VSD I , and a carboxy-terminal domain binds to the III-IV linker. The structure of Na v PaS establishes an important foundation for understanding function and disease mechanism of Na v and related voltage-gated calcium channels. Copyright © 2017, American Association for the Advancement of Science.

Syntheses, structures and redox properties of some complexes containing the Os(dppe)Cp* fragment, including [{Os(dppe)Cp*}2(mu-C triple bondCC triple bond C)].

Science.gov (United States)

Bruce, Michael I; Costuas, Karine; Davin, Thomas; Halet, Jean-François; Kramarczuk, Kathy A; Low, Paul J; Nicholson, Brian K; Perkins, Gary J; Roberts, Rachel L; Skelton, Brian W; Smith, Mark E; White, Allan H

2007-12-14

The sequential conversion of [OsBr(cod)Cp*] (9) to [OsBr(dppe)Cp*] (10), [Os([=C=CH2)(dppe)Cp*]PF6 ([11]PF6), [Os(C triple bond CH)(dppe)Cp*] (12), [{Os(dppe)Cp*}2{mu-(=C=CH-CH=C=)}][PF6]2 ([13](PF6)2) and finally [{Os(dppe)Cp*}(2)(mu-C triple bond CC triple bond C)] (14) has been used to make the third member of the triad [{M(dppe)Cp*}2(mu-C triple bond CC triple bond C)] (M = Fe, Ru, Os). The molecular structures of []PF6, 12 and 14, together with those of the related osmium complexes [Os(NCMe)(dppe)Cp*]PF6 ([15]PF6) and [Os(C triple bond CPh)(dppe)Cp*] (16), have been determined by single-crystal X-ray diffraction studies. Comparison of the redox properties of 14 with those of its iron and ruthenium congeners shows that the first oxidation potential E1 varies as: Fe approximately Os < Ru. Whereas the Fe complex has been shown to undergo three sequential 1-electron oxidation processes within conventional electrochemical solvent windows, the Ru and Os compounds undergo no fewer than four sequential oxidation events giving rise to a five-membered series of redox related complexes [{M(dppe)Cp*}2(mu-C4)]n+ (n = 0, 1, 2, 3 and 4), the osmium derivatives being obtained at considerably lower potentials than the ruthenium analogues. These results are complimented by DFT and DT DFT calculations.

Hypovalency--a kinetic-energy density description of a 4c-2e bond.

Science.gov (United States)

Jacobsen, Heiko

2009-06-07

A bond descriptor based on the kinetic energy density, the localized-orbital locator (LOL), is used to characterize the nature of the chemical bond in electron deficient multi-center bonds. The boranes B(2)H(6), B(4)H(4), B(4)H(10), [B(6)H(6)](2-), and [B(6)H(7)](-) serve as prototypical examples of hypovalent 3c-2e and 4c-2e bonding. The kinetic energy density is derived from a set of Kohn-Sham orbitals obtained from pure density functional calculations (PBE/TZVP), and the topology of LOL is analyzed in terms of (3,-3) attractors (Gamma). The B-B-B and B-H-B 3c-2e, and the B-B-H-B 4c-2e bonding situations are defined by their own characteristic LOL profiles. The presence of one attractor in relation to the three or four atoms that are engaged in electron deficient bonding provides sufficient indication of the type of 3c-2e or 4c-2e bond present. For the 4c-2e bond in [B(6)H(7)](-) the LOL analysis is compared to results from an experimental QTAIM study.

Reliable set-up for in-loop 11C-carboxylations using Grignard reactions for the preparation of [carbonyl-11C]WAY-100635 and [11C]-(+)-PHNO

International Nuclear Information System (INIS)

Rami-Mark, Christina; Ungersboeck, Johanna; Haeusler, Daniela; Nics, Lukas; Philippe, Cecile; Mitterhauser, Markus; Willeit, Matthaeus; Lanzenberger, Rupert; Karanikas, Georgios; Wadsak, Wolfgang

2013-01-01

Aim of this work was the implementation of a generalized in-loop synthesis for 11 C-carboxylations and subsequent 11 C-acylations on the TRACERlab FxC Pro platform. The set-up was tested using [carbonyl- 11 C]WAY-100635 and, for the first time, [ 11 C]-(+)-PHNO. Its general applicability could be demonstrated and both [carbonyl- 11 C]WAY-100635 and [ 11 C]-(+)-PHNO were prepared with high reliability and satisfying outcome. - Highlights: • Generalized method for in-loop 11 C-carboxylations implemented. • Grignard reactions successfully tested. • First in-loop procedure for [ 11 C]-(+)PHNO established. • Satisfactory synthesis outcome for both [carbonyl- 11 C]WAY-100635 and [ 11 C]-(+)PHNO. • No distillation for purification of intermediate required

Biochemical and ultrastructural studies of the C-type lectin bovine conglutinin

DEFF Research Database (Denmark)

Andersen, Ove; Nielsen, E H; Storgaard, P

1992-01-01

at 43 kDa and weaker bands at 86 and 180 kDa. In contrast, reduction with 6-50 mM L-cysteine resulted in 37-kDa subunits indicating the presence of intrachain disulfide bonds within this subunit. Hydroxylamine treatment indicated presence of ester bonds in the 86- and 180-kDa subunits. Collagenase...... digestion and SDS-PAGE under reducing and nonreducing conditions resulted in bands of 20 and 15 kDa, respectively, indicating the presence of intrachain, rather than interchain, disulfide bonds in the carboxy terminus. Deglycosylation and glycan differentiation analysis of BK revealed the presence of O...

DFT Mechanistic Study of the Selective Terminal C-H Activation of n-Pentane with a Tungsten Allyl Nitrosyl Complex

KAUST Repository

Lee, Richmond

2017-01-17

Mechanistic insights into the selective C-H terminal activation of n-pentane with tungsten allyl nitrosyl complex reported by Legzdins were gained by employing density functional theory with B3LYP hybrid functional. Using Bader’s atom in molecules (AIM) analysis on the elementary steps of the hydrogen transfer process, TS1 and TS2, it was observed that the calculated H-transfer models were closely similar to Hall’s metal-assisted σ-bond metathesis through bond critical point (BCP) comparisons. One distinguishable feature was the fact that the formal oxidation state of the W changed in the concerted H-transfer process. To better differentiate, we term these processes as ‘Formal Reductive Hydrogen Transfer’ (FRHT) for TS1 and ‘Formal Oxidative Hydrogen Transfer’ (FOHT) for TS2.

DFT Mechanistic Study of the Selective Terminal C-H Activation of n-Pentane with a Tungsten Allyl Nitrosyl Complex

KAUST Repository

Lee, Richmond; Tan, Davin; Liu, Chaoli; Li, Huaifeng; Guo, Hao; Shyue, Jing-Jong; Huang, Kuo-Wei

2017-01-01

Mechanistic insights into the selective C-H terminal activation of n-pentane with tungsten allyl nitrosyl complex reported by Legzdins were gained by employing density functional theory with B3LYP hybrid functional. Using Bader’s atom in molecules (AIM) analysis on the elementary steps of the hydrogen transfer process, TS1 and TS2, it was observed that the calculated H-transfer models were closely similar to Hall’s metal-assisted σ-bond metathesis through bond critical point (BCP) comparisons. One distinguishable feature was the fact that the formal oxidation state of the W changed in the concerted H-transfer process. To better differentiate, we term these processes as ‘Formal Reductive Hydrogen Transfer’ (FRHT) for TS1 and ‘Formal Oxidative Hydrogen Transfer’ (FOHT) for TS2.

“Invisible” Conformers of an Antifungal Disulfide Protein Revealed by Constrained Cold and Heat Unfolding, CEST-NMR Experiments, and Molecular Dynamics Calculations

Science.gov (United States)

Fizil, Ádám; Gáspári, Zoltán; Barna, Terézia; Marx, Florentine; Batta, Gyula

2015-01-01

Transition between conformational states in proteins is being recognized as a possible key factor of function. In support of this, hidden dynamic NMR structures were detected in several cases up to populations of a few percent. Here, we show by two- and three-state analysis of thermal unfolding, that the population of hidden states may weight 20–40 % at 298 K in a disulfide-rich protein. In addition, sensitive 15N-CEST NMR experiments identified a low populated (0.15 %) state that was in slow exchange with the folded PAF protein. Remarkably, other techniques failed to identify the rest of the NMR “dark matter”. Comparison of the temperature dependence of chemical shifts from experiments and molecular dynamics calculations suggests that hidden conformers of PAF differ in the loop and terminal regions and are most similar in the evolutionary conserved core. Our observations point to the existence of a complex conformational landscape with multiple conformational states in dynamic equilibrium, with diverse exchange rates presumably responsible for the completely hidden nature of a considerable fraction. PMID:25676351

Characterization of Runella slithyformis HD-Pnk, a Bifunctional DNA/RNA End-Healing Enzyme Composed of an N-Terminal 2′,3′-Phosphoesterase HD Domain and a C-Terminal 5′-OH Polynucleotide Kinase Domain

Science.gov (United States)

Munir, Annum

2016-01-01

ABSTRACT 5′- and 3′-end-healing reactions are key steps in nucleic acid break repair in which 5′-OH ends are phosphorylated by a polynucleotide kinase (Pnk) and 3′-PO4 or 2′,3′-cyclic-PO4 ends are hydrolyzed by a phosphoesterase to generate the 5′-PO4 and 3′-OH termini required for sealing by classic polynucleotide ligases. End-healing and sealing enzymes are present in diverse bacterial taxa, often organized as modular units within a single multifunctional polypeptide or as subunits of a repair complex. Here we identify and characterize Runella slithyformis HD-Pnk as a novel bifunctional end-healing enzyme composed of an N-terminal 2′,3′-phosphoesterase HD domain and a C-terminal 5′-OH polynucleotide kinase P-loop domain. HD-Pnk phosphorylates 5′-OH polynucleotides (9-mers or longer) in the presence of magnesium and any nucleoside triphosphate donor. HD-Pnk dephosphorylates RNA 2′,3′-cyclic phosphate, RNA 3′-phosphate, RNA 2′-phosphate, and DNA 3′-phosphate ends in the presence of a transition metal cofactor, which can be nickel, copper, or cobalt. HD-Pnk homologs are present in genera from 11 bacterial phyla and are often encoded in an operon with a putative ATP-dependent polynucleotide ligase. IMPORTANCE The present study provides insights regarding the diversity of nucleic acid repair strategies via the characterization of Runella slithyformis HD-Pnk as the exemplar of a novel clade of dual 5′- and 3′-end-healing enzymes that phosphorylate 5′-OH termini and dephosphorylate 2′,3′-cyclic-PO4, 3′-PO4, and 2′-PO4 ends. The distinctive feature of HD-Pnk is its domain composition, i.e., a fusion of an N-terminal HD phosphohydrolase module and a C-terminal P-loop polynucleotide kinase module. Homologs of Runella HD-Pnk with the same domain composition, same domain order, and similar polypeptide sizes are distributed widely among genera from 11 bacterial phyla. PMID:27895092

Structure of unliganded HSV gD reveals a mechanism for receptor-mediated activation of virus entry

Energy Technology Data Exchange (ETDEWEB)

Krummenacher, Claude; Supekar, Vinit M.; Whitbeck, J. Charles; Lazear, Eric; Connolly, Sarah A.; Eisenberg, Roselyn J.; Cohen, Gary H.; Wiley, Don C.; Carfi, Andrea (UPENN); (IRBM); (CHLMM)

2010-07-19

Herpes simplex virus (HSV) entry into cells requires binding of the envelope glycoprotein D (gD) to one of several cell surface receptors. The 50 C-terminal residues of the gD ectodomain are essential for virus entry, but not for receptor binding. We have determined the structure of an unliganded gD molecule that includes these C-terminal residues. The structure reveals that the C-terminus is anchored near the N-terminal region and masks receptor-binding sites. Locking the C-terminus in the position observed in the crystals by an intramolecular disulfide bond abolished receptor binding and virus entry, demonstrating that this region of gD moves upon receptor binding. Similarly, a point mutant that would destabilize the C-terminus structure was nonfunctional for entry, despite increased affinity for receptors. We propose that a controlled displacement of the gD C-terminus upon receptor binding is an essential feature of HSV entry, ensuring the timely activation of membrane fusion.

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

Science.gov (United States)

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

2018-09-01

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

Bonding and structure of copper nitrenes.

Science.gov (United States)

Cundari, Thomas R; Dinescu, Adriana; Kazi, Abul B

2008-11-03

Copper nitrenes are of interest as intermediates in the catalytic aziridination of olefins and the amination of C-H bonds. However, despite advances in the isolation and study of late-transition-metal multiply bonded complexes, a bona fide structurally characterized example of a terminal copper nitrene has, to our knowledge, not been reported. In anticipation of such a report, terminal copper nitrenes are studied from a computational perspective. The nitrene complexes studied here are of the form (beta-diketiminate)Cu(NPh). Density functional theory (DFT), complete active space self-consistent-field (CASSCF) electronic structure techniques, and hybrid quantum mechanical/molecular mechanical (QM/MM) methods are employed to study such species. While DFT methods indicate that a triplet (S = 1) is the ground state, CASSCF calculations indicate that a singlet (S = 0) is the ground state, with only a small energy gap between the singlet and triplet. Moreover, the ground-state (open-shell) singlet copper nitrene is found to be highly multiconfigurational (i.e., biradical) and to possess a bent geometry about the nitrene nitrogen, contrasting with the linear nitrene geometry of the triplet copper nitrenes. CASSCF calculations also reveal the existence of a closed-shell singlet state with some degree of multiple bonding character for the copper-nitrene bond.

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