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Sample records for non-catalytic exosite binding

  1. A Single-Domain Llama Antibody Potently Inhibits the Enzymatic Activity of Botulinum Neurotoxin by Binding to the Non-Catalytic [alpha]-Exosite Binding Region

    SciTech Connect

    Dong, Jianbo; Thompson, Aaron A.; Fan, Yongfeng; Lou, Jianlong; Conrad, Fraser; Ho, Mengfei; Pires-Alves, Melissa; Wilson, Brenda A.; Stevens, Raymond C.; Marks, James D.

    2010-08-13

    Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K{sub d}) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K{sub d} for BoNT/A Lc of 1.47 x 10{sup -10} M and an IC{sub 50} (50% inhibitory concentration) of 4.7 x 10{sup -10} M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 {angstrom} resolution. The structure reveals that the Aa1 VHH binds in the {alpha}-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc {alpha}-exosite as a target for inhibitor development.

  2. Structural insight into exosite binding and discovery of novel exosite inhibitors of botulinum neurotoxin serotype A through in silico screening.

    PubMed

    Hu, Xin; Legler, Patricia M; Southall, Noel; Maloney, David J; Simeonov, Anton; Jadhav, Ajit

    2014-07-01

    Botulinum neurotoxin serotype A (BoNT/A) is the most lethal toxin among the Tier 1 Select Agents. Development of potent and selective small molecule inhibitors against BoNT/A zinc metalloprotease remains a challenging problem due to its exceptionally large substrate binding surface and conformational plasticity. The exosites of the catalytic domain of BoNT/A are intriguing alternative sites for small molecule intervention, but their suitability for inhibitor design remains largely unexplored. In this study, we employed two recently identified exosite inhibitors, D-chicoric acid and lomofungin, to probe the structural features of the exosites and molecular mechanisms of synergistic inhibition. The results showed that D-chicoric acid favors binding at the α-exosite, whereas lomofungin preferentially binds at the β-exosite by mimicking the substrate β-sheet binding interaction. Molecular dynamics simulations and binding interaction analysis of the exosite inhibitors with BoNT/A revealed key elements and hotspots that likely contribute to the inhibitor binding and synergistic inhibition. Finally, we performed database virtual screening for novel inhibitors of BoNT/A targeting the exosites. Hits C1 and C2 showed non-competitive inhibition and likely target the α- and β-exosites, respectively. The identified exosite inhibitors may provide novel candidates for structure-based development of therapeutics against BoNT/A intoxication. PMID:24958623

  3. Design of highly potent urea-based, exosite-binding inhibitors selective for glutamate carboxypeptidase II.

    PubMed

    Tykvart, Jan; Schimer, Jiří; Jančařík, Andrej; Bařinková, Jitka; Navrátil, Václav; Starková, Jana; Šrámková, Karolína; Konvalinka, Jan; Majer, Pavel; Šácha, Pavel

    2015-05-28

    We present here a structure-aided design of inhibitors targeting the active site as well as exosites of glutamate carboxypeptidase II (GCPII), a prostate cancer marker, preparing potent and selective inhibitors that are more than 1000-fold more active toward GCPII than its closest human homologue, glutamate carboxypeptidase III (GCPIII). Additionally, we demonstrate that the prepared inhibitor conjugate can be used for sensitive and selective imaging of GCPII in mammalian cells. PMID:25923815

  4. Inhibition of thrombin-mediated cellular effects by triabin, a highly potent anion-binding exosite thrombin inhibitor.

    PubMed

    Glusa, E; Bretschneider, E; Daum, J; Noeske-Jungblut, C

    1997-06-01

    Triabin, a 17 kDa protein from the saliva of the assassin bug Triatoma pallidipennis is a potent thrombin inhibitor interfering with the anion-binding exosite of the enzyme. The recombinant protein, produced by the baculovirus/insect cell system, was used to study the inhibitory effect on thrombin-mediated cellular responses. The thrombin (1 nM)-stimulated aggregation of washed human platelets and the rise in cytoplasmic calcium in platelets were inhibited by triabin at nanomolar concentrations. In contrast, the rise in calcium induced by the thrombin receptor-activating peptide (10 microM) was not suppressed by triabin. In isolated porcine pulmonary arteries, preconstricted with PGF 2 alpha thrombin (2 nM) elicited an endothelium-dependent relaxation which was inhibited by triabin in the same concentration range as found for the inhibition of platelet aggregation. Higher concentrations of triabin were required to diminish the contractile response of endotheliumdenuded pulmonary vessels to thrombin (10 nM). In cultured bovine coronary smooth muscle cells, the mitogenic activity of thrombin (3 nM), measured by [3H]thymidine incorporation, was also suppressed by triabin. In all these assays, the inhibitory effect of triabin was dependent on the thrombin concentration used. These studies suggest that the new anion-binding exosite thrombin inhibitor triabin is one of the most potent inhibitors of thrombin-mediated cellular effects. PMID:9241757

  5. The Contribution of Non-catalytic Carbohydrate Binding Modules to the Activity of Lytic Polysaccharide Monooxygenases*

    PubMed Central

    Crouch, Lucy I.; Labourel, Aurore; Walton, Paul H.; Davies, Gideon J.; Gilbert, Harry J.

    2016-01-01

    Lignocellulosic biomass is a sustainable industrial substrate. Copper-dependent lytic polysaccharide monooxygenases (LPMOs) contribute to the degradation of lignocellulose and increase the efficiency of biofuel production. LPMOs can contain non-catalytic carbohydrate binding modules (CBMs), but their role in the activity of these enzymes is poorly understood. Here we explored the importance of CBMs in LPMO function. The family 2a CBMs of two monooxygenases, CfLPMO10 and TbLPMO10 from Cellulomonas fimi and Thermobispora bispora, respectively, were deleted and/or replaced with CBMs from other proteins. The data showed that the CBMs could potentiate and, surprisingly, inhibit LPMO activity, and that these effects were both enzyme-specific and substrate-specific. Removing the natural CBM or introducing CtCBM3a, from the Clostridium thermocellum cellulosome scaffoldin CipA, almost abolished the catalytic activity of the LPMOs against the cellulosic substrates. The deleterious effect of CBM removal likely reflects the importance of prolonged presentation of the enzyme on the surface of the substrate for efficient catalytic activity, as only LPMOs appended to CBMs bound tightly to cellulose. The negative impact of CtCBM3a is in sharp contrast with the capacity of this binding module to potentiate the activity of a range of glycoside hydrolases including cellulases. The deletion of the endogenous CBM from CfLPMO10 or the introduction of a family 10 CBM from Cellvibrio japonicus LPMO10B into TbLPMO10 influenced the quantity of non-oxidized products generated, demonstrating that CBMs can modulate the mode of action of LPMOs. This study demonstrates that engineered LPMO-CBM hybrids can display enhanced industrially relevant oxygenations. PMID:26801613

  6. Structure of the thrombin complex with triabin, a lipocalin-like exosite-binding inhibitor derived from a triatomine bug.

    PubMed

    Fuentes-Prior, P; Noeske-Jungblut, C; Donner, P; Schleuning, W D; Huber, R; Bode, W

    1997-10-28

    Triabin, a 142-residue protein from the saliva of the blood-sucking triatomine bug Triatoma pallidipennis, is a potent and selective thrombin inhibitor. Its stoichiometric complex with bovine alpha-thrombin was crystallized, and its crystal structure was solved by Patterson search methods and refined at 2.6-A resolution to an R value of 0.184. The analysis revealed that triabin is a compact one-domain molecule essentially consisting of an eight-stranded beta-barrel. The eight strands A to H are arranged in the order A-C-B-D-E-F-G-H, with the first four strands exhibiting a hitherto unobserved up-up-down-down topology. Except for the B-C inversion, the triabin fold exhibits the regular up-and-down topology of lipocalins. In contrast to the typical ligand-binding lipocalins, however, the triabin barrel encloses a hydrophobic core intersected by a unique salt-bridge cluster. Triabin interacts with thrombin exclusively via its fibrinogen-recognition exosite. Surprisingly, most of the interface interactions are hydrophobic. A prominent exception represents thrombin's Arg-77A side chain, which extends into a hydrophobic triabin pocket forming partially buried salt bridges with Glu-128 and Asp-135 of the inhibitor. The fully accessible active site of thrombin in this complex is in agreement with its retained hydrolytic activity toward small chromogenic substrates. Impairment of thrombin's fibrinogen converting activity or of its thrombomodulin-mediated protein C activation capacity upon triabin binding is explained by usage of overlapping interaction sites of fibrinogen, thrombomodulin, and triabin on thrombin. These data demonstrate that triabin inhibits thrombin via a novel and unique mechanism that might be of interest in the context of potential therapeutic applications. PMID:9342325

  7. Dabigatran and Argatroban Diametrically Modulate Thrombin Exosite Function

    PubMed Central

    Yeh, Calvin H.; Stafford, Alan R.; Leslie, Beverly A.; Fredenburgh, James C.; Weitz, Jeffrey I.

    2016-01-01

    Thrombin is a highly plastic molecule whose activity and specificity are regulated by exosites 1 and 2, positively-charged domains that flank the active site. Exosite binding by substrates and cofactors regulates thrombin activity by localizing thrombin, guiding substrates, and by inducing allosteric changes at the active site. Although inter-exosite and exosite-to-active-site allostery have been demonstrated, the impact of active site ligation on exosite function has not been examined. To address this gap, we used surface plasmon resonance to determine the effects of dabigatran and argatroban, active site-directed inhibitors, on thrombin binding to immobilized γA/γA-fibrin or glycoprotein Ibα peptide via exosite 1 and 2, respectively, and thrombin binding to γA/γ′-fibrin or factor Va, which is mediated by both exosites. Whereas dabigatran attenuated binding, argatroban increased thrombin binding to γA/γA- and γA/γ′-fibrin and to factor Va. The results with immobilized fibrin were confirmed by examining the binding of radiolabeled thrombin to fibrin clots. Thus, dabigatran modestly accelerated the dissociation of thrombin from γA/γA-fibrin clots, whereas argatroban attenuated dissociation. Dabigatran had no effect on thrombin binding to glycoprotein Ibα peptide, whereas argatroban promoted binding. These findings not only highlight functional effects of thrombin allostery, but also suggest that individual active site-directed thrombin inhibitors uniquely modulate exosite function, thereby identifying potential novel mechanisms of action. PMID:27305147

  8. Dual Exosite-binding Inhibitors of Insulin-degrading Enzyme Challenge Its Role as the Primary Mediator of Insulin Clearance in Vivo.

    PubMed

    Durham, Timothy B; Toth, James L; Klimkowski, Valentine J; Cao, Julia X C; Siesky, Angela M; Alexander-Chacko, Jesline; Wu, Ginger Y; Dixon, Jeffrey T; McGee, James E; Wang, Yong; Guo, Sherry Y; Cavitt, Rachel Nicole; Schindler, John; Thibodeaux, Stefan J; Calvert, Nathan A; Coghlan, Michael J; Sindelar, Dana K; Christe, Michael; Kiselyov, Vladislav V; Michael, M Dodson; Sloop, Kyle W

    2015-08-14

    Insulin-degrading enzyme (IDE, insulysin) is the best characterized catabolic enzyme implicated in proteolysis of insulin. Recently, a peptide inhibitor of IDE has been shown to affect levels of insulin, amylin, and glucagon in vivo. However, IDE(-/-) mice display variable phenotypes relating to fasting plasma insulin levels, glucose tolerance, and insulin sensitivity depending on the cohort and age of animals. Here, we interrogated the importance of IDE-mediated catabolism on insulin clearance in vivo. Using a structure-based design, we linked two newly identified ligands binding at unique IDE exosites together to construct a potent series of novel inhibitors. These compounds do not interact with the catalytic zinc of the protease. Because one of these inhibitors (NTE-1) was determined to have pharmacokinetic properties sufficient to sustain plasma levels >50 times its IDE IC50 value, studies in rodents were conducted. In oral glucose tolerance tests with diet-induced obese mice, NTE-1 treatment improved the glucose excursion. Yet in insulin tolerance tests and euglycemic clamp experiments, NTE-1 did not enhance insulin action or increase plasma insulin levels. Importantly, IDE inhibition with NTE-1 did result in elevated plasma amylin levels, suggesting the in vivo role of IDE action on amylin may be more significant than an effect on insulin. Furthermore, using the inhibitors described in this report, we demonstrate that in HEK cells IDE has little impact on insulin clearance. In total, evidence from our studies supports a minimal role for IDE in insulin metabolism in vivo and suggests IDE may be more important in helping regulate amylin clearance. PMID:26085101

  9. Affinity labeling of lysine-149 in the anion-binding exosite of human. alpha. -thrombin with an N sup. alpha. -(dinitrofluorobenzyl)hirudin C-terminal peptide

    SciTech Connect

    Bourdon, P.; Maraganore, J.M. ); Fenton, J.W. II )

    1990-07-10

    In order to define structural regions in thrombin that interact with hirudin, the N{sup {alpha}}-dinitrofluorobenzyl analogue of an undecapeptide was synthesized corresponding to residues 54-64 of hirudin (GDFEEIPEEY(O{sup 35}SO{sub 3})L (DNFB-({sup 35}S)Hir{sub 54-64})). DNFB-({sup 35}S)Hir{sub 54-64} was reacted at a 10-fold molar excess with human {alpha}-thrombin in phosphate-buffered saline at pH 7.4 and 23{degree}C for 18 h. Autoradiographs of the product in reducing SDS-polyacrylamide gels revealed a single {sup 35}S-labeled band of M{sub r} {approximately}32,500. The labeled product was coincident with a band on Coomassie Blue stained gels migrating slightly above an unlabeled thrombin band at M{sub r} {approximately}31,000. Incorporation of the {sup 35}S affinity reagent peptide was found markedly reduced when reaction with thrombin was performed in the presence of 5- and 20-fold molar excesses of unlabeled hirudin peptide, showing that a specific site was involved in complex formation. The human {alpha}-thrombin-DNFB-Hir{sub 54-64} complex was reduced, S-carboxymethylated, and treated with pepsin. Peptic fragments were separated by reverse-phase HPLC revealing two major peaks containing absorbance at 310 nm. Automated Edman degradation of the peptide fragments allowed identification of Lys-149 of human thrombin as the major site of DNFB-Hir{sub 54-64} derivatization. These data suggest that the anionic C-terminal tail of hirudin interacts with an anion-binding exosite in human thrombin removed 18-20 {angstrom} from the catalytic apparatus.

  10. Dual Exosite-binding Inhibitors of Insulin-degrading Enzyme Challenge Its Role as the Primary Mediator of Insulin Clearance in Vivo*

    PubMed Central

    Durham, Timothy B.; Toth, James L.; Klimkowski, Valentine J.; Cao, Julia X. C.; Siesky, Angela M.; Alexander-Chacko, Jesline; Wu, Ginger Y.; Dixon, Jeffrey T.; McGee, James E.; Wang, Yong; Guo, Sherry Y.; Cavitt, Rachel Nicole; Schindler, John; Thibodeaux, Stefan J.; Calvert, Nathan A.; Coghlan, Michael J.; Sindelar, Dana K.; Christe, Michael; Kiselyov, Vladislav V.; Michael, M. Dodson; Sloop, Kyle W.

    2015-01-01

    Insulin-degrading enzyme (IDE, insulysin) is the best characterized catabolic enzyme implicated in proteolysis of insulin. Recently, a peptide inhibitor of IDE has been shown to affect levels of insulin, amylin, and glucagon in vivo. However, IDE−/− mice display variable phenotypes relating to fasting plasma insulin levels, glucose tolerance, and insulin sensitivity depending on the cohort and age of animals. Here, we interrogated the importance of IDE-mediated catabolism on insulin clearance in vivo. Using a structure-based design, we linked two newly identified ligands binding at unique IDE exosites together to construct a potent series of novel inhibitors. These compounds do not interact with the catalytic zinc of the protease. Because one of these inhibitors (NTE-1) was determined to have pharmacokinetic properties sufficient to sustain plasma levels >50 times its IDE IC50 value, studies in rodents were conducted. In oral glucose tolerance tests with diet-induced obese mice, NTE-1 treatment improved the glucose excursion. Yet in insulin tolerance tests and euglycemic clamp experiments, NTE-1 did not enhance insulin action or increase plasma insulin levels. Importantly, IDE inhibition with NTE-1 did result in elevated plasma amylin levels, suggesting the in vivo role of IDE action on amylin may be more significant than an effect on insulin. Furthermore, using the inhibitors described in this report, we demonstrate that in HEK cells IDE has little impact on insulin clearance. In total, evidence from our studies supports a minimal role for IDE in insulin metabolism in vivo and suggests IDE may be more important in helping regulate amylin clearance. PMID:26085101

  11. Characterization of selective exosite-binding inhibitors of matrix metalloproteinase 13 that prevent articular cartilage degradation in vitro.

    PubMed

    Spicer, Timothy P; Jiang, Jianwen; Taylor, Alexander B; Choi, Jun Yong; Hart, P John; Roush, William R; Fields, Gregg B; Hodder, Peter S; Minond, Dmitriy

    2014-11-26

    Matrix metalloproteinase 13 (MMP-13) has been shown to be the main collagenase responsible for degradation of articular cartilage during osteoarthritis and therefore represents a target for drug development. As a result of high-throughput screening and structure-activity relationship studies, we identified a novel, highly selective class of MMP-13 inhibitors (compounds 1 (Q), 2 (Q1), and 3 (Q2)). Mechanistic characterization revealed a noncompetitive nature of these inhibitors with binding constants in the low micromolar range. Crystallographic analyses revealed two binding modes for compound 2 in the MMP-13 S1' subsite and in an S1/S2* subsite. Type II collagen- and cartilage-protective effects exhibited by compounds 1, 2, and 3 suggested that these compounds might be efficacious in future in vivo studies. Finally, these compounds were also highly selective when tested against a panel of 30 proteases, which, in combination with a good CYP inhibition profile, suggested low off-target toxicity and drug-drug interactions in humans. PMID:25330343

  12. Characterization of Selective Exosite-Binding Inhibitors of Matrix Metalloproteinase 13 That Prevent Articular Cartilage Degradation in Vitro

    PubMed Central

    2015-01-01

    Matrix metalloproteinase 13 (MMP-13) has been shown to be the main collagenase responsible for degradation of articular cartilage during osteoarthritis and therefore represents a target for drug development. As a result of high-throughput screening and structure–activity relationship studies, we identified a novel, highly selective class of MMP-13 inhibitors (compounds 1 (Q), 2 (Q1), and 3 (Q2)). Mechanistic characterization revealed a noncompetitive nature of these inhibitors with binding constants in the low micromolar range. Crystallographic analyses revealed two binding modes for compound 2 in the MMP-13 S1′ subsite and in an S1/S2* subsite. Type II collagen- and cartilage-protective effects exhibited by compounds 1, 2, and 3 suggested that these compounds might be efficacious in future in vivo studies. Finally, these compounds were also highly selective when tested against a panel of 30 proteases, which, in combination with a good CYP inhibition profile, suggested low off-target toxicity and drug–drug interactions in humans. PMID:25330343

  13. Elastin Degradation by Cathepsin V Requires Two Exosites*

    PubMed Central

    Du, Xin; Chen, Nelson L. H.; Wong, Andre; Craik, Charles S.; Brömme, Dieter

    2013-01-01

    Cathepsin V is a highly effective elastase and has been implicated in physiological and pathological extracellular matrix degradation. However, its mechanism of action remains elusive. Whereas human cathepsin V exhibits a potent elastolytic activity, the structurally homologous cathepsin L, which shares a 78% amino acid sequence, has only a minimal proteolytic activity toward insoluble elastin. This suggests that there are distinct structural domains that play an important role in elastinolysis. In this study, a total of 11 chimeras of cathepsins V and L were generated to identify elastin-binding domains in cathepsin V. Evaluation of these chimeras revealed two exosites contributing to the elastolytic activity of cathepsin V that are distant from the active cleft of the protease and are located in surface loop regions. Replacement of exosite 1 or 2 with analogous residues from cathepsin L led to a 75 and 43% loss in the elastolytic activity, respectively. Replacement of both exosites yielded a non-elastase variant similar to that of cathepsin L. Identification of these exosites may contribute to the design of inhibitors that will only affect the elastolytic activity of cysteine cathepsins without interfering with other physiological protease functions. PMID:24121514

  14. Promiscuous, non-catalytic, tandem carbohydrate-binding modules modulate the cell-wall structure and development of transgenic tobacco (Nicotiana tabacum) plants

    PubMed Central

    Obembe, Olawole O.; Jacobsen, Evert; Timmers, Jaap; Gilbert, Harry; Blake, Anthony W.; Knox, J. Paul; Visser, Richard G. F.

    2007-01-01

    We have compared heterologous expression of two types of carbohydrate binding module (CBM) in tobacco cell walls. These are the promiscuous CBM29 modules (a tandem CBM29-1-2 and its single derivative CBM29-2), derived from a non-catalytic protein1, NCP1, of the Piromyces equi cellulase/hemicellulase complex, and the less promiscuous tandem CBM2b-1-2 from the Cellulomonas fimi xylanase 11A. CBM-labelling studies revealed that CBM29-1-2 binds indiscriminately to every tissue of the wild-type tobacco stem whereas binding of CBM2b-1-2 was restricted to vascular tissue. The promiscuous CBM29-1-2 had much more pronounced effects on transgenic tobacco plants than the less promiscuous CBM2b-1-2. Reduced stem elongation and prolonged juvenility, resulting in delayed flower development, were observed in transformants expressing CBM29-1-2 whereas such growth phenotypes were not observed for CBM2b-1-2 plants. Histological examination and electron microscopy revealed layers of collapsed cortical cells in the stems of CBM29-1-2 plants whereas cellular deformation in the stem cortical cells of CBM2b-1-2 transformants was less severe. Altered cell expansion was also observed in most parts of the CBM29-1-2 stem whereas for the CBM2b-1-2 stem this was observed in the xylem cells only. The cellulose content of the transgenic plants was not altered. These results support the hypothesis that CBMs can modify cell wall structure leading to modulation of wall loosening and plant growth. PMID:17622484

  15. Probing BoNT/A protease exosites: implications for inhibitor design and light chain longevity.

    PubMed

    Xue, Song; Javor, Sacha; Hixon, Mark S; Janda, Kim D

    2014-11-01

    Botulinum neurotoxin serotype A (BoNT/A) is one of the most lethal toxins known. Its extreme toxicity is due to its light chain (LC), a zinc protease that cleaves SNAP-25, a synaptosome-associated protein, leading to the inhibition of neuronal activity. Studies on BoNT/A LC have revealed that two regions, termed exosites, can play an important role in BoNT catalytic activity. A clear understanding of how these exosites influence neurotoxin catalytic activity would provide a critical framework for deciphering the mechanism of SNAP-25 cleavage and the design of inhibitors. Herein, based on the crystallographic structure of BoNT/A LC complexed with its substrate, we designed an α-exosite binding probe. Experiments with this unique probe demonstrated that α-exosite binding enhanced both catalytic activity and stability of the LC. These data help delineate why α-exosite binding is needed for SNAP-25 cleavage and also provide new insights into the extended lifetime observed for BoNT/A LC in vivo. PMID:25295706

  16. Structural and evolutionary aspects of two families of non-catalytic domains present in starch and glycogen binding proteins from microbes, plants and animals.

    PubMed

    Janeček, Štefan; Svensson, Birte; MacGregor, E Ann

    2011-10-10

    Starch-binding domains (SBDs) comprise distinct protein modules that bind starch, glycogen or related carbohydrates and have been classified into different families of carbohydrate-binding modules (CBMs). The present review focuses on SBDs of CBM20 and CBM48 found in amylolytic enzymes from several glycoside hydrolase (GH) families GH13, GH14, GH15, GH31, GH57 and GH77, as well as in a number of regulatory enzymes, e.g., phosphoglucan, water dikinase-3, genethonin-1, laforin, starch-excess protein-4, the β-subunit of AMP-activated protein kinase and its homologues from sucrose non-fermenting-1 protein kinase SNF1 complex, and an adaptor-regulator related to the SNF1/AMPK family, AKINβγ. CBM20s and CBM48s of amylolytic enzymes occur predominantly in the microbial world, whereas the non-amylolytic proteins containing these modules are mostly of plant and animal origin. Comparison of amino acid sequences and tertiary structures of CBM20 and CBM48 reveals the close relatedness of these SBDs and, in some cases, glycogen-binding domains (GBDs). The families CBM20 and CBM48 share both an ancestral form and the mode of starch/glycogen binding at one or two binding sites. Phylogenetic analyses demonstrate that they exhibit independent behaviour, i.e. each family forms its own part in an evolutionary tree, with enzyme specificity (protein function) being well represented within each family. The distinction between CBM20 and CBM48 families is not sharp since there are representatives in both CBM families that possess an intermediate character. These are, for example, CBM20s from hypothetical GH57 amylopullulanase (probably lacking the starch-binding site 2) and CBM48s from the GH13 pullulanase subfamily (probably lacking the starch/glycogen-binding site 1). The knowledge gained concerning the occurrence of these SBDs and GBDs through the range of taxonomy will support future experimental research. PMID:22112614

  17. Functional Roles of the Non-Catalytic Calcium-Binding Sites in the N-Terminal Domain of Human Peptidylarginine Deiminase 4

    PubMed Central

    Liu, Yi-Liang; Tsai, I-Chen; Chang, Chia-Wei; Liao, Ya-Fan; Liu, Guang-Yaw; Hung, Hui-Chih

    2013-01-01

    This study investigated the functional roles of the N-terminal Ca2+ ion-binding sites, in terms of enzyme catalysis and stability, of peptidylarginine deiminase 4 (PAD4). Amino acid residues located in the N-terminal Ca2+-binding site of PAD4 were mutated to disrupt the binding of Ca2+ ions. Kinetic data suggest that Asp155, Asp157 and Asp179, which directly coordinate Ca3 and Ca4, are essential for catalysis in PAD4. For D155A, D157A and D179A, the kcat/Km,BAEE values were 0.02, 0.63 and 0.01 s−1mM−1 (20.8 s−1mM−1 for WT), respectively. Asn153 and Asp176 are directly coordinated with Ca3 and indirectly coordinated with Ca5 via a water molecule. However, N153A displayed low enzymatic activity with a kcat value of 0.3 s−1 (13.3 s−1 for wild-type), whereas D176A retained some catalytic power with a kcat of 9.7 s−1. Asp168 is the direct ligand for Ca5, and Ca5 coordination by Glu252 is mediated by two water molecules. However, mutation of these two residues to Ala did not cause a reduction in the kcat/Km,BAEE values, which indicates that the binding of Ca5 may not be required for PAD4 enzymatic activity. The possible conformational changes of these PAD4 mutants were examined. Thermal stability analysis of the PAD4 mutants in the absence or presence of Ca2+ indicated that the conformational stability of the enzyme is highly dependent on Ca2+ ions. In addition, the results of urea-induced denaturation for the N153, D155, D157 and D179 series mutants further suggest that the binding of Ca2+ ions in the N-terminal Ca2+-binding site stabilizes the overall conformational stability of PAD4. Therefore, our data strongly suggest that the N-terminal Ca2+ ions play critical roles in the full activation of the PAD4 enzyme. PMID:23382808

  18. Non-catalytic recuperative reformer

    SciTech Connect

    Khinkis, Mark J.; Kozlov, Aleksandr P.; Kurek, Harry

    2015-12-22

    A non-catalytic recuperative reformer has a flue gas flow path for conducting hot flue gas from a thermal process and a reforming mixture flow path for conducting a reforming mixture. At least a portion of the reforming mixture flow path is embedded in the flue gas flow path to permit heat transfer from the hot flue gas to the reforming mixture. The reforming mixture flow path contains substantially no material commonly used as a catalyst for reforming hydrocarbon fuel (e.g., nickel oxide, platinum group elements or rhenium), but instead the reforming mixture is reformed into a higher calorific fuel via reactions due to the heat transfer and residence time. In a preferred embodiment, extended surfaces of metal material such as stainless steel or metal alloy that are high in nickel content are included within at least a portion of the reforming mixture flow path.

  19. Leptospira interrogans reduces fibrin clot formation by modulating human thrombin activity via exosite I.

    PubMed

    Fernandes, Luis G; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

    2015-06-01

    Pathogenic bacteria of the genus Leptospira are the etiological agents of leptospirosis, a disease that affects humans and animals worldwide. Although there are an increasing number of studies on the biology of Leptospira, the mechanisms of pathogenesis are not yet understood. We report in this work that Leptospira interrogans FIOCRUZ L1-130 virulent, M20 culture attenuated and the saprophyte L. biflexa Patoc 1 strains do not bind prothrombin. Leptospiral binding to thrombin was detected with the virulent, followed by culture-attenuated M20, and practically none was observed with the saprophyte strain. The interaction of Leptospira with thrombin mostly occurs via exosite I, with a minor participation of catalytic site, as determined by employing the thrombin inhibitors hirugen, hirudin and argatroban. Leptospira interrogans binding to thrombin inhibits its catalytic activity reducing fibrin clot formation in thrombin-catalyzed reaction of fibrinogen. This inhibition was more efficient with the virulent FIOCRUZ L1-130 than with the M20 culture attenuated, while none was seen with the saprophyte strain, suggesting that this binding might be important for bacterial virulence. This is the first study reporting the binding of pathogenic Leptospira to thrombin promoting a decrease in fibrin clotting that could lead to hemorrhage, helping bacteria dissemination. PMID:25834144

  20. Non-catalytic steam hydrolysis of fats

    SciTech Connect

    Deibert, M.C.

    1992-08-28

    Hydrolysis of fats and oils produces fatty acid and glycerol. The catalyzed, liquid phase Colgate-Emry process, state-of-the-art, produces impure products that require extensive energy investment for their purification to commercial grade. Non-catalytic steam hydrolysis may produce products more easily purified. A bench-scale hydrolyzer was designed and constructed to contact descending liquid fat or oil with rising superheated steam. Each of the five stages in the reactor was designed similar to a distillation column stage to promote intimate liquid-gas contact. Degree of hydrolysis achieved in continuous tests using tallow feed were 15% at 280C and 35% at 300C at a tallow-to-steam mass feed ratio of 4.2. At a feed ratio of 9.2, the degree of hydrolysis was 21% at 300C. Decomposition was strongly evident at 325C but not at lower temperatures. Soybean oil rapidly polymerized under reaction conditions. Batch tests at 320C produced degrees of hydrolyses of between 44% and 63% using tallow and palm oil feeds. Over 95% fatty acids were present in a clean, readily separated organic portion of the overhead product from most tests. The test reactor had serious hydraulic resistance to liquid down-flow which limited operation to very long liquid residence times. These times are in excess of those that tallow and palm oil are stable at the reaction temperature. Little glycerol and extensive light organics were produced indicating that unexplained competing reactions to hydrolysis occurred in the experimental system. Further tests using an improved reactor will be required.

  1. Antibody-based exosite inhibitors of ADAMTS-5 (aggrecanase-2)

    PubMed Central

    Santamaria, Salvatore; Yamamoto, Kazuhiro; Botkjaer, Kenneth; Tape, Christopher; Dyson, Michael R.; McCafferty, John; Murphy, Gillian; Nagase, Hideaki

    2015-01-01

    Adamalysin-like metalloproteinases with thrombospondin (TS) motifs (ADAMTS)-5 is the multi-domain metalloproteinase that most potently degrades aggrecan proteoglycan in the cartilage and its activity is implicated in the development of osteoarthritis (OA). To generate specific exosite inhibitors for it, we screened a phage display antibody library in the presence of the zinc-chelating active site-directed inhibitor GM6001 (Ilomastat) and isolated four highly selective inhibitory antibodies. Two antibodies were mapped to react with exosites in the catalytic/disintegrin domains (Cat/Dis) of the enzyme, one in the TS domain and one in the spacer domain (Sp). The antibody reacting with the Sp blocked the enzyme action only when aggrecan or the Escherichia coli-expressed aggrecan core protein were substrates, but not against a peptide substrate. The study with this antibody revealed the importance of the Sp for effective aggrecanolytic activity of ADAMTS-5 and that this domain does not interact with sulfated glycosaminoglycans (GAGs) but with the protein moiety of the proteoglycan. An antibody directed against the Cat/Dis of ADAMTS-5 was effective in a cell-based model of aggrecan degradation; however, the anti-Sp antibody was ineffective. Western blot analysis of endogenous ADAMTS-5 expressed by human chondrocytes showed the presence largely of truncated forms of ADAMTS-5, thus explaining the lack of efficacy of the anti-Sp antibody. The possibility of ADAMTS-5 truncation must then be taken into account when considering developing anti-ancillary domain antibodies for therapeutic purposes. PMID:26303525

  2. Cooperative exosite-dependent cleavage of synaptobrevin by tetanus toxin light chain.

    PubMed

    Cornille, F; Martin, L; Lenoir, C; Cussac, D; Roques, B P; Fournie-Zaluski, M C

    1997-02-01

    The light chain (L chain) of tetanus neurotoxin (TeNT) has been shown to have been endowed with zinc endopeptidase activity, selectively directed toward the Gln76-Phe77 bond of synaptobrevin, a vesicle-associated membrane protein (VAMP) critically involved in neuroexocytosis. In previous reports, truncations at the NH2 and COOH terminus of synaptobrevin have shown that the sequence 39-88 of synaptobrevin is the minimum substrate of TeNT, suggesting either the requirement of a well defined three-dimensional structure of synaptobrevin or a role in the mechanism of substrate hydrolysis for residues distal from the cleavage site. In this study, the addition of NH2- and COOH-terminal peptides of synaptobrevin, S 27-55 (S1) and S 82-93 (S2), to the synaptobrevin fragment S 56-81 allowed the cleavage of this latter peptide by TeNT to occur. This appears to result from an activation process mediated by the simultaneous binding of S1 and S2 with complementary sites present on TeNT as shown by surface plasmon resonance experiments and the determination of kinetic constants. All these results favor an exosite-controlled hydrolysis of synaptobrevin by TeNT, probably involving a conformational change of the toxin. This could account for the high degree of substrate specificity of TeNT and, probably, botulinum neurotoxins. PMID:9013591

  3. Evaluation of a non-catalytic coating for metallic TPS

    NASA Technical Reports Server (NTRS)

    Pittman, C. M.; Brown, R. D.; Shideler, J. L.

    1984-01-01

    A commercially available ceramic coating was evaluated for application to metallic heat shields for Shuttle-type entry vehicles. Coated Inconel 617 specimens were subjected to thermal shock cycles, surface emittances were measured, and surface equilibrium temperatures were measured for coated and oxidized specimens exposed to an arc-tunnel environment. The coating adhered very well to the metal and appeared to be very non-catalytic.

  4. Monoclonal Antibodies Targeting the Alpha-Exosite of Botulinum Neurotoxin Serotype/A Inhibit Catalytic Activity

    PubMed Central

    Fan, Yongfeng; Geren, Isin N.; Dong, Jianbo; Lou, Jianlong; Wen, Weihua; Conrad, Fraser; Smith, Theresa J.; Smith, Leonard A.; Ho, Mengfei; Pires-Alves, Melissa; Wilson, Brenda A.; Marks, James D.

    2015-01-01

    The paralytic disease botulism is caused by botulinum neurotoxins (BoNT), multi-domain proteins containing a zinc endopeptidase that cleaves the cognate SNARE protein, thereby blocking acetylcholine neurotransmitter release. Antitoxins currently used to treat botulism neutralize circulating BoNT but cannot enter, bind to or neutralize BoNT that has already entered the neuron. The light chain endopeptidase domain (LC) of BoNT serotype A (BoNT/A) was targeted for generation of monoclonal antibodies (mAbs) that could reverse paralysis resulting from intoxication by BoNT/A. Single-chain variable fragment (scFv) libraries from immunized humans and mice were displayed on the surface of yeast, and 19 BoNT/A LC-specific mAbs were isolated by using fluorescence-activated cell sorting (FACS). Affinities of the mAbs for BoNT/A LC ranged from a KD value of 9.0×10−11 M to 3.53×10−8 M (mean KD 5.38×10−9 M and median KD 1.53×10−9 M), as determined by flow cytometry analysis. Eleven mAbs inhibited BoNT/A LC catalytic activity with IC50 values ranging from 8.3 ~73×10−9 M. The fine epitopes of selected mAbs were also mapped by alanine-scanning mutagenesis, revealing that the inhibitory mAbs bound the α-exosite region remote from the BoNT/A LC catalytic center. The results provide mAbs that could prove useful for intracellular reversal of paralysis post-intoxication and further define epitopes that could be targeted by small molecule inhibitors. PMID:26275214

  5. Monoclonal Antibodies Targeting the Alpha-Exosite of Botulinum Neurotoxin Serotype/A Inhibit Catalytic Activity.

    PubMed

    Fan, Yongfeng; Geren, Isin N; Dong, Jianbo; Lou, Jianlong; Wen, Weihua; Conrad, Fraser; Smith, Theresa J; Smith, Leonard A; Ho, Mengfei; Pires-Alves, Melissa; Wilson, Brenda A; Marks, James D

    2015-01-01

    The paralytic disease botulism is caused by botulinum neurotoxins (BoNT), multi-domain proteins containing a zinc endopeptidase that cleaves the cognate SNARE protein, thereby blocking acetylcholine neurotransmitter release. Antitoxins currently used to treat botulism neutralize circulating BoNT but cannot enter, bind to or neutralize BoNT that has already entered the neuron. The light chain endopeptidase domain (LC) of BoNT serotype A (BoNT/A) was targeted for generation of monoclonal antibodies (mAbs) that could reverse paralysis resulting from intoxication by BoNT/A. Single-chain variable fragment (scFv) libraries from immunized humans and mice were displayed on the surface of yeast, and 19 BoNT/A LC-specific mAbs were isolated by using fluorescence-activated cell sorting (FACS). Affinities of the mAbs for BoNT/A LC ranged from a KD value of 9.0×10-11 M to 3.53×10-8 M (mean KD 5.38×10-9 M and median KD 1.53×10-9 M), as determined by flow cytometry analysis. Eleven mAbs inhibited BoNT/A LC catalytic activity with IC50 values ranging from 8.3 ~73×10-9 M. The fine epitopes of selected mAbs were also mapped by alanine-scanning mutagenesis, revealing that the inhibitory mAbs bound the α-exosite region remote from the BoNT/A LC catalytic center. The results provide mAbs that could prove useful for intracellular reversal of paralysis post-intoxication and further define epitopes that could be targeted by small molecule inhibitors. PMID:26275214

  6. Non-catalytic hydropyrolysis of microalgae to produce liquid biofuels.

    PubMed

    Duan, Peigao; Bai, Xiujun; Xu, Yuping; Zhang, Aiyun; Wang, Feng; Zhang, Lei; Miao, Juan

    2013-05-01

    Non-catalytic hydropyrolysis of Chlorella pyrenoidosa was studied by using a stainless-steel batch reactor at different temperature (150-450 °C), time (5-120 min) and initial hydrogen pressure (1 atm-8 MPa), aiming to find how these parameters affect the product (oil, gas and solid) yields and properties of the hydropyrolysis oil (HPO). Temperature was the most influential factor to the relative amount of each product and properties of the HPOs. The hydrogen favored the stabilization of the active intermediates but cannot guarantee to produce HPOs in higher hydrogen at its higher initial pressure. The HPO, which showed much difference in component strongly depending on the reaction conditions, mainly consisted of aromatics and straight-chain hydrocarbons, amides, amines, nitriles and carboxylic acids at moderate temperatures. The main gas products detected during the hydropyrolysis were unreacted H2, CO2, CO and CH4. About 85% of energy originally present in the microalgae was recovered as oil under the optimal conditions. PMID:23567740

  7. Non-Catalytic Roles of Presenilin Throughout Evolution.

    PubMed

    Otto, Grant P; Sharma, Devdutt; Williams, Robin S B

    2016-04-12

    Research into Alzheimer's disease pathology and treatment has often focused on presenilin proteins. These proteins provide the key catalytic activity of the γ-secretase complex in the cleavage of amyloid-β precursor protein and resultant amyloid tangle deposition. Over the last 25 years, screening novel drugs to control this aberrant proteolytic activity has yet to identify effective treatments for the disease. In the search for other mechanisms of presenilin pathology, several studies have demonstrated that mammalian presenilin proteins also act in a non-proteolytic role as a scaffold to co-localize key signaling proteins. This role is likely to represent an ancestral presenilin function, as it has been described in genetically distant species including non-mammalian animals, plants, and a simple eukaryotic amoeba Dictyostelium that diverged from the human lineage over a billion years ago. Here, we review the non-catalytic scaffold role of presenilin, from mammalian models to other biomedical models, and include recent insights using Dictyostelium, to suggest that this role may provide an early evolutionary function of presenilin proteins. PMID:27079701

  8. Non-Catalytic Roles of Presenilin Throughout Evolution

    PubMed Central

    Otto, Grant P.; Sharma, Devdutt; Williams, Robin S.B.

    2016-01-01

    Research into Alzheimer’s disease pathology and treatment has often focused on presenilin proteins. These proteins provide the key catalytic activity of the γ-secretase complex in the cleavage of amyloid-β precursor protein and resultant amyloid tangle deposition. Over the last 25 years, screening novel drugs to control this aberrant proteolytic activity has yet to identify effective treatments for the disease. In the search for other mechanisms of presenilin pathology, several studies have demonstrated that mammalian presenilin proteins also act in a non-proteolytic role as a scaffold to co-localize key signaling proteins. This role is likely to represent an ancestral presenilin function, as it has been described in genetically distant species including non-mammalian animals, plants, and a simple eukaryotic amoeba Dictyostelium that diverged from the human lineage over a billion years ago. Here, we review the non-catalytic scaffold role of presenilin, from mammalian models to other biomedical models, and include recent insights using Dictyostelium, to suggest that this role may provide an early evolutionary function of presenilin proteins. PMID:27079701

  9. Non-catalytic steam hydrolysis of fats. Final report

    SciTech Connect

    Deibert, M.C.

    1992-08-28

    Hydrolysis of fats and oils produces fatty acid and glycerol. The catalyzed, liquid phase Colgate-Emry process, state-of-the-art, produces impure products that require extensive energy investment for their purification to commercial grade. Non-catalytic steam hydrolysis may produce products more easily purified. A bench-scale hydrolyzer was designed and constructed to contact descending liquid fat or oil with rising superheated steam. Each of the five stages in the reactor was designed similar to a distillation column stage to promote intimate liquid-gas contact. Degree of hydrolysis achieved in continuous tests using tallow feed were 15% at 280C and 35% at 300C at a tallow-to-steam mass feed ratio of 4.2. At a feed ratio of 9.2, the degree of hydrolysis was 21% at 300C. Decomposition was strongly evident at 325C but not at lower temperatures. Soybean oil rapidly polymerized under reaction conditions. Batch tests at 320C produced degrees of hydrolyses of between 44% and 63% using tallow and palm oil feeds. Over 95% fatty acids were present in a clean, readily separated organic portion of the overhead product from most tests. The test reactor had serious hydraulic resistance to liquid down-flow which limited operation to very long liquid residence times. These times are in excess of those that tallow and palm oil are stable at the reaction temperature. Little glycerol and extensive light organics were produced indicating that unexplained competing reactions to hydrolysis occurred in the experimental system. Further tests using an improved reactor will be required.

  10. Esterification of glycerol from biodiesel production to glycerol carbonate in non-catalytic supercritical dimethyl carbonate.

    PubMed

    Ilham, Zul; Saka, Shiro

    2016-01-01

    Conversion of glycerol from biodiesel production to glycerol carbonate was studied by esterification with dimethyl carbonate in a non-catalytic supercritical condition. It was found that in a non-catalytic supercritical condition, glycerol at higher purity gave higher yield of glycerol carbonate at 98 wt% after reaction at 300 °C/20-40 MPa/15 min. The yield of glycerol carbonate was observed to increase with molar ratio, temperature, pressure and time until a certain equilibrium limit. The existence of impurities such as water and remnants of alkaline catalyst in crude glycerol will direct the reaction to produce glycidol. Although impurities might not be desirable, the non-catalytic supercritical dimethyl carbonate could be an alternative method for conversion of glycerol from biodiesel production to value-added glycerol carbonate.Graphical abstractPlausible reaction scheme for conversion of glycerol to glycerol carbonate in non-catalytic supercritical dimethyl carbonate. PMID:27386367

  11. Diversity between mammalian tolloid proteinases: Oligomerisation and non-catalytic domains influence activity and specificity

    PubMed Central

    Bayley, Christopher P.; Ruiz Nivia, Hilda D.; Dajani, Rana; Jowitt, Thomas A.; Collins, Richard F.; Rada, Heather; Bird, Louise E.; Baldock, Clair

    2016-01-01

    The mammalian tolloid family of metalloproteinases is essential for tissue patterning and extracellular matrix assembly. The four members of the family: bone morphogenetic protein-1 (BMP-1), mammalian tolloid (mTLD), tolloid-like (TLL)-1 and TLL-2 differ in their substrate specificity and activity levels, despite sharing similar domain organization. We have previously described a model of substrate exclusion by dimerisation to explain differences in the activities of monomeric BMP-1 and dimers of mTLD and TLL-1. Here we show that TLL-2, the least active member of the tolloid family, is predominantly monomeric in solution, therefore it appears unlikely that substrate exclusion via dimerisation is a mechanism for regulating TLL-2 activity. X-ray scattering and electron microscopy structural and biophysical analyses reveal an elongated shape for the monomer and flexibility in the absence of calcium. Furthermore, we show that TLL-2 can cleave chordin in vitro, similar to other mammalian tolloids, but truncated forms of TLL-2 mimicking BMP-1 are unable to cleave chordin. However, both the N- and C-terminal non-catalytic domains from all mammalian tolloids bind chordin with high affinity. The mechanisms underlying substrate specificity and activity in the tolloid family are complex with variation between family members and depend on both multimerisation and substrate interaction. PMID:26902455

  12. The Importance of Exosite Interactions for Substrate Cleavage by Human Thrombin

    PubMed Central

    Chahal, Gurdeep; Thorpe, Michael; Hellman, Lars

    2015-01-01

    Thrombin is a serine protease of the chymotrypsin family that acts both as a procoagulant and as an anticoagulant by cleaving either factor VIII, factor V and fibrinogen or protein C, respectively. Numerous previous studies have shown that electropositive regions at a distance from the active site, so called exosites, are of major importance for the cleavage by human thrombin. Upstream of all the known major cleavage sites for thrombin in factor VIII, factor V and fibrinogen are clusters of negatively charged amino acids. To study the importance of these sites for the interaction with the exosites and thereby the cleavage by thrombin, we have developed a new type of recombinant substrate. We have compared the cleavage rate of the minimal cleavage site, involving only 8-9 amino acids (typically the P4-P4’ positions) surrounding the cleavage site, with the substrates also containing the negatively charged regions upstream of the cleavage sites. The results showed that addition of these regions enhanced the cleavage rate by more than fifty fold. However, the enhancement was highly dependent on the sequence of the actual cleavage site. A minimal site that showed poor activity by itself could be cleaved as efficiently as an optimal cleavage site when presented together with these negatively charged regions. Whereas sites conforming closely to the optimal site were only minimally enhanced by the addition of these regions. The possibility to mimic this interaction for the sites in factor V and factor VIII by recombinant substrates, which do not have the same folding as the full size target, indicates that the enhancement was primarily dependent on a relatively simple electrostatic interaction. However, the situation was very different for fibrinogen and protein C where other factors than only charge is of major importance. PMID:26110612

  13. Designing Allosteric Regulators of Thrombin. Exosite 2 Features Multiple Sub-Sites That Can Be Targeted By Sulfated Small Molecules for Inducing Inhibition

    PubMed Central

    Sidhu, Preetpal Singh; Abdel Aziz, May H.; Sarkar, Aurijit; Mehta, Akul Y.; Zhou, Qibing; Desai, Umesh R.

    2013-01-01

    We recently designed a group of novel exosite 2-directed, sulfated, small, allosteric inhibitors of thrombin. To develop more potent inhibitors, monosulfated benzofuran tri- and tetrameric homologs of the parent designed dimers were synthesized in 7–8 steps and found to exhibit a wide range of potencies. Among these, trimer 9a was found to be nearly 10-fold more potent than the first generation molecules. Michaelis-Menten studies indicated an allosteric mechanism of inhibition. Competitive studies using a hirudin peptide (exosite 1 ligand) and, unfractionated heparin, heparin octasaccharide and γ′-fibrinogen peptide (exosite 2 ligands), demonstrated exosite 2 recognition in a manner different from the parent dimers. Alanine scanning mutagenesis of 12 Arg/Lys residues of exosite 2 revealed a defect in 9a potency for Arg233Ala thrombin only confirming the major difference in site of recognition between the two structurally related sulfated benzofurans. The results suggest that multiple avenues are available within exosite 2 for inducing thrombin inhibition. PMID:23718540

  14. The Catalytic and Non-catalytic Functions of the Brahma Chromatin-Remodeling Protein Collaborate to Fine-Tune Circadian Transcription in Drosophila

    PubMed Central

    Kwok, Rosanna S.; Li, Ying H.; Lei, Anna J.; Edery, Isaac; Chiu, Joanna C.

    2015-01-01

    Daily rhythms in gene expression play a critical role in the progression of circadian clocks, and are under regulation by transcription factor binding, histone modifications, RNA polymerase II (RNAPII) recruitment and elongation, and post-transcriptional mechanisms. Although previous studies have shown that clock-controlled genes exhibit rhythmic chromatin modifications, less is known about the functions performed by chromatin remodelers in animal clockwork. Here we have identified the Brahma (Brm) complex as a regulator of the Drosophila clock. In Drosophila, CLOCK (CLK) is the master transcriptional activator driving cyclical gene expression by participating in an auto-inhibitory feedback loop that involves stimulating the expression of the main negative regulators, period (per) and timeless (tim). BRM functions catalytically to increase nucleosome density at the promoters of per and tim, creating an overall restrictive chromatin landscape to limit transcriptional output during the active phase of cycling gene expression. In addition, the non-catalytic function of BRM regulates the level and binding of CLK to target promoters and maintains transient RNAPII stalling at the per promoter, likely by recruiting repressive and pausing factors. By disentangling its catalytic versus non-catalytic functions at the promoters of CLK target genes, we uncovered a multi-leveled mechanism in which BRM fine-tunes circadian transcription. PMID:26132408

  15. The Non-Catalytic Domains of Drosophila Katanin Regulate Its Abundance and Microtubule-Disassembly Activity

    PubMed Central

    Grode, Kyle D.; Rogers, Stephen L.

    2015-01-01

    Microtubule severing is a biochemical reaction that generates an internal break in a microtubule and regulation of microtubule severing is critical for cellular processes such as ciliogenesis, morphogenesis, and meiosis and mitosis. Katanin is a conserved heterodimeric ATPase that severs and disassembles microtubules, but the molecular determinants for regulation of microtubule severing by katanin remain poorly defined. Here we show that the non-catalytic domains of Drosophila katanin regulate its abundance and activity in living cells. Our data indicate that the microtubule-interacting and trafficking (MIT) domain and adjacent linker region of the Drosophila katanin catalytic subunit Kat60 cooperate to regulate microtubule severing in two distinct ways. First, the MIT domain and linker region of Kat60 decrease its abundance by enhancing its proteasome-dependent degradation. The Drosophila katanin regulatory subunit Kat80, which is required to stabilize Kat60 in cells, conversely reduces the proteasome-dependent degradation of Kat60. Second, the MIT domain and linker region of Kat60 augment its microtubule-disassembly activity by enhancing its association with microtubules. On the basis of our data, we propose that the non-catalytic domains of Drosophila katanin serve as the principal sites of integration of regulatory inputs, thereby controlling its ability to sever and disassemble microtubules. PMID:25886649

  16. Non-Catalytic Site HIV-1 Integrase Inhibitors Disrupt Core Maturation and Induce a Reverse Transcription Block in Target Cells

    PubMed Central

    Tsai, Luong; O’Sullivan, Christopher; Bam, Rujuta A.; Tsai, Angela; Niedziela-Majka, Anita; Stray, Kirsten M.; Sakowicz, Roman; Cihlar, Tomas

    2013-01-01

    HIV-1 integrase (IN) is the target for two classes of antiretrovirals: i) the integrase strand-transfer inhibitors (INSTIs) and ii) the non-catalytic site integrase inhibitors (NCINIs). NCINIs bind at the IN dimer interface and are thought to interfere primarily with viral DNA (vDNA) integration in the target cell by blocking IN-vDNA assembly as well as the IN-LEDGF/p75 interaction. Herein we show that treatment of virus-producing cells, but not of mature virions or target cells, drives NCINI antiviral potency. NCINIs target an essential late-stage event in HIV replication that is insensitive to LEDGF levels in the producer cells. Virus particles produced in the presence of NCINIs displayed normal Gag-Pol processing and endogenous reverse transcriptase activity, but were defective at initiating vDNA synthesis following entry into the target cell. NCINI-resistant virus carrying a T174I mutation in the IN dimer interface was less sensitive to the compound-induced late-stage effects, including the reverse transcription block. Wild-type, but not T174I virus, produced in the presence of NCINIs exhibited striking defects in core morphology and an increased level of IN oligomers that was not observed upon treatment of mature cell-free particles. Collectively, these results reveal that NCINIs act through a novel mechanism that is unrelated to the previously observed inhibition of IN activity or IN-LEDGF interaction, and instead involves the disruption of an IN function during HIV-1 core maturation and assembly. PMID:24040198

  17. Non-catalytic site HIV-1 integrase inhibitors disrupt core maturation and induce a reverse transcription block in target cells.

    PubMed

    Balakrishnan, Mini; Yant, Stephen R; Tsai, Luong; O'Sullivan, Christopher; Bam, Rujuta A; Tsai, Angela; Niedziela-Majka, Anita; Stray, Kirsten M; Sakowicz, Roman; Cihlar, Tomas

    2013-01-01

    HIV-1 integrase (IN) is the target for two classes of antiretrovirals: i) the integrase strand-transfer inhibitors (INSTIs) and ii) the non-catalytic site integrase inhibitors (NCINIs). NCINIs bind at the IN dimer interface and are thought to interfere primarily with viral DNA (vDNA) integration in the target cell by blocking IN-vDNA assembly as well as the IN-LEDGF/p75 interaction. Herein we show that treatment of virus-producing cells, but not of mature virions or target cells, drives NCINI antiviral potency. NCINIs target an essential late-stage event in HIV replication that is insensitive to LEDGF levels in the producer cells. Virus particles produced in the presence of NCINIs displayed normal Gag-Pol processing and endogenous reverse transcriptase activity, but were defective at initiating vDNA synthesis following entry into the target cell. NCINI-resistant virus carrying a T174I mutation in the IN dimer interface was less sensitive to the compound-induced late-stage effects, including the reverse transcription block. Wild-type, but not T174I virus, produced in the presence of NCINIs exhibited striking defects in core morphology and an increased level of IN oligomers that was not observed upon treatment of mature cell-free particles. Collectively, these results reveal that NCINIs act through a novel mechanism that is unrelated to the previously observed inhibition of IN activity or IN-LEDGF interaction, and instead involves the disruption of an IN function during HIV-1 core maturation and assembly. PMID:24040198

  18. Dimethyl carbonate as potential reactant in non-catalytic biodiesel production by supercritical method.

    PubMed

    Ilham, Zul; Saka, Shiro

    2009-03-01

    In this study, the non-catalytic supercritical method has been studied in utilizing dimethyl carbonate. It was demonstrated that, the supercritical dimethyl carbonate process without any catalysts applied, converted triglycerides to fatty acid methyl esters with glycerol carbonate and citramalic acid as by-products, while free fatty acids were converted to fatty acid methyl esters with glyoxal. After 12 min of reaction at 350 degrees C/20 MPa, rapeseed oil treated with supercritical dimethyl carbonate reached 94% (w/w) yield of fatty acid methyl ester. The by-products from this process which are glycerol carbonate and citramalic acid are much higher in value than glycerol produced by the conventional process. In addition, the yield of the fatty acid methyl esters as biodiesel was almost at par with supercritical methanol method. Therefore, supercritical dimethyl carbonate process can be a good candidate as an alternative biodiesel production process. PMID:18990561

  19. SWI/SNF-mutant cancers depend on catalytic and non-catalytic activity of EZH2.

    PubMed

    Kim, Kimberly H; Kim, Woojin; Howard, Thomas P; Vazquez, Francisca; Tsherniak, Aviad; Wu, Jennifer N; Wang, Weishan; Haswell, Jeffrey R; Walensky, Loren D; Hahn, William C; Orkin, Stuart H; Roberts, Charles W M

    2015-12-01

    Human cancer genome sequencing has recently revealed that genes that encode subunits of SWI/SNF chromatin remodeling complexes are frequently mutated across a wide variety of cancers, and several subunits of the complex have been shown to have bona fide tumor suppressor activity. However, whether mutations in SWI/SNF subunits result in shared dependencies is unknown. Here we show that EZH2, a catalytic subunit of the polycomb repressive complex 2 (PRC2), is essential in all tested cancer cell lines and xenografts harboring mutations of the SWI/SNF subunits ARID1A, PBRM1, and SMARCA4, which are several of the most frequently mutated SWI/SNF subunits in human cancer, but that co-occurrence of a Ras pathway mutation is correlated with abrogation of this dependence. Notably, we demonstrate that SWI/SNF-mutant cancer cells are primarily dependent on a non-catalytic role of EZH2 in the stabilization of the PRC2 complex, and that they are only partially dependent on EZH2 histone methyltransferase activity. These results not only reveal a shared dependency of cancers with genetic alterations in SWI/SNF subunits, but also suggest that EZH2 enzymatic inhibitors now in clinical development may not fully suppress the oncogenic activity of EZH2. PMID:26552009

  20. A non-catalytic histidine residue influences the function of the metalloprotease of Listeria monocytogenes.

    PubMed

    Forster, Brian M; Bitar, Alan Pavinski; Marquis, Hélène

    2014-01-01

    Mpl, a thermolysin-like metalloprotease, and PC-PLC, a phospholipase C, are synthesized as proenzymes by the intracellular bacterial pathogen Listeria monocytogenes. During intracellular growth, L. monocytogenes is temporarily confined in a membrane-bound vacuole whose acidification leads to Mpl autolysis and Mpl-mediated cleavage of the PC-PLC N-terminal propeptide. Mpl maturation also leads to the secretion of both Mpl and PC-PLC across the bacterial cell wall. Previously, we identified negatively charged and uncharged amino acid residues within the N terminus of the PC-PLC propeptide that influence the ability of Mpl to mediate the maturation of PC-PLC, suggesting that these residues promote the interaction of the PC-PLC propeptide with Mpl. In the present study, we identified a non-catalytic histidine residue (H226) that influences Mpl secretion across the cell wall and its ability to process PC-PLC. Our results suggest that a positive charge at position 226 is required for Mpl functions other than autolysis. Based on the charge requirement at this position, we hypothesize that this residue contributes to the interaction of Mpl with the PC-PLC propeptide. PMID:24140648

  1. Kinetics of Non-Catalytic Esterification of Free Fatty Acids Present in Jatropha Oil.

    PubMed

    Prasanna Rani, Karna Narayana; Ramana Neeharika, Tulasi Sri Venkata; Kumar, Thella Prathap; Satyavathi, Bankupalli; Sailu, Chintha

    2016-05-01

    Non-catalytic esterfication of free fatty acids (FFA) present in vegetable oils is an alternative pretreatment process for the biodiesel production. Biodiesel, consists of long-chain fatty acid methyl esters (FAME) and is obtained from renewable sources such as vegetable oils or animal fat. This study presents kinetics of thermal esterification of free fatty acids present in jatropha oil with methanol. The effect of process parameters like reaction time (1-5 h), temperature (170-190°C) and oil to methanol ratio (1:3-1:5) at constant pressure was investigated. The optimal conditions were found to be oil to methanol ratio of 1:4, 190°C, at 27.1 bar and 5 h which gave a maximum conversion of 95.1%. A second order kinetic model for both forward and backward reactions was proposed to study the reaction system. A good agreement was observed between the experimental data and the model values. The activation energy for forward reaction and the heat of reaction were found to be 36.364 Kcal/mol and 1.74 Kcal/mol respectively. PMID:27086997

  2. Non-catalytic transfer hydrogenation in supercritical CO2 for coal liquefaction

    NASA Astrophysics Data System (ADS)

    Elhussien, Hussien

    This thesis presents the results of the investigation on developing and evaluating a low temperature (<150°C) non - catalytic process using a hydrogen transfer agent (instead of molecu-lar hydrogen) for coal dissolution in supercritical CO2. The main idea behind the thesis was that one hydrogen atom from water and one hydrogen atom from the hydrogen transfer agent (HTA) were used to hydrogenate the coal. The products of coal dissolution were non-polar and polar while the supercritical CO2, which enhanced the rates of hydrogenation and dissolution of the non-polar molecules and removal from the reaction site, was non-polar. The polar modifier (PM) for CO2 was added to the freed to aid in the dissolution and removal of the polar components. The addition of a phase transfer agent (PTA) allowed a seamless transport of the ions and by-product between the aqueous and organic phases. DDAB, used as the PTA, is an effective phase transfer catalyst and showed enhancement to the coal dissolution process. COAL + DH- +H 2O → COAL.H2 + DHO-- This process has a great feature due to the fact that the chemicals were obtained without requir-ing to first convert coal to CO and H2 units as in indirect coal liquefaction. The experiments were conducted in a unique reactor set up that can be connected through two lines. one line to feed the reactor with supercritical CO 2 and the other connected to gas chromatograph. The use of the supercritical CO2 enhanced the solvent option due to the chemical extraction, in addition to the low environmental impact and energy cost. In this thesis the experiment were conducted at five different temperatures from atmos-pheric to 140°C, 3000 - 6000 psi with five component of feed mixture, namely water, HTA, PTA, coal, and PM in semi batch vessels reactor system with a volume of 100 mL. The results show that the chemicals were obtained without requiring to first convert coal to CO and H2 units as in indirect coal liquefaction. The results show that

  3. Non-catalytic alcoholysis process for production of biodiesel fuel by using bubble column reactor

    NASA Astrophysics Data System (ADS)

    Hagiwara, S.; Nabetani, H.; Nakajima, M.

    2015-04-01

    -edible lipids by use of the SMV reactor has not been examined yet. Therefore, this study aims to investigate the productivity of biodiesel produced from waste vegetable oils using the SMV reactor. Biodiesel fuel is a replacement for diesel as a fuel produced from biomass resources. It is generally produced as a FAME derived from vegetable oil by using alkaline catalyzed alcoholysis process. This alkaline method requires deacidification process prior to the reaction process and the alkaline catalyst removal process after the reaction. Those process increases the total cost of biodiesel fuel production. In order to solve the problems in the conventional alkaline catalyzed alcoholysis process, the authors proposed a non-catalytic alcoholysis process called the Superheated Methanol Vapor (SMV) method with bubble column reactor. So, this study aims to investigate the productivity of biodiesel produced from vegetable oils and other lipids using the SMV method with bubble column reactor.

  4. Deletion of a non-catalytic region increases the enzymatic activity of a β-agarase from Flammeovirga sp. MY04

    NASA Astrophysics Data System (ADS)

    Han, Wenjun; Gu, Jingyan; Liu, Huihui; Li, Fuchuan; Wu, Zhihong; Li, Yuezhong

    2015-10-01

    A Glycoside hydrolase (GH) typically contains one catalytic module and varied non-catalytic regions (NCRs). However, effects of the NCRs to the catalytic modules remain mostly unclear except the carbohydrate-binding modules (CBMs). AgaG4 is a GH16 endo- β-agarase of the agarolytic marine bacterium Flammeovirga sp. MY04. The enzyme consists of an extra sugar-binding peptide within the catalytic module, with no predictable CBMs but function-unknown sequences in the NCR, which is a new characteristic of agarase sequences. In this study, we deleted the NCR sequence, a 140-amino acid peptide at the C-terminus and expressed the truncated gene, agaG4-T140, in Escherichia coli. After purification and refolding, the truncated agarase rAgaG4-T140 retained the same catalytic temperature and pH value as rAgaG4. Using combined fluorescent labeling, HPLC and MS/MS techniques, we identified the end-products of agarose degradation by rAgaG4-T140 as neoagarotetraose and neoagarohexaose, with a final molar ratio of 1.53:1 and a conversion ratio of approximately 70%, which were similar to those of rAgaG4. However, the truncated agarase rAgaG4-T140 markedly decreased in protein solubility by 15 times and increased in enzymatic activities by 35 times. The oligosaccharide production of rAgaG4-T140 was approximately 25 times the weight of that produced by equimolar rAgaG4. This study provides some insights into the influences of NCR on the biochemical characteristics of agarase AgaG4 and implies some new strategies to improve the properties of a GH enzyme.

  5. Biodiesel production with continuous supercritical process: non-catalytic transesterification and esterification with or without carbon dioxide.

    PubMed

    Tsai, Yu-Ting; Lin, Ho-mu; Lee, Ming-Jer

    2013-10-01

    The non-catalytic transesterification of refined sunflower oil with supercritical methanol, in the presence of carbon dioxide, was conducted in a tubular reactor at temperatures from 553.2 to 593.2K and pressures up to 25.0 MPa. The FAME yield can be achieved up to about 0.70 at 593.2 K and 10.0 MPa in 23 min with methanol:oil of 25:1 in molar ratio. The effect of adding CO2 on the FAME yield is insignificant. The kinetic behavior of the non-catalytic esterification and transesterification of oleic acid or waste cooking oil (WCO) with supercritical methanol was also investigated. By using the supercritical process, the presence of free fatty acid (FFA) in WCO gives positive contribution to FAME production. The FAME yield of 0.90 from WCO can be achieved in 13 min at 573.2K. The kinetic data of supercritical transesterification and esterifaication were correlated well with a power-law model. PMID:23339904

  6. Hypoxia-induced carbonic anhydrase IX facilitates lactate flux in human breast cancer cells by non-catalytic function

    PubMed Central

    Jamali, Somayeh; Klier, Michael; Ames, Samantha; Felipe Barros, L.; McKenna, Robert; Deitmer, Joachim W.; Becker, Holger M.

    2015-01-01

    The most aggressive tumour cells, which often reside in hypoxic environments, rely on glycolysis for energy production. Thereby they release vast amounts of lactate and protons via monocarboxylate transporters (MCTs), which exacerbates extracellular acidification and supports the formation of a hostile environment. We have studied the mechanisms of regulated lactate transport in MCF-7 human breast cancer cells. Under hypoxia, expression of MCT1 and MCT4 remained unchanged, while expression of carbonic anhydrase IX (CAIX) was greatly enhanced. Our results show that CAIX augments MCT1 transport activity by a non-catalytic interaction. Mutation studies in Xenopus oocytes indicate that CAIX, via its intramolecular H+-shuttle His200, functions as a “proton-collecting/distributing antenna” to facilitate rapid lactate flux via MCT1. Knockdown of CAIX significantly reduced proliferation of cancer cells, suggesting that rapid efflux of lactate and H+, as enhanced by CAIX, contributes to cancer cell survival under hypoxic conditions. PMID:26337752

  7. Targeting the GPIbα Binding Site of Thrombin To Simultaneously Induce Dual Anticoagulant and Antiplatelet Effects

    PubMed Central

    2015-01-01

    Exosite 2 of human thrombin contributes to two opposing pathways, the anticoagulant pathway and the platelet aggregation pathway. We reasoned that an exosite 2 directed allosteric thrombin inhibitor should simultaneously induce anticoagulant and antiplatelet effects. To assess this, we synthesized SbO4L based on the sulfated tyrosine-containing sequence of GPIbα. SbO4L was synthesized in three simple steps in high yield and found to be a highly selective, direct inhibitor of thrombin. Michelis–Menten kinetic studies indicated a noncompetitive mechanism of inhibition. Competitive inhibition studies suggested ideal competition with heparin and glycoprotein Ibα, as predicted. Studies with site-directed mutants of thrombin indicated that SbO4L binds to Arg233, Lys235, and Lys236 of exosite 2. SbO4L prevented thrombin-mediated platelet activation and aggregation as expected on the basis of competition with GPIbα. SbO4L presents a novel paradigm of simultaneous dual anticoagulant and antiplatelet effects achieved through the GPIbα binding site of thrombin. PMID:24635452

  8. Catalytic and non-catalytic roles of DNA polymerase κ in the protection of human cells against genotoxic stresses.

    PubMed

    Kanemaru, Yuki; Suzuki, Tetsuya; Niimi, Naoko; Grúz, Petr; Matsumoto, Kyomu; Adachi, Noritaka; Honma, Masamitsu; Nohmi, Takehiko

    2015-10-01

    DNA polymerase κ (Pol κ) is a specialized DNA polymerase involved in translesion DNA synthesis. Although its bypass activities across lesions are well characterized in biochemistry, its cellular protective roles against genotoxic insults are still elusive. To better understand the in vivo protective roles, we have established a human cell line deficient in the expression of Pol κ (KO) and another expressing catalytically dead Pol κ (CD), to examine the cytotoxic sensitivity to 11 genotoxins including ultraviolet C light (UV). These cell lines were established in a genetic background of Nalm-6-MSH+, a human lymphoblastic cell line that has high efficiency for gene targeting, and functional p53 and mismatch repair activities. We classified the genotoxins into four groups. Group 1 includes benzo[a]pyrene diolepoxide, mitomycin C, and bleomycin, where the sensitivity was equally higher in KO and CD than in the cell line expressing wild-type Pol κ (WT). Group 2 includes hydrogen peroxide and menadione, where hypersensitivity was observed only in KO. Group 3 includes methyl methanesulfonate and ethyl methanesulfonate, where hypersensitivity was observed only in CD. Group 4 includes UV and three chemicals, where the chemicals exhibited similar cytotoxicity to all three cell lines. The results suggest that Pol κ not only protects cells from genotoxic DNA lesions via DNA polymerase activities, but also contributes to genome integrity by acting as a non-catalytic protein against oxidative damage caused by hydrogen peroxide and menadione. The non-catalytic roles of Pol κ in protection against oxidative damage by hydrogen peroxide are discussed. PMID:26031400

  9. Selection and evolution of enzymes from a partially randomized non-catalytic scaffold.

    PubMed

    Seelig, Burckhard; Szostak, Jack W

    2007-08-16

    Enzymes are exceptional catalysts that facilitate a wide variety of reactions under mild conditions, achieving high rate-enhancements with excellent chemo-, regio- and stereoselectivities. There is considerable interest in developing new enzymes for the synthesis of chemicals and pharmaceuticals and as tools for molecular biology. Methods have been developed for modifying and improving existing enzymes through screening, selection and directed evolution. However, the design and evolution of truly novel enzymes has relied on extensive knowledge of the mechanism of the reaction. Here we show that genuinely new enzymatic activities can be created de novo without the need for prior mechanistic information by selection from a naive protein library of very high diversity, with product formation as the sole selection criterion. We used messenger RNA display, in which proteins are covalently linked to their encoding mRNA, to select for functional proteins from an in vitro translated protein library of >10(12 )independent sequences without the constraints imposed by any in vivo step. This technique has been used to evolve new peptides and proteins that can bind a specific ligand, from both random-sequence libraries and libraries based on a known protein fold. We now describe the isolation of novel RNA ligases from a library that is based on a zinc finger scaffold, followed by in vitro directed evolution to further optimize these enzymes. The resulting ligases exhibit multiple turnover with rate enhancements of more than two-million-fold. PMID:17700701

  10. Catalytic and non-catalytic pyrolysis of biomass in non-inert environments for production of deoxygenated bio-oil and chemicals

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fast pyrolysis processes are among the most effective methods for liquefaction of lignocellulosic biomass. Catalytic fast pyrolysis (CFP) over HZSM-5 or other zeolites and/or utilization of reactive atmospheres such as in the non-catalytic Tail Gas Reactive Pyrolysis (TRGP) process, a recent patent...

  11. Evidence for beta-lactoglobulin involvement in vitamin D transport in vivo--role of the gamma-turn (Leu-Pro-Met) of beta-lactoglobulin in vitamin D binding.

    PubMed

    Yang, Ming Chi; Chen, Nai Chi; Chen, Chun-Jung; Wu, Chin Yun; Mao, Simon J T

    2009-04-01

    Beta-lactoglobulin (LG) is a major bovine milk protein, containing a central calyx and a second exosite beyond the calyx to bind vitamin D; however, the biological function of LG in transporting vitamin D remains elusive. Crystallographic findings from our previous study showed the exosite to be located at the pocket between the alpha-helix and beta-strand I. In the present study, using site-directed mutagenesis, we demonstrate that residues Leu143, Pro144 and Met145 in the gamma-turn loop play a crucial role in the binding. Further evidence is provided by the ability of vitamin D(3) to block the binding of a specific mAb in the gamma-turn loop. Using the mouse (n = 95) as an animal model, we initially demonstrated that LG is a major fraction of milk proteins responsible for uptake of vitamin D. Most interestingly, dosing mice with LG supplemented with vitamin D(3) revealed that native LG containing two binding sites gave a saturated concentration of plasma 25-hydroxyvitamin D at a dose ratio of 2 : 1 (vitamin D(3)/LG), whereas heated LG containing one exosite (lacking a central calyx) gave a ratio of 1 : 1. We have demonstrated for the first time that LG has a functional advantage in the transport of vitamin D, indicating that supplementing milk with vitamin D effectively enhances its uptake. PMID:19298386

  12. Non-ionic Surfactants and Non-Catalytic Protein Treatment on Enzymatic Hydrolysis of Pretreated Creeping Wild Ryegrass

    NASA Astrophysics Data System (ADS)

    Zheng, Yi; Pan, Zhongli; Zhang, Ruihong; Wang, Donghai; Jenkins, Bryan

    Our previous research has shown that saline Creeping Wild Ryegrass (CWR), Leymus triticoides, has a great potential to be used for bioethanol production because of its high fermentable sugar yield, up to 85% cellulose conversion of pretreated CWR. However, the high cost of enzyme is still one of the obstacles making large-scale lignocellulosic bioethanol production economically difficult. It is desirable to use reduced enzyme loading to produce fermentable sugars with high yield and low cost. To reduce the enzyme loading, the effect of addition of non-ionic surfactants and non-catalytic protein on the enzymatic hydrolysis of pretreated CWR was investigated in this study. Tween 20, Tween 80, and bovine serum albumin (BSA) were used as additives to improve the enzymatic hydrolysis of dilute sulfuric-acid-pretreated CWR. Under the loading of 0.1 g additives/g dry solid, Tween 20 was the most effective additive, followed by Tween 80 and BSA. With the addition of Tween 20 mixed with cellulase loading of 15 FPU/g cellulose, the cellulose conversion increased 14% (from 75 to 89%), which was similar to that with cellulase loading of 30 FPU/g cellulose and without additive addition. The results of cellulase and BSA adsorption on the Avicel PH101, pretreated CWR, and lignaceous residue of pretreated CWR support the theory that the primary mechanism behind the additives is prevention of non-productive adsorption of enzymes on lignaceous material of pretreated CWR. The addition of additives could be a promising technology to improve the enzymatic hydrolysis by reducing the enzyme activity loss caused by non-productive adsorption.

  13. Boosting the value of biodiesel byproduct by the non-catalytic transesterification of dimethyl carbonate via a continuous flow system under ambient pressure.

    PubMed

    Kwon, Eilhann E; Yi, Haakrho; Jeon, Young Jae

    2014-10-01

    Transformation of coconut oil into biodiesel by using dimethyl carbonate (DMC) via a non-catalytic transesterification reaction under ambient pressure was investigated in this study. The non-catalytic transformation to biodiesel was achieved by means of a heterogeneous reaction between liquid triglycerides and gas phase DMC. The reaction was enhanced in the presence of porous material due to its intrinsic physical properties such as tortuosity and absorption/adsorption. The numerous pores in the material served as micro reaction chambers and ensured that there was enough contact time between the liquid triglycerides and the gaseous DMC, which enabled the completion of the transesterification. The highest fatty acid methyl esters (FAMEs) yield achieved was 98±0.5% within 1-2min at a temperature of 360-450°C under ambient pressure. The fast reaction rates made it possible to convert the lipid feedstock into biodiesel via a continuous flow system without the application of increased pressure. This suggested that the commonly used supercritical conditions could be avoided, resulting in huge cost benefits for biodiesel production. In addition, the high value of the byproduct from the transesterification of the lipid feedstock with DMC suggested that the production biodiesel using this method could be more economically competitive. Finally, the basic properties of biodiesel derived from the non-catalytic conversion of rapeseed oil with DMC were summarised. PMID:25065794

  14. An experimental study of catalytic and non-catalytic reaction in heat recirculating reactors and applications to power generation

    NASA Astrophysics Data System (ADS)

    Ahn, Jeongmin

    An experimental study of the performance of a Swiss roll heat exchanger and reactor was conducted, with emphasis on the extinction limits and comparison of results with and without Pt catalyst. At Re<40, the catalyst was required to sustain reaction; with the catalyst self-sustaining reaction could be obtained at Re less than 1. Both lean and rich extinction limits were extended with the catalyst, though rich limits were extended much further. At low Re, the lean extinction limit was rich of stoichiometric and rich limit had equivalence ratios 80 in some cases. Non-catalytic reaction generally occurred in a flameless mode near the center of the reactor. With or without catalyst, for sufficiently robust conditions, a visible flame would propagate out of the center, but this flame could only be re-centered with catalyst. Gas chromatography indicated that at low Re, CO and non-C3 H8 hydrocarbons did not form. For higher Re, catalytic limits were slightly broader but had much lower limit temperatures. At sufficiently high Re, catalytic and gas-phase limits merged. Experiments with titanium Swiss rolls have demonstrated reducing wall thermal conductivity and thickness leads to lower heat losses and therefore increases operating temperatures and extends flammability limits. By use of Pt catalysts, reaction of propane-air mixtures at temperatures 54°C was sustained. Such low temperatures suggest that polymers may be employed as a reactor material. A polyimide reactor was built and survived prolonged testing at temperatures up to 500°C. Polymer reactors may prove more practical for microscale devices due to their lower thermal conductivity and ease of manufacturing. Since the ultimate goal of current efforts is to develop combustion driven power generation devices at MEMS like scales, a thermally self-sustaining miniature power generation device was developed utilizing a single-chamber solid-oxide-fuel-cell (SOFC) placed in a Swiss roll. With the single-chamber design

  15. Recombinant expression of the precursor of the hemorrhagic metalloproteinase HF3 and its non-catalytic domains using a cell-free synthesis system.

    PubMed

    Menezes, Milene C; Imbert, Lionel; Kitano, Eduardo S; Vernet, Thierry; Serrano, Solange M T

    2016-09-01

    Snake venom metalloproteinases (SVMPs) participate in snakebite pathology such as hemorrhage, inflammation, and necrosis. They are synthesized as latent multi-domain precursors whose processing generates either catalytically active enzymes or free non-enzymatic domains. Recombinant expression of the precursor of P-III class SVMPs has failed due to the instability of the multi-domain polypeptide structure. Conversely, functional recombinant non-catalytic domains were obtained by prokaryotic expression systems. Here, we show for the first time the recombinant expression of the precursor of HF3, a highly hemorrhagic SVMP from Bothrops jararaca, and its non-catalytic domains, using an E. coli-based cell-free synthesis system. The precursor of HF3, composed of pro-, metalloproteinase-, disintegrin-like-, and cysteine-rich domains, and containing 38 Cys residues, was successfully expressed and purified. A protein composed of the disintegrin-like and cysteine-rich domains (DC protein) and the cysteine-rich domain alone (C protein) were expressed in vitro individually and purified. Both proteins were shown to be functional in assays monitoring the interaction with matrix proteins and in modulating the cleavage of fibrinogen by HF3. These data indicate that recombinant expression using prokaryotic-based cell-free synthesis emerges as an attractive alternative for the study of the structure and function of multi-domain proteins with a high content of Cys residues. PMID:27209197

  16. Non-catalytic Roles for XPG with BRCA1 and BRCA2 in Homologous Recombination and Genome Stability.

    PubMed

    Trego, Kelly S; Groesser, Torsten; Davalos, Albert R; Parplys, Ann C; Zhao, Weixing; Nelson, Michael R; Hlaing, Ayesu; Shih, Brian; Rydberg, Björn; Pluth, Janice M; Tsai, Miaw-Sheue; Hoeijmakers, Jan H J; Sung, Patrick; Wiese, Claudia; Campisi, Judith; Cooper, Priscilla K

    2016-02-18

    XPG is a structure-specific endonuclease required for nucleotide excision repair, and incision-defective XPG mutations cause the skin cancer-prone syndrome xeroderma pigmentosum. Truncating mutations instead cause the neurodevelopmental progeroid disorder Cockayne syndrome, but little is known about how XPG loss results in this devastating disease. We identify XPG as a partner of BRCA1 and BRCA2 in maintaining genomic stability through homologous recombination (HRR). XPG depletion causes DNA double-strand breaks, chromosomal abnormalities, cell-cycle delays, defective HRR, inability to overcome replication fork stalling, and replication stress. XPG directly interacts with BRCA2, RAD51, and PALB2, and XPG depletion reduces their chromatin binding and subsequent RAD51 foci formation. Upstream in HRR, XPG interacts directly with BRCA1. Its depletion causes BRCA1 hyper-phosphorylation and persistent chromatin binding. These unexpected findings establish XPG as an HRR protein with important roles in genome stability and suggest how XPG defects produce severe clinical consequences including cancer and accelerated aging. PMID:26833090

  17. Behavior of chars from Bursa Mustafa Kemal Pasa Alpagut and Balkesir Dursunbey Cakiirca Lignite (Turkey) during non-catalytic and catalytic gasification

    SciTech Connect

    Bozkurt, Y.; Misirlioglu, Z.; Sinag, A.; Tekes, A.T.; Canel, M.

    2008-07-01

    The reactivities of chars obtained by pyrolysis of Bursa Mustafa Kemal Pasa Alpagut lignite and Balkesir Dursunbey Cakiirca lignite (Turkey) at different temperatures were determined by CO{sub 2} gasification and by combustion with O{sub 2}. Catalytic effect of Na{sub 2}CO{sub 3} on the CO{sub 2} and O{sub 2} gasification reactivity of chars was investigated. Gasification tests were performed in the fixed bed reactors operating at ambient pressure. Reactivity of chars during the CO{sub 2} gasification reactions was determined by calculating the reaction rate constants and reactivity of chars during the O{sub 2} gasification was determined by using ignition temperatures of the samples. Activation energies and Arrhenius constants of the chars on the CO{sub 2} gasification reactions were also calculated by the help of Arrhenius curves. The activation energy for CO{sub 2} gasification was generally decreased with pyrolysis temperature, due to the different surface characteristics and different nature of carbon atoms gasified as the gasification reactions proceed. Generally, the increase in pyrolysis temperature leads to an increase in gasification reactivity with CO{sub 2}. The reactivity of chars in catalytic gasification was higher than the corresponding non-catalytic reactivity of the same chars. Ignition temperature increased with increasing pyrolysis temperature.

  18. Effects of gas compositions on NOx reduction by selective non-catalytic reduction with ammonia in a simulated cement precalciner atmosphere.

    PubMed

    Fan, Weiyi; Zhu, Tianle; Sun, Yifei; Lv, Dong

    2014-10-01

    The effects of gas compositions on NOx reduction and NH3 slip by selective non-catalytic reduction (SNCR) with NH3 were investigated in a simulated cement precalciner atmosphere. The results show that the presence of H2O improves NOx reduction and widens the reduction temperature window significantly. O2 is indispensable for reducing NOx. The optimum reduction temperature decreases and the temperature window widens to a lower temperature with the increase of O2 content. In addition, the increase of O2 content also results in a decrease of the maximum NOx reduction efficiency. The effect of SO2 on NOx reduction is negligible in the simulated precalciner atmosphere. To increase CO concentration makes NO reduction take place at relatively low temperatures. However, NH3 will tend to be oxidized into NO instead of reducing NO after entering the stream containing O2 at high temperatures if it is initially blended with a high concentration of CO in an oxygen-free environment. The increase of H2O, O2, SO2 or CO concentration is helpful to reduce NH3 slip in the temperature region below 900°C. These effects are resulted from the fact that the generation and consumption of O and OH radicals which are crucial to NO reduction and formation can be influenced by the four gas compositions. In industrial operation of SNCR for cement precalciner, these effects should be taken into account to increase NOx reduction efficiency and avoid NH3 slip. PMID:25065808

  19. Effect of defects in oxide templates on Non-catalytic growth of GaN nanowires for high-efficiency light-emitting diodes

    NASA Astrophysics Data System (ADS)

    Hwang, Sung Won; Choi, Suk-Ho

    2016-04-01

    Two kinds of oxide templates, one with and one without undercuts, are employed to study the effect of defects in oxide templates on non-catalytic growth of GaN nanowires (NWs). GaN NWs abnormally grown from the templates containing undercuts exhibit two types of patterns: earlystage growth of premature NWs and abnormally-overgrown (~2 μm) NWs. GaN NWs grown on perfectly-symmetric template patterns are highly crystalline and have high aspect ratios (2 ~ 5), and their tops are shaped as pyramids with semipolar facets, clearly indicating hexagonal symmetry. The internal quantum efficiency of the well-grown NWs is 10% larger than that of the deformed NWs, as estimated by using photoluminescence. These results suggest that our technique is an effective approach for growing large-area-patterned, vertically-aligned, hexagonal GaN NWs without catalysts, in strong contrast to catalytic vapor-liquid-solid growth, and that good formation of the oxide templates is crucial for the growth of high-quality GaN NWs.

  20. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Protein-Protein Interaction Inhibitor Reveals a Non-catalytic Role for GAPDH Oligomerization in Cell Death.

    PubMed

    Qvit, Nir; Joshi, Amit U; Cunningham, Anna D; Ferreira, Julio C B; Mochly-Rosen, Daria

    2016-06-24

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an important glycolytic enzyme, has a non-catalytic (thus a non-canonical) role in inducing mitochondrial elimination under oxidative stress. We recently demonstrated that phosphorylation of GAPDH by δ protein kinase C (δPKC) inhibits this GAPDH-dependent mitochondrial elimination. δPKC phosphorylation of GAPDH correlates with increased cell injury following oxidative stress, suggesting that inhibiting GAPDH phosphorylation should decrease cell injury. Using rational design, we identified pseudo-GAPDH (ψGAPDH) peptide, an inhibitor of δPKC-mediated GAPDH phosphorylation that does not inhibit the phosphorylation of other δPKC substrates. Unexpectedly, ψGAPDH decreased mitochondrial elimination and increased cardiac damage in an animal model of heart attack. Either treatment with ψGAPDH or direct phosphorylation of GAPDH by δPKC decreased GAPDH tetramerization, which corresponded to reduced GAPDH glycolytic activity in vitro and ex vivo Taken together, our study identified the potential mechanism by which oxidative stress inhibits the protective GAPDH-mediated elimination of damaged mitochondria. Our study also identified a pharmacological tool, ψGAPDH peptide, with interesting properties. ψGAPDH peptide is an inhibitor of the interaction between δPKC and GAPDH and of the resulting phosphorylation of GAPDH by δPKC. ψGAPDH peptide is also an inhibitor of GAPDH oligomerization and thus an inhibitor of GAPDH glycolytic activity. Finally, we found that ψGAPDH peptide is an inhibitor of the elimination of damaged mitochondria. We discuss how this unique property of increasing cell damage following oxidative stress suggests a potential use for ψGAPDH peptide-based therapy. PMID:27129213

  1. Life cycle assessment of selective non-catalytic reduction (SNCR) of nitrous oxides in a full-scale municipal solid waste incinerator.

    PubMed

    Møller, Jacob; Munk, Bjarne; Crillesen, Kim; Christensen, Thomas H

    2011-06-01

    Selective non-catalytic reduction (SNCR) of nitrous oxides in a full-scale municipal solid waste incinerator was investigated using LCA. The relationship between NO(x)-cleaning and ammonia dosage was measured at the plant. Un-reacted ammonia - the ammonia slip - leaving the flue-gas cleaning system adsorbed to fly-ash or in the effluent of the acidic scrubber was quantified from the stoichiometric reaction of NO(x) and ammonia assuming no other reaction products was formed. Of the ammonia slip, 37% was associated with the fly-ash and 63% was in the effluent of the acidic scrubber. Based on NO(x)-cleaning efficiency, the fate of the ammonia slip as well as the environmental impact from ammonia production, the potential acidification and nutrient enrichment from NO(x)-cleaning was calculated as a function of ammonia dosage. Since the exact fate of the ammonia slip could not be measured directly, a number of scenarios were set up ranging from "best case" with no ammonia from the slip ending up in the environment to "worst case" where all the ammonia slip eventually ended up in the environment and contributed to environmental pollution. In the "best case" scenario the highest ammonia dosage was most beneficial demonstrating that the environmental load associated with ammonia production is of minor importance. In contrast, in a "worst case" scenario" NO(x)-cleaning using SNCR is not recommendable at all, since the impacts from the ammonia slip exceed the saved impacts from the NO(x) removal. Increased dosage of ammonia for removal of NO(x) is recommendable as long as less than 10-20% of the ammonia slip to the effluent of the acidic scrubber ends up in the environment and less than 40% of the slip to the fly-ash ends up in the environment. The study suggests that the actual fate of the ammonia slip is crucial, but since the release of the ammonia may take place during transport and at the facilities that treat the wastewater and treat the fly-ash this factor depends

  2. Integrating low-NO{sub x} burners, overfire air, and selective non-catalytic reduction on a utility coal-fired boiler

    SciTech Connect

    Hunt, T.; Muzio, L.; Smith, R.

    1995-05-01

    Public Service Company of Colorado (PSCo), in cooperation with the US Department of Energy (DOE) and the Electric Power Research Institute (EPRI), is testing the Integrated Dry NO{sub x}/SO{sub 2} Emissions Control system. This system combines low-NO{sub x} burners, overfire air, selective non-catalytic reduction (SNCR), and dry sorbent injection with humidification to reduce by up to 70% both NO{sub x} and SO{sub 2} emissions from a 100 MW coal-fired utility boiler. The project is being conducted at PSCo`s Arapahoe Unit 4 located in Denver, Colorado as part of the DOE`s Clean Coal Technology Round 3 program. The urea-based SNCR system, supplied by Noell, Inc., was installed in late 1991 and was tested with the unmodified boiler in 1992. At full load, it reduced NO{sub x} emissions by about 35% with an associated ammonia slip limit of 10 ppm. Babcock & Wilcox XLS{reg_sign} burners and a dual-zone overfire air system were retrofit to the top-fired boiler in mid-1992 and demonstrated a NO{sub x} reduction of nearly 70% across the load range. Integrated testing of the combustion modifications and the SNCR system were conducted in 1993 and showed that the SNCR system could reduce NO{sub x} emissions by an additional 45% while maintaining 10 ppm of ammonia slip limit at full load. Lower than expect4ed flue-gas temperatures caused low-load operation to be less effective than at high loads. NO{sub x} reduction decreased to as low as 11% at 60 MWe at an ammonia slip limit of 10 ppm. An ammonia conversion system was installed to improve performance at low loads. Other improvements to increase NO{sub x} removal at low-loads are planned. The combined system of combustion modifications and SNCR reduced NO{sub x} emissions by over 80% from the original full-load baseline. 11 figs.

  3. HYBRID SELECTIVE NON-CATALYTIC REDUCTION (SNCR)/SELECTIVE CATALYTIC REDUCTION (SCR) DEMONSTRATION FOR THE REMOVAL OF NOx FROM BOILER FLUE GASES

    SciTech Connect

    Jerry B. Urbas

    1999-05-01

    The U. S. Department of Energy (DOE), Electric Power Research Institute (EPRI), Pennsylvania Electric Energy Research Council, (PEERC), New York State Electric and Gas and GPU Generation, Inc. jointly funded a demonstration to determine the capabilities for Hybrid SNCR/SCR (Selective Non-Catalytic Reduction/Selective Catalytic Reduction) technology. The demonstration site was GPU Generation's Seward Unit No.5 (147MW) located in Seward Pennsylvania. The demonstration began in October of 1997 and ended in December 1998. DOE funding was provided through Grant No. DE-FG22-96PC96256 with T. J. Feeley as the Project Manager. EPRI funding was provided through agreements TC4599-001-26999 and TC4599-002-26999 with E. Hughes as the Project Manager. This project demonstrated the operation of the Hybrid SNCR/SCR NO{sub x} control process on a full-scale coal fired utility boiler. The hybrid technology was expected to provide a cost-effective method of reducing NO{sub x} while balancing capital and operation costs. An existing urea based SNCR system was modified with an expanded-duct catalyst to provide increased NO{sub x} reduction efficiency from the SNCR while producing increased ammonia slip levels to the catalyst. The catalyst was sized to reduce the ammonia slip to the air heaters to less than 2 ppm while providing equivalent NO{sub x} reductions. The project goals were to demonstrate hybrid technology is capable of achieving at least a 55% reduction in NO{sub x} emissions while maintaining less than 2ppm ammonia slip to the air heaters, maintain flyash marketability, verify the cost benefit and applicability of Hybrid post combustion technology, and reduce forced outages due to ammonium bisulfate (ABS) fouling of the air heaters. Early system limitations, due to gas temperature stratification, restricted the Hybrid NO{sub x} reduction capabilities to 48% with an ammonia slip of 6.1 mg/Nm{sup 3} (8 ppm) at the catalyst inlet. After resolving the stratification problem

  4. Recognition of xyloglucan by the crystalline cellulose-binding site of a family 3a carbohydrate-binding module.

    PubMed

    Hernandez-Gomez, Mercedes C; Rydahl, Maja G; Rogowski, Artur; Morland, Carl; Cartmell, Alan; Crouch, Lucy; Labourel, Aurore; Fontes, Carlos M G A; Willats, William G T; Gilbert, Harry J; Knox, J Paul

    2015-08-19

    Type A non-catalytic carbohydrate-binding modules (CBMs), exemplified by CtCBM3acipA, are widely believed to specifically target crystalline cellulose through entropic forces. Here we have tested the hypothesis that type A CBMs can also bind to xyloglucan (XG), a soluble β-1,4-glucan containing α-1,6-xylose side chains. CtCBM3acipA bound to xyloglucan in cell walls and arrayed on solid surfaces. Xyloglucan and cellulose were shown to bind to the same planar surface on CBM3acipA. A range of type A CBMs from different families were shown to bind to xyloglucan in solution with ligand binding driven by enthalpic changes. The nature of CBM-polysaccharide interactions is discussed. PMID:26193423

  5. Development of a specific affinity-matured exosite inhibitor to MT1-MMP that efficiently inhibits tumor cell invasion in vitro and metastasis in vivo

    PubMed Central

    Botkjaer, Kenneth A.; Kwok, Hang Fai; Terp, Mikkel G.; Karatt-Vellatt, Aneesh; Santamaria, Salvatore; McCafferty, John; Andreasen, Peter A.; Itoh, Yoshifumi; Ditzel, Henrik J.; Murphy, Gillian

    2016-01-01

    The membrane-associated matrix metalloproteinase-14, MT1-MMP, has been implicated in pericellular proteolysis with an important role in cellular invasion of collagenous tissues. It is substantially upregulated in various cancers and rheumatoid arthritis, and has been considered as a potential therapeutic target. Here, we report the identification of antibody fragments to MT1-MMP that potently and specifically inhibit its cell surface functions. Lead antibody clones displayed inhibitory activity towards pro-MMP-2 activation, collagen-film degradation and gelatin-film degradation, and were shown to bind to the MT1-MMP catalytic domain outside the active site cleft, inhibiting binding to triple helical collagen. Affinity maturation using CDR3 randomization created a second generation of antibody fragments with dissociation constants down to 0.11 nM, corresponding to an improved affinity of 332-fold with the ability to interfere with cell-surface MT1-MMP functions, displaying IC50 values down to 5 nM. Importantly, the new inhibitors were able to inhibit collagen invasion by tumor-cells in vitro and in vivo primary tumor growth and metastasis of MDA-MB-231 cells in a mouse orthotopic xenograft model. Herein is the first demonstration that an inhibitory antibody targeting sites outside the catalytic cleft of MT1-MMP can effectively abrogate its in vivo activity during tumorigenesis and metastasis. PMID:26934448

  6. Xylanase B from Neocallimastix patriciarum contains a non-catalytic 455-residue linker sequence comprised of 57 repeats of an octapeptide.

    PubMed Central

    Black, G W; Hazlewood, G P; Xue, G P; Orpin, C G; Gilbert, H J

    1994-01-01

    A Neocallimastix patriciarum cDNA library was screened for xylanase-expressing clones, which were distinct from the previously characterized N. patriciarum xynA cDNA encoding xylanase A. A single cDNA, designated xynB, which did not exhibit homology with xynA, was isolated. Northern-blot analysis of mRNA from Avicel-grown N. patriciarum showed that xynB hybridized to a 3.4 kb mRNA species. The nucleotide sequence of xynB revealed a single open reading frame of 2580 bp coding for a protein designated xylanase B (XYLB), of M(r) 88,066. The primary structure of XYLB was comprised of a 21-residue N-terminal signal peptide, followed by a 304-amino acid sequence that exhibited substantial homology with the catalytic domains of family F xylanases. The N-terminal domain was linked to a C-terminal 70-residue sequence by a putative linker region, comprising 12 tandem repeats of a sequence containing TLPG as the core sequence, followed by an octapeptide XSKTLPGG where X can be S, K or N, which was repeated in tandem 45 times. Truncated derivatives of xynB encoding the N-terminal 338 residues directed the synthesis of a functional xylanase, confirming that the region of XYLB, which exhibited homology with family F xylanases, constitutes the catalytic domain. To investigate the catalytic properties of XYLB, the catalytic domain was fused to the Escherichia coli maltose-binding protein, and the fusion protein purified by amylose affinity chromatography. The purified enzyme hydrolysed oat, rye and wheat arabinoxylan releasing primarily xylobiose, xylotriose and some xylose. The XYLB fusion did not cleave any cellulosic substrates. The data presented in this report suggest that the multiple xylanases of N. patriciarum arose, not through the duplication of a single gene, but by the transfer of distinct xylanase-encoding DNA sequences into the anaerobic fungus. The possible origin of the xynB gene is discussed. Images Figure 1 Figure 4 Figure 5 PMID:8172598

  7. Investigation of the selectivity of thrombin-binding aptamers for thrombin titration in murine plasma.

    PubMed

    Trapaidze, Ana; Hérault, Jean-Pascal; Herbert, Jean-Marc; Bancaud, Aurélien; Gué, Anne-Marie

    2016-04-15

    Detection of thrombin in plasma raises timely challenges to enable therapeutic management of thrombosis in patients under vital threat. Thrombin binding aptamers represent promising candidates as sensing elements for the development of real-time thrombin biosensors; however implementation of such biosensor requires the clear understanding of thrombin-aptamer interaction properties in real-like environment. In this study, we used Surface Plasmon Resonance technique to answer the questions of specificity and sensitivity of thrombin detection by the thrombin-binding aptamers HD1, NU172 and HD22. We systematically characterized their properties in the presence of thrombin, as well as interfering molecular species such as the thrombin precursor prothrombin, thrombin in complex with some of its natural inhibitors, nonspecific serum proteins, and diluted plasma. Kinetic experiments show the multiple binding modes of HD1 and NU172, which both interact with multiple sites of thrombin with low nanomolar affinities and show little specificity of interaction for prothrombin vs. thrombin. HD22, on the other hand, binds specifically to thrombin exosite II and has no affinity to prothrombin at all. While thrombin in complex with some of its inhibitors could not be recognized by any aptamer, the binding of HD1 and NU172 properties is compromised by thrombin inhibitors alone, as well as with serum albumin. Finally, the complex nature of plasma was overwhelming for HD1, but we define conditions for the thrombin detection at 10nM range in 100-fold diluted plasma by HD22. Consequently HD22 showed key advantage over HD1 and NU172, and appears as the only alternative to design an aptasensor. PMID:26594887

  8. Rapid Binding of Plasminogen to Streptokinase in a Catalytic Complex Reveals a Three-step Mechanism*

    PubMed Central

    Verhamme, Ingrid M.; Bock, Paul E.

    2014-01-01

    Rapid kinetics demonstrate a three-step pathway of streptokinase (SK) binding to plasminogen (Pg), the zymogen of plasmin (Pm). Formation of a fluorescently silent encounter complex is followed by two conformational tightening steps reported by fluorescence quenches. Forward reactions were defined by time courses of biphasic quenching during complex formation between SK or its COOH-terminal Lys414 deletion mutant (SKΔK414) and active site-labeled [Lys]Pg ([5-(acetamido)fluorescein]-d-Phe-Phe-Arg-[Lys]Pg ([5F]FFR-[Lys]Pg)) and by the SK dependences of the quench rates. Active site-blocked Pm rapidly displaced [5F]FFR-[Lys]Pg from the complex. The encounter and final SK·[5F]FFR-[Lys]Pg complexes were weakened similarly by SK Lys414 deletion and blocking of lysine-binding sites (LBSs) on Pg kringles with 6-aminohexanoic acid or benzamidine. Forward and reverse rates for both tightening steps were unaffected by 6-aminohexanoic acid, whereas benzamidine released constraints on the first conformational tightening. This indicated that binding of SK Lys414 to Pg kringle 4 plays a role in recognition of Pg by SK. The substantially lower affinity of the final SK·Pg complex compared with SK·Pm is characterized by a ∼25-fold weaker encounter complex and ∼40-fold faster off-rates for the second conformational step. The results suggest that effective Pg encounter requires SK Lys414 engagement and significant non-LBS interactions with the protease domain, whereas Pm binding additionally requires contributions of other lysines. This difference may be responsible for the lower affinity of the SK·Pg complex and the expression of a weaker “pro”-exosite for binding of a second Pg in the substrate mode compared with SK·Pm. PMID:25138220

  9. Rapid binding of plasminogen to streptokinase in a catalytic complex reveals a three-step mechanism.

    PubMed

    Verhamme, Ingrid M; Bock, Paul E

    2014-10-01

    Rapid kinetics demonstrate a three-step pathway of streptokinase (SK) binding to plasminogen (Pg), the zymogen of plasmin (Pm). Formation of a fluorescently silent encounter complex is followed by two conformational tightening steps reported by fluorescence quenches. Forward reactions were defined by time courses of biphasic quenching during complex formation between SK or its COOH-terminal Lys(414) deletion mutant (SKΔK414) and active site-labeled [Lys]Pg ([5-(acetamido)fluorescein]-D-Phe-Phe-Arg-[Lys]Pg ([5F]FFR-[Lys]Pg)) and by the SK dependences of the quench rates. Active site-blocked Pm rapidly displaced [5F]FFR-[Lys]Pg from the complex. The encounter and final SK ·[5F]FFR-[Lys]Pg complexes were weakened similarly by SK Lys(414) deletion and blocking of lysine-binding sites (LBSs) on Pg kringles with 6-aminohexanoic acid or benzamidine. Forward and reverse rates for both tightening steps were unaffected by 6-aminohexanoic acid, whereas benzamidine released constraints on the first conformational tightening. This indicated that binding of SK Lys(414) to Pg kringle 4 plays a role in recognition of Pg by SK. The substantially lower affinity of the final SK · Pg complex compared with SK · Pm is characterized by a ∼ 25-fold weaker encounter complex and ∼ 40-fold faster off-rates for the second conformational step. The results suggest that effective Pg encounter requires SK Lys(414) engagement and significant non-LBS interactions with the protease domain, whereas Pm binding additionally requires contributions of other lysines. This difference may be responsible for the lower affinity of the SK · Pg complex and the expression of a weaker "pro"-exosite for binding of a second Pg in the substrate mode compared with SK · Pm. PMID:25138220

  10. Family 42 carbohydrate-binding modules display multiple arabinoxylan-binding interfaces presenting different ligand affinities.

    PubMed

    Ribeiro, Teresa; Santos-Silva, Teresa; Alves, Victor D; Dias, Fernando M V; Luís, Ana S; Prates, José A M; Ferreira, Luís M A; Romão, Maria J; Fontes, Carlos M G A

    2010-10-01

    Enzymes that degrade plant cell wall polysaccharides display a modular architecture comprising a catalytic domain bound to one or more non-catalytic carbohydrate-binding modules (CBMs). CBMs display considerable variation in primary structure and are grouped into 59 sequence-based families organized in the Carbohydrate-Active enZYme (CAZy) database. Here we report the crystal structure of CtCBM42A together with the biochemical characterization of two other members of family 42 CBMs from Clostridium thermocellum. CtCBM42A, CtCBM42B and CtCBM42C bind specifically to the arabinose side-chains of arabinoxylans and arabinan, suggesting that various cellulosomal components are targeted to these regions of the plant cell wall. The structure of CtCBM42A displays a beta-trefoil fold, which comprises 3 sub-domains designated as alpha, beta and gamma. Each one of the three sub-domains presents a putative carbohydrate-binding pocket where an aspartate residue located in a central position dominates ligand recognition. Intriguingly, the gamma sub-domain of CtCBM42A is pivotal for arabinoxylan binding, while the concerted action of beta and gamma sub-domains of CtCBM42B and CtCBM42C is apparently required for ligand sequestration. Thus, this work reveals that the binding mechanism of CBM42 members is in contrast with that of homologous CBM13s where recognition of complex polysaccharides results from the cooperative action of three protein sub-domains presenting similar affinities. PMID:20637315

  11. Binding Procurement

    NASA Technical Reports Server (NTRS)

    Rao, Gopalakrishna M.; Vaidyanathan, Hari

    2007-01-01

    This viewgraph presentation reviews the use of the binding procurement process in purchasing Aerospace Flight Battery Systems. NASA Engineering and Safety Center (NESC) requested NASA Aerospace Flight Battery Systems Working Group to develop a set of guideline requirements document for Binding Procurement Contracts.

  12. Functional characterization of the Cdc42p binding domain of yeast Ste20p protein kinase.

    PubMed Central

    Leberer, E; Wu, C; Leeuw, T; Fourest-Lieuvin, A; Segall, J E; Thomas, D Y

    1997-01-01

    Ste20p from Saccharomyces cerevisiae belongs to the Ste20p/p65PAK family of protein kinases which are highly conserved from yeast to man and regulate conserved mitogen-activated protein kinase pathways. Ste20p fulfills multiple roles in pheromone signaling, morphological switching and vegetative growth and binds Cdc42p, a Rho-like small GTP binding protein required for polarized morphogenesis. We have analyzed the functional consequences of mutations that prevent binding of Cdc42p to Ste20p. The complete amino-terminal, non-catalytic half of Ste20p, including the conserved Cdc42p binding domain, was dispensable for heterotrimeric G-protein-mediated pheromone signaling. However, the Cdc42p binding domain was necessary for filamentous growth in response to nitrogen starvation and for an essential function that Ste20p shares with its isoform Cla4p during vegetative growth. Moreover, the Cdc42p binding domain was required for cell-cell adhesion during conjugation. Subcellular localization of wild-type and mutant Ste20p fused to green fluorescent protein showed that the Cdc42p binding domain is needed to direct localization of Ste20p to regions of polarized growth. These results suggest that Ste20p is regulated in different developmental pathways by different mechanisms which involve heterotrimeric and small GTP binding proteins. PMID:9009270

  13. Potent Glycosidase Inhibition with Heterovalent Fullerenes: Unveiling the Binding Modes Triggering Multivalent Inhibition.

    PubMed

    Abellán Flos, Marta; García Moreno, M Isabel; Ortiz Mellet, Carmen; García Fernández, Jose Manuel; Nierengarten, Jean-Francois; Vincent, Stéphane P

    2016-08-01

    Glycosidases are key enzymes in metabolism, pathogenic/antipathogenic mechanisms and normal cellular functions. Recently, a novel approach for glycosidase inhibition that conveys multivalent glycomimetic conjugates has emerged. Many questions regarding the mechanism(s) of multivalent enzyme inhibition remain unanswered. Herein we report the synthesis of a collection of novel homo- and heterovalent glyco(mimetic)-fullerenes purposely conceived for probing the contribution of non-catalytic pockets in glysosidases to the multivalent inhibitory effect. Their affinities towards selected glycosidases were compared with data from homovalent fullerene conjugates. An original competitive glycosidase-lectin binding assay demonstrated that the multivalent derivatives and the substrate compete for low affinity non-glycone binding sites of the enzyme, leading to inhibition by a "recognition and blockage" mechanism. Most notably, this work provides evidence for enzyme inhibition by multivalent glycosystems, which will likely have a strong impact in the glycosciences given the utmost relevance of multivalency in Nature. PMID:27374430

  14. A single residue mutation abolishes attachment of the CBM26 starch-binding domain from Lactobacillus amylovorus alpha-amylase.

    PubMed

    Rodríguez-Sanoja, Romina; Oviedo, N; Escalante, L; Ruiz, B; Sánchez, S

    2009-03-01

    Starch is degraded by amylases that frequently have a modular structure composed of a catalytic domain and at least one non-catalytic domain that is involved in polysaccharide binding. The C-terminal domain from the Lactobacillus amylovorus alpha-amylase has an unusual architecture composed of five tandem starch-binding domains (SBDs). These domains belong to family 26 in the carbohydrate-binding modules (CBM) classification. It has been reported that members of this family have only one site for starch binding, where aromatic amino acids perform the binding function. In SBDs, fold similarities are better conserved than sequences; nevertheless, it is possible to identify in CBM26 members at least two aromatic residues highly conserved. We attempt to explain polysaccharide recognition for the L. amylovorus alpha-amylase SBD through site-directed mutagenesis of aromatic amino acids. Three amino acids were identified as essential for binding, two tyrosines and one tryptophan. Y18L and Y20L mutations were found to decrease the SBD binding capacity, but unexpectedly, the mutation at W32L led to a total loss of affinity, either with linear or ramified substrates. The critical role of Trp 32 in substrate binding confirms the presence of just one binding site in each alpha-amylase SBD. PMID:19052787

  15. An autoantibody epitope comprising residues R660, Y661, and Y665 in the ADAMTS13 spacer domain identifies a binding site for the A2 domain of VWF

    PubMed Central

    Pos, Wouter; Crawley, James T. B.; Fijnheer, Rob; Voorberg, Jan; Lane, David A.

    2010-01-01

    In the majority of patients with acquired thrombotic thrombocytopenic purpura (TTP), antibodies are directed toward the spacer domain of ADAMTS13. We have previously shown that region Y658-Y665 is involved. We now show that replacement of R660, Y661, or Y665 with alanine in ADAMTS13 reduced/abolished the binding of 2 previously isolated human monoclonal antibodies and polyclonal antibodies derived from plasma of 6 patients with acquired TTP. We investigated whether these residues also influenced cleavage of short von Willebrand factor (VWF) fragment substrate VWF115. An ADAMTS13 variant (R660A/Y661A/Y665A, ADAMTS13-RYY) showed a 12-fold reduced catalytic efficiency (kcat/Km) arising from greatly reduced (> 25-fold) binding, demonstrated by surface plasmon resonance. The influence of these residue changes on full-length VWF was determined with denaturing and flow assays. ADAMTS13-RYY had reduced activity in both, with proteolysis of VWF unaffected by autoantibody. Binding of ADAMTS13-RYY mutant to VWF was, however, similar to normal. Our results demonstrate that residues within Y658-Y665 of the ADAMTS13 spacer domain that are targeted by autoantibodies in TTP directly interact with a complementary exosite (E1660-R1668) within the VWF A2 domain. Residues R660, Y661, and Y665 are critical for proteolysis of short VWF substrates, but wider domain interactions also make important contributions to cleavage of full-length VWF. PMID:20032502

  16. Carbohydrate-binding modules: fine-tuning polysaccharide recognition

    PubMed Central

    2004-01-01

    The enzymic degradation of insoluble polysaccharides is one of the most important reactions on earth. Despite this, glycoside hydrolases attack such polysaccharides relatively inefficiently as their target glycosidic bonds are often inaccessible to the active site of the appropriate enzymes. In order to overcome these problems, many of the glycoside hydrolases that utilize insoluble substrates are modular, comprising catalytic modules appended to one or more non-catalytic CBMs (carbohydrate-binding modules). CBMs promote the association of the enzyme with the substrate. In view of the central role that CBMs play in the enzymic hydrolysis of plant structural and storage polysaccharides, the ligand specificity displayed by these protein modules and the mechanism by which they recognize their target carbohydrates have received considerable attention since their discovery almost 20 years ago. In the last few years, CBM research has harnessed structural, functional and bioinformatic approaches to elucidate the molecular determinants that drive CBM–carbohydrate recognition. The present review summarizes the impact structural biology has had on our understanding of the mechanisms by which CBMs bind to their target ligands. PMID:15214846

  17. Spastin's microtubule-binding properties and comparison to katanin.

    PubMed

    Eckert, Thomas; Le, Doan Tuong-Van; Link, Susanne; Friedmann, Lena; Woehlke, Günther

    2012-01-01

    Spastin and katanin are ring-shaped hexameric AAA ATPases that sever microtubules, and thus crucially depend on a physical interaction with microtubules. For the first time, we report here the microtubule binding properties of spastin at the single-molecule level, and compare them to katanin. Microscopic fluorescence assays showed that human spastin bound to microtubules by ionic interactions, and diffused along microtubules with a diffusion coefficient comparable to katanin. The microscopic measurement of landing and dissociation rates demonstrated the ionic character of the interaction, which could be mapped to a patch of three lysine residues outside of the catalytic domain of human spastin. This motif is not conserved in Drosophila spastin or katanin, which also bound by non-catalytic parts of the protein. The binding affinities of spastin and katanin were nucleotide-sensitive, with the lowest affinities under ADP,, the highest under ATP-γS conditions. These changes correlated with the formation of higher oligomeric states, as shown in biochemical experiments and electron microscopic images. Vice versa, the artificial dimerization of human spastin by addition of a coiled coil led to a constitutively active enzyme. These observations suggest that dimer formation is a crucial step in the formation of the active complex, and thus the severing process by spastin. PMID:23272056

  18. Identification of a novel family of carbohydrate-binding modules with broad ligand specificity

    PubMed Central

    Duan, Cheng-Jie; Feng, Yu-Liang; Cao, Qi-Long; Huang, Ming-Yue; Feng, Jia-Xun

    2016-01-01

    Most enzymes that act on carbohydrates include non-catalytic carbohydrate-binding modules (CBMs) that recognize and target carbohydrates. CBMs bring their appended catalytic modules into close proximity with the target substrate and increase the hydrolytic rate of enzymes acting on insoluble substrates. We previously identified a novel CBM (CBMC5614-1) at the C-terminus of endoglucanase C5614-1 from an uncultured microorganism present in buffalo rumen. In the present study, that the functional region of CBMC5614-1 involved in ligand binding was localized to 134 amino acids. Two representative homologs of CBMC5614-1, sharing the same ligand binding profile, targeted a range of β-linked polysaccharides that adopt very different conformations. Targeted substrates included soluble and insoluble cellulose, β-1,3/1,4-mixed linked glucans, xylan, and mannan. Mutagenesis revealed that three conserved aromatic residues (Trp-380, Tyr-411, and Trp-423) play an important role in ligand recognition and targeting. These results suggest that CBMC5614-1 and its homologs form a novel CBM family (CBM72) with a broad ligand-binding specificity. CBM72 members can provide new insight into CBM-ligand interactions and may have potential in protein engineering and biocatalysis. PMID:26765840

  19. Interaction of the 268-282 region of glycoprotein Ibalpha with the heparin-binding site of thrombin inhibits the enzyme activation of factor VIII.

    PubMed Central

    De Cristofaro, R; De Filippis, V

    2003-01-01

    Activation of factor VIII (FVIII) by thrombin plays a fundamental role in the amplification of the coagulation cascade and takes place through specific proteolytic cleavages at Arg(372), Arg(740) and Arg(1689). Full FVIII activation requires cleavage at Arg(372), a process involving the alpha-thrombin exosite-II; referred to as heparin-binding site (HBS). The present study was aimed at investigating the effect of glycoprotein Ibalpha (GpIbalpha; 1-282 fragment) binding to thrombin HBS on FVIII activation. Similar experiments were also performed using a synthetic peptide modelled on the 268-282 sequence of GpIbalpha, and sulphated successfully at all tyrosine residues present along its sequence, at positions 276, 278 and 279. Both GpIbalpha 1-282 and the sulphated GpIb 268-282 peptides induced a progressive decrease (up to 70%) in activated FVIII generation, assessed by coagulation and FXa-generation assays. Furthermore, SDS/PAGE and Western-blot experiments showed that the specific appearance of the 44 kDa A2 domain on cleavage of the FVIII Arg(372)-Ser(373) peptide bond was delayed significantly in the presence of either GpIbalpha 1-282 or GpIb 268-282 peptide. Moreover, the effect of the latter on thrombin-mediated hydrolysis of a peptide having the sequence 341-376 of FVIII was investigated using reverse-phase HPLC. The k (cat)/ K (m) values of the FVIII 341-376 peptide hydrolysis by thrombin decreased linearly as a function of the GpIbalpha 268-282 peptide concentration, according to a competitive inhibition effect. Taken together, these experiments suggest that the sulphated 268-282 region of GpIbalpha binds to thrombin HBS, and is responsible for the inhibition of the Arg(372)-Ser(373) bond cleavage and activation of FVIII. PMID:12689334

  20. Inhibition of Mycobacterium tuberculosis PknG by non-catalytic rubredoxin domain specific modification: reaction of an electrophilic nitro-fatty acid with the Fe–S center

    PubMed Central

    Gil, Magdalena; Graña, Martín; Schopfer, Francisco J.; Wagner, Tristan; Denicola, Ana; Freeman, Bruce A.; Alzari, Pedro M.; Batthyány, Carlos; Durán, Rosario

    2014-01-01

    PknG from Mycobacterium tuberculosis is a Ser/Thr protein kinase that regulates key metabolic processes within the bacterial cell as well as signaling pathways from the infected host cell. This multidomain protein has a conserved canonical kinase domain with N- and C-terminal flanking regions of unclear functional roles. The N-terminus harbors a rubredoxin-like domain (Rbx), a bacterial protein module characterized by an iron ion coordinated by four cysteine residues. Disruption of the Rbx-metal binding site by simultaneous mutations of all the key cysteine residues significantly impairs PknG activity. This encouraged us to evaluate the effect of a nitro-fatty acid (9- and 10-nitro-octadeca-9-cis-enoic acid; OA-NO2) on PknG activity. Fatty acid nitroalkenes are electrophilic species produced during inflammation and metabolism that react with nucleophilic residues of target proteins (i.e., Cys and His), modulating protein function and subcellular distribution in a reversible manner. Here, we show that OA-NO2 inhibits kinase activity by covalently adducting PknG remote from the catalytic domain. Mass spectrometry-based analysis established that cysteines located at Rbx are the specific targets of the nitroalkene. Cys-nitroalkylation is a Michael addition reaction typically reverted by thiols. However, the reversible OA-NO2-mediated nitroalkylation of the kinase results in an irreversible inhibition of PknG. Cys adduction by OA-NO2 induced iron release from the Rbx domain, revealing a new strategy for the specific inhibition of PknG. These results affirm the relevance of the Rbx domain as a target for PknG inhibition and support that electrophilic lipid reactions of Rbx-Cys may represent a new drug strategy for specific PknG inhibition. PMID:23792274

  1. Mechanistic basis of Nek7 activation through Nek9 binding and induced dimerization

    NASA Astrophysics Data System (ADS)

    Haq, Tamanna; Richards, Mark W.; Burgess, Selena G.; Gallego, Pablo; Yeoh, Sharon; O'Regan, Laura; Reverter, David; Roig, Joan; Fry, Andrew M.; Bayliss, Richard

    2015-11-01

    Mitotic spindle assembly requires the regulated activities of protein kinases such as Nek7 and Nek9. Nek7 is autoinhibited by the protrusion of Tyr97 into the active site and activated by the Nek9 non-catalytic C-terminal domain (CTD). CTD binding apparently releases autoinhibition because mutation of Tyr97 to phenylalanine increases Nek7 activity independently of Nek9. Here we find that self-association of the Nek9-CTD is needed for Nek7 activation. We map the minimal Nek7 binding region of Nek9 to residues 810-828. A crystal structure of Nek7Y97F bound to Nek9810-828 reveals a binding site on the C-lobe of the Nek7 kinase domain. Nek7Y97F crystallizes as a back-to-back dimer between kinase domain N-lobes, in which the specific contacts within the interface are coupled to the conformation of residue 97. Hence, we propose that the Nek9-CTD activates Nek7 through promoting back-to-back dimerization that releases the autoinhibitory tyrosine residue, a mechanism conserved in unrelated kinase families.

  2. Probing the mechanism of ligand recognition in family 29 carbohydrate-binding modules.

    PubMed

    Flint, James; Bolam, David N; Nurizzo, Didier; Taylor, Edward J; Williamson, Michael P; Walters, Christopher; Davies, Gideon J; Gilbert, Harry J

    2005-06-24

    The recycling of photosynthetically fixed carbon, by the action of microbial plant cell wall hydrolases, is integral to one of the major geochemical cycles and is of considerable industrial importance. Non-catalytic carbohydrate-binding modules (CBMs) play a key role in this degradative process by targeting hydrolytic enzymes to their cognate substrate within the complex milieu of polysaccharides that comprise the plant cell wall. Family 29 CBMs have, thus far, only been found in an extracellular multienzyme plant cell wall-degrading complex from the anaerobic fungus Piromyces equi, where they exist as a CBM29-1:CBM29-2 tandem. Here we present both the structure of the CBM29-1 partner, at 1.5 A resolution, and examine the importance of hydrophobic stacking interactions as well as direct and solvent-mediated hydrogen bonds in the binding of CBM29-2 to different polysaccharides. CBM29 domains display unusual binding properties, exhibiting specificity for both beta-manno- and beta-gluco-configured ligands such as mannan, cellulose, and glucomannan. Mutagenesis reveals that "stacking" of tryptophan residues in the n and n+2 subsites plays a critical role in ligand binding, whereas the loss of tyrosine-mediated stacking in the n+4 subsite reduces, but does not abrogate, polysaccharide recognition. Direct hydrogen bonds to ligand, such as those provided by Arg-112 and Glu-78, play a pivotal role in the interaction with both mannan and cellulose, whereas removal of water-mediated interactions has comparatively little effect on carbohydrate binding. The interactions of CBM29-2 with the O2 of glucose or mannose contribute little to binding affinity, explaining why this CBM displays dual gluco/manno specificity. PMID:15784618

  3. Short-time dynamics of pH-dependent conformation and substrate binding in the active site of beta-glucosidases: A computational study.

    PubMed

    Flannelly, David F; Aoki, Thalia G; Aristilde, Ludmilla

    2015-09-01

    The complete degradation of cellulose to glucose is essential to carbon turnover in terrestrial ecosystems and to engineered biofuel production. A rate-limiting step in this pathway is catalyzed by beta-glucosidase (BG) enzymes, which convert cellulobiose into two glucose molecules. The activity of these enzymes has been shown to vary with solution pH. However, it is not well understood how pH influences the enzyme conformation required for catalytic action on the substrate. A structural understanding of this pH effect is important for predicting shifts in BG activity in bioreactors and environmental matrices, in addition to informing targeted protein engineering. Here we applied molecular dynamics simulations to explore conformational and substrate binding dynamics in two well-characterized BGs of bacterial (Clostridium cellulovorans) and fungal (Trichoderma reesei) origins as a function of pH. The enzymes were simulated in an explicit solvated environment, with NaCl as electrolytes, at their prominent ionization states obtained at pH 5, 6, 7, and 7.5. Our findings indicated that pH-dependent changes in the ionization states of non-catalytic residues localized outside of the immediate active site led to pH-dependent disruption of the active site conformation. This disruption interferes with favorable H-bonding interactions with catalytic residues required to initiate catalysis on the substrate. We also identified specific non-catalytic residues that are involved in stabilizing the substrate at the optimal pH for enzyme activity. The simulations further revealed the dynamics of water-bridging interactions both outside and inside the substrate binding cleft during structural changes in the enzyme-substrate complex. These findings provide new structural insights into the pH-dependent substrate binding specificity in BGs. PMID:26160737

  4. SERCA mutant E309Q binds two Ca2+ ions but adopts a catalytically incompetent conformation

    PubMed Central

    Clausen, Johannes D; Bublitz, Maike; Arnou, Bertrand; Montigny, Cédric; Jaxel, Christine; Møller, Jesper Vuust; Nissen, Poul; Andersen, Jens Peter; le Maire, Marc

    2013-01-01

    The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) couples ATP hydrolysis to transport of Ca2+. This directed energy transfer requires cross-talk between the two Ca2+ sites and the phosphorylation site over 50 Å distance. We have addressed the mechano-structural basis for this intramolecular signal by analysing the structure and the functional properties of SERCA mutant E309Q. Glu309 contributes to Ca2+ coordination at site II, and a consensus has been that E309Q only binds Ca2+ at site I. The crystal structure of E309Q in the presence of Ca2+ and an ATP analogue, however, reveals two occupied Ca2+ sites of a non-catalytic Ca2E1 state. Ca2+ is bound with micromolar affinity by both Ca2+ sites in E309Q, but without cooperativity. The Ca2+-bound mutant does phosphorylate from ATP, but at a very low maximal rate. Phosphorylation depends on the correct positioning of the A-domain, requiring a shift of transmembrane segment M1 into an ‘up and kinked position'. This transition is impaired in the E309Q mutant, most likely due to a lack of charge neutralization and altered hydrogen binding capacities at Ca2+ site II. PMID:24270570

  5. Non-catalytic coatings for hypersonic vehicle applications

    NASA Technical Reports Server (NTRS)

    Cunnington, G. R.; Robinson, J. C.; Clark, R. K.

    1990-01-01

    CVD coatings of boron-oxygen-silicon and aluminum-boron-oxygen and silicon compositions were applied to alpha 2 and gamma titanium-aluminide alloys, and tested under simulated earth reentry conditions to evaluate oxidation protection, recombination effifiency and total emittance under cyclic exposures between temperatures of 300 and 1255 K. The degree of oxidation protection afforded to the alloys was evaluated from the amount of specimen weight gain after a 100 hour exposure to air at atmospheric pressure and at a temperature of 1255 K. Data are presented for two types of coatings, a monolithic and a multilayer, which have recombination efficiencies from 0.005 to 0.020 after ten one-half hour long simulated reentry cycles. Total normal emittance was 0.80 or greater and weight gain was less than 10 g/sq m both coating types. Compositional and morphological changes resulting from the exposures are discussed in relation to time-dependent variations in recombination efficiency.

  6. Cord Wood Testing in a Non-Catalytic Wood Stove

    SciTech Connect

    Butcher, T.; Trojanowski, R.; Wei, G.

    2014-06-30

    EPA Method 28 and the current wood stove regulations have been in-place since 1988. Recently, EPA proposed an update to the existing NSPS for wood stove regulations which includes a plan to transition from the current crib wood fuel to cord wood fuel for certification testing. Cord wood is seen as generally more representative of field conditions while the crib wood is seen as more repeatable. In any change of certification test fuel, there are questions about the impact on measured results and the correlation between tests with the two different fuels. The purpose of the work reported here is to provide data on the performance of a noncatalytic stove with cord wood. The stove selected has previously been certified with crib wood which provides a basis for comparison with cord wood. Overall, particulate emissions were found to be considerably higher with cord wood.

  7. A Non-catalytic Deep Desulphurization Process using Hydrodynamic Cavitation

    PubMed Central

    Suryawanshi, Nalinee B.; Bhandari, Vinay M.; Sorokhaibam, Laxmi Gayatri; Ranade, Vivek V.

    2016-01-01

    A novel approach is developed for desulphurization of fuels or organics without use of catalyst. In this process, organic and aqueous phases are mixed in a predefined manner under ambient conditions and passed through a cavitating device. Vapor cavities formed in the cavitating device are then collapsed which generate (in-situ) oxidizing species which react with the sulphur moiety resulting in the removal of sulphur from the organic phase. In this work, vortex diode was used as a cavitating device. Three organic solvents (n-octane, toluene and n-octanol) containing known amount of a model sulphur compound (thiophene) up to initial concentrations of 500 ppm were used to verify the proposed method. A very high removal of sulphur content to the extent of 100% was demonstrated. The nature of organic phase and the ratio of aqueous to organic phase were found to be the most important process parameters. The results were also verified and substantiated using commercial diesel as a solvent. The developed process has great potential for deep of various organics, in general, and for transportation fuels, in particular. PMID:27605492

  8. A Non-catalytic Deep Desulphurization Process using Hydrodynamic Cavitation.

    PubMed

    Suryawanshi, Nalinee B; Bhandari, Vinay M; Sorokhaibam, Laxmi Gayatri; Ranade, Vivek V

    2016-01-01

    A novel approach is developed for desulphurization of fuels or organics without use of catalyst. In this process, organic and aqueous phases are mixed in a predefined manner under ambient conditions and passed through a cavitating device. Vapor cavities formed in the cavitating device are then collapsed which generate (in-situ) oxidizing species which react with the sulphur moiety resulting in the removal of sulphur from the organic phase. In this work, vortex diode was used as a cavitating device. Three organic solvents (n-octane, toluene and n-octanol) containing known amount of a model sulphur compound (thiophene) up to initial concentrations of 500 ppm were used to verify the proposed method. A very high removal of sulphur content to the extent of 100% was demonstrated. The nature of organic phase and the ratio of aqueous to organic phase were found to be the most important process parameters. The results were also verified and substantiated using commercial diesel as a solvent. The developed process has great potential for deep of various organics, in general, and for transportation fuels, in particular. PMID:27605492

  9. Enhanced exo-inulinase activity and stability by fusion of an inulin-binding module.

    PubMed

    Zhou, Shun-Hua; Liu, Yuan; Zhao, Yu-Juan; Chi, Zhe; Chi, Zhen-Ming; Liu, Guang-Lei

    2016-09-01

    In this study, an inulin-binding module from Bacillus macerans was successfully fused to an exo-inulinase from Kluyveromyces marxianus, creating a hybrid functional enzyme. The recombinant exo-inulinase (rINU), the hybrid enzyme (rINUIBM), and the recombinant inulin-binding module (rIBM) were, respectively, heterologously expressed and biochemically characterized. It was found that both the inulinase activity and the catalytic efficiency (k cat/K m(app)) of the rINUIBM were considerably higher than those of rINU. Though the rINU and the rINUIBM shared the same optimum pH of 4.5, the optimum temperature of the rINUIBM (60 °C) was 5 °C higher than that of the rINU. Notably, the fused IBM significantly enhanced both the pH stability and the thermostability of the rINUIBM, suggesting that the rINUIBM obtained would have more extensive potential applications. Furthermore, the fusion of the IBM could substantially improve the inulin-binding capability of the rINUIBM, which was consistent with the determination of the K m(app). This meant that the fused IBM could play a critical role in the recognition of polysaccharides and enhanced the hydrolase activity of the associated inulinase by increasing enzyme-substrate proximity. Besides, the extra supplement of the independent non-catalytic rIBM could also improve the inulinase activity of the rINU. However, this improvement was much better in case of the fusion. Consequently, the IBM could be designated as a multifunctional domain that was responsible for the activity enhancement, the stabilization, and the substrate binding of the rINUIBM. All these features obtained in this study make the rINUIBM become an attractive candidate for an efficient inulin hydrolysis. PMID:27164865

  10. Protein Binding Pocket Dynamics.

    PubMed

    Stank, Antonia; Kokh, Daria B; Fuller, Jonathan C; Wade, Rebecca C

    2016-05-17

    The dynamics of protein binding pockets are crucial for their interaction specificity. Structural flexibility allows proteins to adapt to their individual molecular binding partners and facilitates the binding process. This implies the necessity to consider protein internal motion in determining and predicting binding properties and in designing new binders. Although accounting for protein dynamics presents a challenge for computational approaches, it expands the structural and physicochemical space for compound design and thus offers the prospect of improved binding specificity and selectivity. A cavity on the surface or in the interior of a protein that possesses suitable properties for binding a ligand is usually referred to as a binding pocket. The set of amino acid residues around a binding pocket determines its physicochemical characteristics and, together with its shape and location in a protein, defines its functionality. Residues outside the binding site can also have a long-range effect on the properties of the binding pocket. Cavities with similar functionalities are often conserved across protein families. For example, enzyme active sites are usually concave surfaces that present amino acid residues in a suitable configuration for binding low molecular weight compounds. Macromolecular binding pockets, on the other hand, are located on the protein surface and are often shallower. The mobility of proteins allows the opening, closing, and adaptation of binding pockets to regulate binding processes and specific protein functionalities. For example, channels and tunnels can exist permanently or transiently to transport compounds to and from a binding site. The influence of protein flexibility on binding pockets can vary from small changes to an already existent pocket to the formation of a completely new pocket. Here, we review recent developments in computational methods to detect and define binding pockets and to study pocket dynamics. We introduce five

  11. Evolving nucleotide binding surfaces

    NASA Technical Reports Server (NTRS)

    Kieber-Emmons, T.; Rein, R.

    1981-01-01

    An analysis is presented of the stability and nature of binding of a nucleotide to several known dehydrogenases. The employed approach includes calculation of hydrophobic stabilization of the binding motif and its intermolecular interaction with the ligand. The evolutionary changes of the binding motif are studied by calculating the Euclidean deviation of the respective dehydrogenases. Attention is given to the possible structural elements involved in the origin of nucleotide recognition by non-coded primordial polypeptides.

  12. Melanin-binding radiopharmaceuticals

    SciTech Connect

    Packer, S; Fairchild, R G; Watts, K P; Greenberg, D; Hannon, S J

    1980-01-01

    The scope of this paper is limited to an analysis of the factors that are important to the relationship of radiopharmaceuticals to melanin. While the authors do not attempt to deal with differences between melanin-binding vs. melanoma-binding, a notable variance is assumed. (PSB)

  13. The nuclear fraction of protein kinase CK2 binds to the upstream stimulatory factors (USFs) in the absence of DNA.

    PubMed

    Spohrer, Sarah; Dimova, Elitsa Y; Kietzmann, Thomas; Montenarh, Mathias; Götz, Claudia

    2016-02-01

    The functions of the upstream stimulatory factors USF1 and USF2 are, like those of other transcription factors, regulated by reversible phosphorylation. Besides many other kinases also protein kinase CK2 phosphorylates USF1 but not USF2. In a yeast-two-hybrid screen, however, the non-catalytic CK2β subunit of CK2 was identified as a binding partner of USF2. This surprising observation prompted us to investigate the CK2/USF interaction in more detail in the present study. By using immunofluorescence analyses as well as co-immunoprecipitations we found that USF1 and USF2 bound to CK2α and CK2β exclusively in the nucleus, though CK2β and to a minor amount CK2α were also present in the cytoplasm. Furthermore, we found that unlike other substrates the phosphorylation of USF1 required the presence of the regulatory CK2β subunit; the catalytic α-subunit of CK2 alone was not able to phosphorylate USF1. Thus, the correct phosphorylation of USF1 is only guaranteed and strictly controlled in particular by nuclear CK2β. Although the data indicated that a nuclear subfraction of CK2 subunits associated with USF proteins, DNA pull down experiments revealed that the CK2 subunits did not co-localize with DNA bound USF proteins indicating that the USF/CK2 interaction has a pre- or post DNA binding function. PMID:26577526

  14. Positron binding to molecules

    NASA Astrophysics Data System (ADS)

    Danielson, J. R.

    2011-05-01

    While there is theoretical evidence that positrons can bind to atoms, calculations for molecules are much less precise. Unfortunately, there have been no measurements of positron-atom binding, due primarily to the difficulty in forming positron-atom bound states in two-body collisions. In contrast, positrons attach to molecules via Feshbach resonances (VFR) in which a vibrational mode absorbs the excess energy. Using a high-resolution positron beam, this VFR process has been studied to measure binding energies for more than 40 molecules. New measurements will be described in two areas: positron binding to relatively simple molecules, for which theoretical calculations appear to be possible; and positron binding to molecules with large permanent dipole moments, which can be compared to analogous, weakly bound electron-molecule (negative-ion) states. Binding energies range from 75 meV for CS2 (no dipole moment) to 180 meV for acetonitrile (CH3CN). Other species studied include aldehydes and ketones, which have permanent dipole moments in the range 2.5 - 3.0 debye. The measured binding energies are surprisingly large (by a factor of 10 to 100) compared to those for the analogous negative ions, and these differences will be discussed. New theoretical calculations for positron-molecule binding are in progress, and a recent result for acetonitrile will be discussed. This ability to compare theory and experiment represents a significant step in attempts to understand positron binding to matter. In collaboration with A. C. L. Jones, J. J. Gosselin, and C. M. Surko, and supported by NSF grant PHY 07-55809.

  15. ¹H, ¹³C and ¹⁵N backbone and side-chain resonance assignments of a family 36 carbohydrate binding module of xylanase from Paenibacillus campinasensis.

    PubMed

    Wang, Yu-Sheng; Ko, Chun-Han; Chang, Hao-Ting; Yang, Kai-Jay; Chen, Yu-Jen; Huang, Shing-Jong; Fang, Pei-Ju; Chang, Chi-Fon; Tzou, Der-Lii M

    2014-10-01

    Paenibacillus campinasensis BL11 isolated from black liquor secretes multiple glycoside hydrolases (GHs) against all kinds of polysaccharides. GH consists of a catalytic module and non-catalytic carbohydrate-binding modules (CBMs), in which CBMs append to the catalytic module, mediating specific interactions with insoluble carbohydrates to promote the hydrolysis efficiency of the cognate enzyme. Endo-β-1,4-xylanase (XylX) is one of the GHs reveals high enzymatic activity in a wide range of pH and thermal endurance, suitable for bioconversion and bio-refinement applications. In this work, we report the resonance assignments of a family 36 CBM (characterized as CBM36) derived from XylX. Our investigations will facilitate molecular structure determination and molecular dynamics analysis of CBMs. PMID:23835623

  16. Metallochaperones: bind and deliver

    SciTech Connect

    Rosenzweig, A.C.

    2010-03-08

    Metallochaperones deliver metal ions directly to target proteins via specific protein-protein interactions. Recent research has led to a molecular picture of how some metallochaperones bind metal ions, recognize their partner proteins, and accomplish metal ion transfer.

  17. The Conformational Changes Induced by Ubiquinone Binding in the Na+-pumping NADH:Ubiquinone Oxidoreductase (Na+-NQR) Are Kinetically Controlled by Conserved Glycines 140 and 141 of the NqrB Subunit*

    PubMed Central

    Strickland, Madeleine; Juárez, Oscar; Neehaul, Yashvin; Cook, Darcie A.; Barquera, Blanca; Hellwig, Petra

    2014-01-01

    Na+-pumping NADH:ubiquinone oxidoreductase (Na+-NQR) is responsible for maintaining a sodium gradient across the inner bacterial membrane. This respiratory enzyme, which couples sodium pumping to the electron transfer between NADH and ubiquinone, is not present in eukaryotes and as such could be a target for antibiotics. In this paper it is shown that the site of ubiquinone reduction is conformationally coupled to the NqrB subunit, which also hosts the final cofactor in the electron transport chain, riboflavin. Previous work showed that mutations in conserved NqrB glycine residues 140 and 141 affect ubiquinone reduction and the proper functioning of the sodium pump. Surprisingly, these mutants did not affect the dissociation constant of ubiquinone or its analog HQNO (2-n-heptyl-4-hydroxyquinoline N-oxide) from Na+-NQR, which indicates that these residues do not participate directly in the ubiquinone binding site but probably control its accessibility. Indeed, redox-induced difference spectroscopy showed that these mutations prevented the conformational change involved in ubiquinone binding but did not modify the signals corresponding to bound ubiquinone. Moreover, data are presented that demonstrate the NqrA subunit is able to bind ubiquinone but with a low non-catalytically relevant affinity. It is also suggested that Na+-NQR contains a single catalytic ubiquinone binding site and a second site that can bind ubiquinone but is not active. PMID:25006248

  18. Inhibition of selectin binding

    DOEpatents

    Nagy, Jon O.; Spevak, Wayne R.; Dasgupta, Falguni; Bertozzi, Caroline

    1999-01-01

    This invention provides compositions for inhibiting the binding between two cells, one expressing P- or L-selectin on the surface and the other expressing the corresponding ligand. A covalently crosslinked lipid composition is prepared having saccharides and acidic group on separate lipids. The composition is then interposed between the cells so as to inhibit binding. Inhibition can be achieved at an effective oligosaccharide concentration as low as 10.sup.6 fold below that of the free saccharide. Since selectins are involved in recruiting cells to sites of injury, these composition scan be used to palliate certain inflammatory and immunological conditions.

  19. Inhibition of selectin binding

    DOEpatents

    Nagy, Jon O.; Spevak, Wayne R.; Dasgupta, Falguni; Bertozzi, Carolyn

    1999-10-05

    This invention provides a system for inhibiting the binding between two cells, one expressing P- or L-selectin on the surface and the other expressing the corresponding ligand. A covalently crosslinked lipid composition is prepared having saccharides and acidic group on separate lipids. The composition is then interposed between the cells so as to inhibit binding. Inhibition can be achieved at an effective oligosaccharide concentration as low as 10.sup.6 fold below that of the free saccharide. Since selectins are involved in recruiting cells to sites of injury, this system can be used to palliate certain inflammatory and immunological conditions.

  20. Inhibition of selectin binding

    DOEpatents

    Nagy, Jon O.; Spevak, Wayne R.; Dasgupta, Falguni; Bertozzi, Caroline

    2001-10-09

    This invention provides compositions for inhibiting the binding between two cells, one expressing P- or L-selectin on the surface and the other expressing the corresponding ligand. A covalently crosslinked lipid composition is prepared having saccharides and acidic group on separate lipids. The composition is then interposed between the cells so as to inhibit binding. Inhibition can be achieved at an effective oligosaccharide concentration as low as 10.sup.6 fold below that of the free saccharide. Since selectins are involved in recruiting cells to sites of injury, these composition scan be used to palliate certain inflammatory and immunological conditions.

  1. Cellulose binding domain proteins

    DOEpatents

    Shoseyov, O.; Shpiegl, I.; Goldstein, M.; Doi, R.

    1998-11-17

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  2. Cellulose binding domain proteins

    DOEpatents

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc; Doi, Roy

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  3. MD-2 binds cholesterol.

    PubMed

    Choi, Soo-Ho; Kim, Jungsu; Gonen, Ayelet; Viriyakosol, Suganya; Miller, Yury I

    2016-02-19

    Cholesterol is a structural component of cellular membranes, which is transported from liver to peripheral cells in the form of cholesterol esters (CE), residing in the hydrophobic core of low-density lipoprotein. Oxidized CE (OxCE) is often found in plasma and in atherosclerotic lesions of subjects with cardiovascular disease. Our earlier studies have demonstrated that OxCE activates inflammatory responses in macrophages via toll-like receptor-4 (TLR4). Here we demonstrate that cholesterol binds to myeloid differentiation-2 (MD-2), a TLR4 ancillary molecule, which is a binding receptor for bacterial lipopolysaccharide (LPS) and is indispensable for LPS-induced TLR4 dimerization and signaling. Cholesterol binding to MD-2 was competed by LPS and by OxCE-modified BSA. Furthermore, soluble MD-2 in human plasma and MD-2 in mouse atherosclerotic lesions carried cholesterol, the finding supporting the biological significance of MD-2 cholesterol binding. These results help understand the molecular basis of TLR4 activation by OxCE and mechanisms of chronic inflammation in atherosclerosis. PMID:26806306

  4. Sequential memory: Binding dynamics

    NASA Astrophysics Data System (ADS)

    Afraimovich, Valentin; Gong, Xue; Rabinovich, Mikhail

    2015-10-01

    Temporal order memories are critical for everyday animal and human functioning. Experiments and our own experience show that the binding or association of various features of an event together and the maintaining of multimodality events in sequential order are the key components of any sequential memories—episodic, semantic, working, etc. We study a robustness of binding sequential dynamics based on our previously introduced model in the form of generalized Lotka-Volterra equations. In the phase space of the model, there exists a multi-dimensional binding heteroclinic network consisting of saddle equilibrium points and heteroclinic trajectories joining them. We prove here the robustness of the binding sequential dynamics, i.e., the feasibility phenomenon for coupled heteroclinic networks: for each collection of successive heteroclinic trajectories inside the unified networks, there is an open set of initial points such that the trajectory going through each of them follows the prescribed collection staying in a small neighborhood of it. We show also that the symbolic complexity function of the system restricted to this neighborhood is a polynomial of degree L - 1, where L is the number of modalities.

  5. Sequential memory: Binding dynamics.

    PubMed

    Afraimovich, Valentin; Gong, Xue; Rabinovich, Mikhail

    2015-10-01

    Temporal order memories are critical for everyday animal and human functioning. Experiments and our own experience show that the binding or association of various features of an event together and the maintaining of multimodality events in sequential order are the key components of any sequential memories-episodic, semantic, working, etc. We study a robustness of binding sequential dynamics based on our previously introduced model in the form of generalized Lotka-Volterra equations. In the phase space of the model, there exists a multi-dimensional binding heteroclinic network consisting of saddle equilibrium points and heteroclinic trajectories joining them. We prove here the robustness of the binding sequential dynamics, i.e., the feasibility phenomenon for coupled heteroclinic networks: for each collection of successive heteroclinic trajectories inside the unified networks, there is an open set of initial points such that the trajectory going through each of them follows the prescribed collection staying in a small neighborhood of it. We show also that the symbolic complexity function of the system restricted to this neighborhood is a polynomial of degree L - 1, where L is the number of modalities. PMID:26520084

  6. Library Binding Manual. Revised Edition.

    ERIC Educational Resources Information Center

    Lakhanpal, S. K.

    This procedural manual is designed to be used in bindery sections in public, university and special libraries. It briefly discusses these general matters: administrative control; selection of a binder; when and what to bind; conventional binding; routines; missing issues; schedule for shipments; temporary binding; rare books, maps and newspapers;…

  7. Alcohol binding to liposomes by 2H NMR and radiolabel binding assays: does partitioning describe binding?

    PubMed Central

    Dubey, A K; Eryomin, V A; Taraschi, T F; Janes, N

    1996-01-01

    Implicit within the concept of membrane-buffer partition coefficients of solutes is a nonspecific solvation mechanism of solute binding. However, (2)H NMR studies of the binding of (2)H(6)-ethanol and [1-(2)H(2)] n-hexanol to phosphatidylcholine vesicles have been interpreted as evidence for two distinct alcohol binding modes. One binding mode was reported to be at the membrane surface. The second mode was reported to be within the bilayer interior. An examination of the (2)H NMR binding studies, together with direct radiolabel binding assays, shows that other interpretations of the data are more plausible. The results are entirely consistent with partitioning (nonspecific binding) as the sole mode of alcohol binding to liposomes, in accord with our previous thermodynamic interpretation of alcohol action in phosphatidylcholine liposomes. PMID:9172754

  8. Carboplatin binding to histidine

    SciTech Connect

    Tanley, Simon W. M.; Diederichs, Kay; Kroon-Batenburg, Loes M. J.; Levy, Colin; Schreurs, Antoine M. M.; Helliwell, John R.

    2014-08-29

    An X-ray crystal structure showing the binding of purely carboplatin to histidine in a model protein has finally been obtained. This required extensive crystallization trials and various novel crystal structure analyses. Carboplatin is a second-generation platinum anticancer agent used for the treatment of a variety of cancers. Previous X-ray crystallographic studies of carboplatin binding to histidine (in hen egg-white lysozyme; HEWL) showed the partial conversion of carboplatin to cisplatin owing to the high NaCl concentration used in the crystallization conditions. HEWL co-crystallizations with carboplatin in NaBr conditions have now been carried out to confirm whether carboplatin converts to the bromine form and whether this takes place in a similar way to the partial conversion of carboplatin to cisplatin observed previously in NaCl conditions. Here, it is reported that a partial chemical transformation takes place but to a transplatin form. Thus, to attempt to resolve purely carboplatin binding at histidine, this study utilized co-crystallization of HEWL with carboplatin without NaCl to eliminate the partial chemical conversion of carboplatin. Tetragonal HEWL crystals co-crystallized with carboplatin were successfully obtained in four different conditions, each at a different pH value. The structural results obtained show carboplatin bound to either one or both of the N atoms of His15 of HEWL, and this particular variation was dependent on the concentration of anions in the crystallization mixture and the elapsed time, as well as the pH used. The structural details of the bound carboplatin molecule also differed between them. Overall, the most detailed crystal structure showed the majority of the carboplatin atoms bound to the platinum centre; however, the four-carbon ring structure of the cyclobutanedicarboxylate moiety (CBDC) remained elusive. The potential impact of the results for the administration of carboplatin as an anticancer agent are described.

  9. An olive pollen protein with allergenic activity, Ole e 10, defines a novel family of carbohydrate-binding modules and is potentially implicated in pollen germination

    PubMed Central

    2005-01-01

    CBMs (carbohydrate-binding modules) are the most common non-catalytic modules associated with enzymes active in plant cell-wall hydrolysis. They have been frequently identified by amino acid sequence alignments, but only a few have been experimentally established to have a carbohydrate-binding activity. A small olive pollen protein, Ole e 10 (10 kDa), has been described as a major inducer of type I allergy in humans. In the present study, the ability of Ole e 10 to bind several polysaccharides has been analysed by affinity gel electrophoresis, which demonstrated that the protein bound 1,3-β-glucans preferentially. Analytical ultracentrifugation studies confirmed binding to laminarin, at a protein/ligand ratio of 1:1. The interaction of Ole e 10 with laminarin induced a conformational change in the protein, as detected by CD and fluorescence analyses, and an increase of 3.6 °C in the thermal denaturation temperature of Ole e 10 in the presence of the glycan. These results, and the absence of alignment of the sequence of Ole e 10 with that of any classified CBM, indicate that this pollen protein defines a novel family of CBMs, which we propose to name CBM43. Immunolocalization of Ole e 10 in mature and germinating pollen by transmission electron microscopy and confocal laser scanning microscopy demonstrated the co-localization of Ole e 10 and callose (1,3-β-glucan) in the growing pollen tube, suggesting a role for this protein in the metabolism of carbohydrates and in pollen tube wall re-formation during germination. PMID:15882149

  10. A new family of rhamnogalacturonan lyases contains an enzyme that binds to cellulose.

    PubMed Central

    McKie, V A; Vincken, J P; Voragen, A G; van den Broek, L A; Stimson, E; Gilbert, H J

    2001-01-01

    Pseudomonas cellulosa is an aerobic bacterium that synthesizes an extensive array of modular cellulases and hemicellulases, which have a modular architecture consisting of catalytic domains and distinct non-catalytic carbohydrate-binding modules (CBMs). To investigate whether the main-chain-cleaving pectinases from this bacterium also have a modular structure, a library of P. cellulosa genomic DNA, constructed in lambdaZAPII, was screened for pectinase-encoding sequences. A recombinant phage that attacked arabinan, galactan and rhamnogalacturonan was isolated. The encoded enzyme, designated Rgl11A, had a modular structure comprising an N-terminal domain that exhibited homology to Bacillus and Streptomyces proteins of unknown function, a middle domain that exhibited sequence identity to fibronectin-3 domains, and a C-terminal domain that was homologous to family 2a CBMs. Expression of the three modules of the Pseudomonas protein in Escherichia coli showed that its C-terminal module was a functional cellulose-binding domain, and the N-terminal module consisted of a catalytic domain that hydrolysed rhamnogalacturonan-containing substrates. The activity of Rgl11A against apple- and potato-derived rhamnogalacturonan substrates indicated that the enzyme had a strong preference for rhamnogalacturonans that contained galactose side chains, and which were not esterified. The enzyme had an absolute requirement for calcium, a high optimum pH, and catalysis was associated with an increase in absorbance at 235 nm, indicating that glycosidic bond cleavage was mediated via a beta-elimination mechanism. These data indicate that Rgl11A is a rhamnogalacturonan lyase and, together with the homologous Bacillus and Streptomyces proteins, comprise a new family of polysaccharide lyases. The presence of a family 2a CBM in Rgl11A, and in a P. cellulosa pectate lyase described in the accompanying paper [Brown, Mallen, Charnock, Davies and Black (2001) Biochem. J. 355, 155-165] suggests that

  11. Cold Spots in Protein Binding.

    PubMed

    Shirian, Jason; Sharabi, Oz; Shifman, Julia M

    2016-09-01

    Understanding the energetics and architecture of protein-binding interfaces is important for basic research and could potentially facilitate the design of novel binding domains for biotechnological applications. It is well accepted that a few key residues at binding interfaces (binding hot spots) are responsible for contributing most to the free energy of binding. In this opinion article, we introduce a new concept of 'binding cold spots', or interface positions occupied by suboptimal amino acids. Such positions exhibit a potential for affinity enhancement through various mutations. We give several examples of cold spots from different protein-engineering studies and argue that identification of such positions is crucial for studies of protein evolution and protein design. PMID:27477052

  12. Quarkonium binding and entropic force

    NASA Astrophysics Data System (ADS)

    Satz, Helmut

    2015-05-01

    A bound state represents a balance between repulsive kinetic and attractive potential energy. In a hot quark-gluon plasma, the interaction potential experiences medium effects. Color screening modifies the attractive binding force between the quarks, while the increase of entropy with separation gives rise to a growing repulsion. We study the role of these phenomena for in-medium binding and dissociation. It is found that the relevant potential for binding is the free energy ; with increasing separation, further binding through the internal energy is compensated by repulsive entropic effects.

  13. Identification of consensus binding sites clarifies FMRP binding determinants.

    PubMed

    Anderson, Bart R; Chopra, Pankaj; Suhl, Joshua A; Warren, Stephen T; Bassell, Gary J

    2016-08-19

    Fragile X mental retardation protein (FMRP) is a multifunctional RNA-binding protein with crucial roles in neuronal development and function. Efforts aimed at elucidating how FMRP target mRNAs are selected have produced divergent sets of target mRNA and putative FMRP-bound motifs, and a clear understanding of FMRP's binding determinants has been lacking. To clarify FMRP's binding to its target mRNAs, we produced a shared dataset of FMRP consensus binding sequences (FCBS), which were reproducibly identified in two published FMRP CLIP sequencing datasets. This comparative dataset revealed that of the various sequence and structural motifs that have been proposed to specify FMRP binding, the short sequence motifs TGGA and GAC were corroborated, and a novel TAY motif was identified. In addition, the distribution of the FCBS set demonstrates that FMRP preferentially binds to the coding region of its targets but also revealed binding along 3' UTRs in a subset of target mRNAs. Beyond probing these putative motifs, the FCBS dataset of reproducibly identified FMRP binding sites is a valuable tool for investigating FMRP targets and function. PMID:27378784

  14. Reversible calcitonin binding to solubilized sheep brain binding sites.

    PubMed Central

    Sexton, P M; Schneider, H G; D'Santos, C S; Mendelsohn, F A; Kemp, B E; Moseley, J M; Martin, T J; Findlay, D M

    1991-01-01

    In this study we have solubilized and characterized binding sites for calcitonin (CT) from sheep brainstem. Autoradiography of 125I-labelled salmon CT (125I-sCT) binding to sheep diencephalon revealed a similar pattern of binding to that seen in other species, although the extent of distribution was greater in the sheep. CT binding activity could be extracted from membranes with either CHAPS or digitonin, but not with beta-octyl glucoside, 125I-sCT binding was saturable, with a dissociation constant for CHAPS-solubilized membranes of 2.8 +/- 0.5 nM and a maximum binding site concentration of 6.2 +/- 1.6 pmol/mg of protein. In competition binding studies, various CTs and their analogues demonstrated a similar rank order of potency to that seen in other CT receptor systems, Optimal binding occurred in the pH range 6.5-7.5, and was decreased in the presence of NaCl concentrations greater than 200 mM. In contrast with most other CT receptor binding systems, in which binding is poorly reversible, the binding of 125I-sCT to sheep brain binding sites underwent substantial dissociation upon addition of excess unlabelled sCT, with 40% and 46% dissociation after 2 h at 4 degree C in particulate and solubilized membranes respectively. Photoaffinity labelling of the binding site with the biologically active analogue 125I-[Arg11,18,4-azidobenzoyl-Lys14]sCT and analysis on SDS/PAGE under reducing conditions revealed a specific protein band of Mr approximately solubilized and particulate brain membranes. This is in accordance with the molecular size of CT receptors in other tissues where two species of receptor have been identified. one of Mr approximately 71,000 and another of Mr approximately 88,000. These results demonstrate the presence of high concentrations of CT binding sites in sheep brain which display different kinetic properties to those of CT receptors found in other tissues. Images Fig. 1. Fig. 6. PMID:1846527

  15. Binding Energy and Enzymatic Catalysis.

    ERIC Educational Resources Information Center

    Hansen, David E.; Raines, Ronald T.

    1990-01-01

    Discussed is the fundamental role that the favorable free energy of binding of the rate-determining transition state plays in catalysis. The principle that all of the catalytic factors discussed are realized by the use of this binding energy is reviewed. (CW)

  16. Cholinergic influences on feature binding.

    PubMed

    Botly, Leigh C P; De Rosa, Eve

    2007-04-01

    The binding problem refers to the fundamental challenge of the central nervous system to integrate sensory information registered by multiple brain regions to form a unified neural representation of a stimulus. Human behavioral, neuropsychological, and functional neuroimaging evidence suggests a fundamental role for attention in feature binding; however, its neurochemical basis is currently unknown. This study examined whether acetylcholine (ACh), a neuromodulator that has been implicated in attentional processes, plays a critical role in feature binding. Using a within-subjects pharmacological design and the cholinergic muscarinic antagonist scopolamine, the present experiments demonstrate, in a rat model, a critical role for the cortical muscarinic cholinergic system in feature binding. Specifically, ACh and the attentional resources that it supports are essential for the initial feature binding process but are not required to maintain neural representations of bound stimuli. PMID:17469916

  17. Cooperative binding: a multiple personality.

    PubMed

    Martini, Johannes W R; Diambra, Luis; Habeck, Michael

    2016-06-01

    Cooperative binding has been described in many publications and has been related to or defined by several different properties of the binding behavior of the ligand to the target molecule. In addition to the commonly used Hill coefficient, other characteristics such as a sigmoidal shape of the overall titration curve in a linear plot, a change of ligand affinity of the other binding sites when a site of the target molecule becomes occupied, or complex roots of the binding polynomial have been used to define or to quantify cooperative binding. In this work, we analyze how the different properties are related in the most general model for binding curves based on the grand canonical partition function and present several examples which highlight differences between the cooperativity characterizing properties which are discussed. Our results mainly show that among the presented definitions there are not two which fully coincide. Moreover, this work poses the question whether it can make sense to distinguish between positive and negative cooperativity based on the macroscopic binding isotherm only. This article shall emphasize that scientists who investigate cooperative effects in biological systems could help avoiding misunderstandings by stating clearly which kind of cooperativity they discuss. PMID:26319983

  18. (/sup 3/)tetrahydrotrazodone binding. Association with serotonin binding sites

    SciTech Connect

    Kendall, D.A.; Taylor, D.P.; Enna, S.J.

    1983-05-01

    High (17 nM) and low (603 nM) affinity binding sites for (/sup 3/)tetrahydrotrazodone ((/sup 3/) THT), a biologically active analogue of trazodone, have been identified in rat brain membranes. The substrate specificity, concentration, and subcellular and regional distributions of these sites suggest that they may represent a component of the serotonin transmitter system. Pharmacological analysis of (/sup 3/)THT binding, coupled with brain lesion and drug treatment experiments, revealed that, unlike other antidepressants, (/sup 3/) THT does not attach to either a biogenic amine transporter or serotonin binding sites. Rather, it would appear that (/sup 3/)THT may be an antagonist ligand for the serotonin binding site. This probe may prove of value in defining the mechanism of action of trazodone and in further characterizing serotonin receptors.

  19. Benzodiazepine binding to bovine retina.

    PubMed

    Osborne, N N

    1980-02-01

    [3H]Diazepam binds to membrane preparations of the retina, suggesting that benzodiazepine receptors exist in this tissue. The binding characteristics are similar to those known to occur in the brain, with affinity constants in the same range. Unlike the finding in the brain, [3H]diazepam binding in the retina is not stimulated by GABA and other GABA agonists. These findings indicate that benzodiazepine receptors may have a more general function and not only be associated with anxiety or emotional behaviour. PMID:6302572

  20. Mercury binding on activated carbon

    SciTech Connect

    Bihter Padak; Michael Brunetti; Amanda Lewis; Jennifer Wilcox

    2006-11-15

    Density functional theory has been employed for the modeling of activated carbon (AC) using a fused-benzene ring cluster approach. Oxygen functional groups have been investigated for their promotion of effective elemental mercury binding on AC surface sites. Lactone and carbonyl functional groups yield the highest mercury binding energies. Further, the addition of halogen atoms has been considered to the modeled surface, and has been found to increase the AC's mercury adsorption capacity. The mercury binding energies increase with the addition of the following halogen atoms, F {gt} Cl {gt} Br {gt} I, with the fluorine addition being the most promising halogen for increasing mercury adsorption.

  1. Superresolution microscopy with transient binding.

    PubMed

    Molle, Julia; Raab, Mario; Holzmeister, Susanne; Schmitt-Monreal, Daniel; Grohmann, Dina; He, Zhike; Tinnefeld, Philip

    2016-06-01

    For single-molecule localization based superresolution, the concentration of fluorescent labels has to be thinned out. This is commonly achieved by photophysically or photochemically deactivating subsets of molecules. Alternatively, apparent switching of molecules can be achieved by transient binding of fluorescent labels. Here, a diffusing dye yields bright fluorescent spots when binding to the structure of interest. As the binding interaction is weak, the labeling is reversible and the dye ligand construct diffuses back into solution. This approach of achieving superresolution by transient binding (STB) is reviewed in this manuscript. Different realizations of STB are discussed and compared to other localization-based superresolution modalities. We propose the development of labeling strategies that will make STB a highly versatile tool for superresolution microscopy at highest resolution. PMID:26773299

  2. When is protein binding important?

    PubMed

    Heuberger, Jules; Schmidt, Stephan; Derendorf, Hartmut

    2013-09-01

    The present paper is an ode to a classic citation by Benet and Hoener (2002. Clin Pharm Ther 71(3):115-121). The now classic paper had a huge impact on drug development and the way the issue of protein binding is perceived and interpreted. Although the authors very clearly pointed out the limitations and underlying assumptions for their delineations, these are too often overlooked and the classic paper's message is misinterpreted by broadening to cases that were not intended. Some members of the scientific community concluded from the paper that protein binding is not important. This was clearly not intended by the authors, as they finished their paper with a paragraph entitled: "When is protein binding important?" Misinterpretation of the underlying assumptions in the classic work can result in major pitfalls in drug development. Therefore, we revisit the topic of protein binding with the intention of clarifying when clinically relevant changes should be considered during drug development. PMID:23650013

  3. Cholesterol binding to ion channels

    PubMed Central

    Levitan, Irena; Singh, Dev K.; Rosenhouse-Dantsker, Avia

    2014-01-01

    Numerous studies demonstrated that membrane cholesterol is a major regulator of ion channel function. The goal of this review is to discuss significant advances that have been recently achieved in elucidating the mechanisms responsible for cholesterol regulation of ion channels. The first major insight that comes from growing number of studies that based on the sterol specificity of cholesterol effects, show that several types of ion channels (nAChR, Kir, BK, TRPV) are regulated by specific sterol-protein interactions. This conclusion is supported by demonstrating direct saturable binding of cholesterol to a bacterial Kir channel. The second major advance in the field is the identification of putative cholesterol binding sites in several types of ion channels. These include sites at locations associated with the well-known cholesterol binding motif CRAC and its reversed form CARC in nAChR, BK, and TRPV, as well as novel cholesterol binding regions in Kir channels. Notably, in the majority of these channels, cholesterol is suggested to interact mainly with hydrophobic residues in non-annular regions of the channels being embedded in between transmembrane protein helices. We also discuss how identification of putative cholesterol binding sites is an essential step to understand the mechanistic basis of cholesterol-induced channel regulation. Clearly, however, these are only the first few steps in obtaining a general understanding of cholesterol-ion channels interactions and their roles in cellular and organ functions. PMID:24616704

  4. Water binding in legume seeds

    NASA Technical Reports Server (NTRS)

    Vertucci, C. W.; Leopold, A. C.

    1987-01-01

    The physical status of water in seeds has a pivotal role in determining the physiological reactions that can take place in the dry state. Using water sorption isotherms from cotyledon and axis tissue of five leguminous seeds, the strength of water binding and the numbers of binding sites have been estimated using van't Hoff analyses and the D'Arcy/Watt equation. These parameters of water sorption are calculated for each of the three regions of water binding and for a range of temperatures. Water sorption characteristics are reflective of the chemical composition of the biological materials as well as the temperature at which hydration takes place. Changes in the sorption characteristics with temperature and hydration level may suggest hydration-induced structural changes in cellular components.

  5. Computational Prediction of RNA-Binding Proteins and Binding Sites

    PubMed Central

    Si, Jingna; Cui, Jing; Cheng, Jin; Wu, Rongling

    2015-01-01

    Proteins and RNA interaction have vital roles in many cellular processes such as protein synthesis, sequence encoding, RNA transfer, and gene regulation at the transcriptional and post-transcriptional levels. Approximately 6%–8% of all proteins are RNA-binding proteins (RBPs). Distinguishing these RBPs or their binding residues is a major aim of structural biology. Previously, a number of experimental methods were developed for the determination of protein–RNA interactions. However, these experimental methods are expensive, time-consuming, and labor-intensive. Alternatively, researchers have developed many computational approaches to predict RBPs and protein–RNA binding sites, by combining various machine learning methods and abundant sequence and/or structural features. There are three kinds of computational approaches, which are prediction from protein sequence, prediction from protein structure, and protein-RNA docking. In this paper, we review all existing studies of predictions of RNA-binding sites and RBPs and complexes, including data sets used in different approaches, sequence and structural features used in several predictors, prediction method classifications, performance comparisons, evaluation methods, and future directions. PMID:26540053

  6. Unusual monoclonal DNA binding immunoglobulin.

    PubMed

    Sawada, S; Iijima, S; Kuwana, K; Nishinarita, S; Takeuchi, J; Shida, M; Karasaki, M; Amaki, I

    1983-03-01

    The monoclonal antibodies directed against DNA were produced by somatic cell hybridization with parental cells (SP-2) and spleen cells from nonimmunized autoimmune MRL/lpr mice. The immunoglobulins were recovered from the culture supernatant from hybridoma by a solid immunoadsorbent and antibody immunoprecipitation. The results from the specificities of DNA binding monoclonal immunoglobulins suggest that the antibodies to DNA have the antibody combining sites for both epitope of double stranded helix and base of DNA and support the concept of the multiple antigen binding potentials of the hybridoma autoantibodies. PMID:6857646

  7. RNA Bind-n-Seq: Measuring the Binding Affinity Landscape of RNA-Binding Proteins.

    PubMed

    Lambert, Nicole J; Robertson, Alex D; Burge, Christopher B

    2015-01-01

    RNA-binding proteins (RBPs) coordinate post-transcriptional control of gene expression, often through sequence-specific recognition of primary transcripts or mature messenger RNAs. Hundreds of RBPs are encoded in the human genome, most with undefined or incompletely defined biological roles. Understanding the function of these factors will require the identification of each RBP's distinct RNA binding specificity. RNA Bind-n-Seq (RBNS) is a high-throughput, cost-effective in vitro method capable of resolving sequence and secondary structure preferences of RBPs. Dissociation constants can also be inferred from RBNS data when provided with additional experimental information. Here, we describe the experimental procedures to perform RBNS and discuss important parameters of the method and ways that the experiment can be tailored to the specific RBP under study. Additionally, we present the conceptual framework and execution of the freely available RBNS computational pipeline and describe the outputs of the pipeline. Different approaches to quantify binding specificity, quality control metrics, and estimation of binding constants are also covered. PMID:26068750

  8. Synthetic heparin-binding growth factor analogs

    DOEpatents

    Pena, Louis A.; Zamora, Paul; Lin, Xinhua; Glass, John D.

    2007-01-23

    The invention provides synthetic heparin-binding growth factor analogs having at least one peptide chain that binds a heparin-binding growth factor receptor, covalently bound to a hydrophobic linker, which is in turn covalently bound to a non-signaling peptide that includes a heparin-binding domain. The synthetic heparin-binding growth factor analogs are useful as soluble biologics or as surface coatings for medical devices.

  9. Sucrose Octasulfate Selectively Accelerates Thrombin Inactivation by Heparin Cofactor II*

    PubMed Central

    Sarilla, Suryakala; Habib, Sally Y.; Kravtsov, Dmitri V.; Matafonov, Anton; Gailani, David; Verhamme, Ingrid M.

    2010-01-01

    Inactivation of thrombin (T) by the serpins heparin cofactor II (HCII) and antithrombin (AT) is accelerated by a heparin template between the serpin and thrombin exosite II. Unlike AT, HCII also uses an allosteric interaction of its NH2-terminal segment with exosite I. Sucrose octasulfate (SOS) accelerated thrombin inactivation by HCII but not AT by 2000-fold. SOS bound to two sites on thrombin, with dissociation constants (KD) of 10 ± 4 μm and 400 ± 300 μm that were not kinetically resolvable, as evidenced by single hyperbolic SOS concentration dependences of the inactivation rate (kobs). SOS bound HCII with KD 1.45 ± 0.30 mm, and this binding was tightened in the T·SOS·HCII complex, characterized by Kcomplex of ∼0.20 μm. Inactivation data were incompatible with a model solely depending on HCII·SOS but fit an equilibrium linkage model employing T·SOS binding in the pathway to higher order complex formation. Hirudin-(54–65)(SO3−) caused a hyperbolic decrease of the inactivation rates, suggesting partial competitive binding of hirudin-(54–65)(SO3−) and HCII to exosite I. Meizothrombin(des-fragment 1), binding SOS with KD = 1600 ± 300 μm, and thrombin were inactivated at comparable rates, and an exosite II aptamer had no effect on the inactivation, suggesting limited exosite II involvement. SOS accelerated inactivation of meizothrombin 1000-fold, reflecting the contribution of direct exosite I interaction with HCII. Thrombin generation in plasma was suppressed by SOS, both in HCII-dependent and -independent processes. The ex vivo HCII-dependent process may utilize the proposed model and suggests a potential for oversulfated disaccharides in controlling HCII-regulated thrombin generation. PMID:20053992

  10. Al(+)-ligand binding energies

    NASA Technical Reports Server (NTRS)

    Sodupe, M.; Bauschlicher, Charles W., Jr.

    1991-01-01

    Ab initio calculations are used to optimize the structure and determine the binding energies of Al(+) to a series of ligands. For Al(+)-CN, the bonding was found to have a large covalent component. For the remaining ligands, the bonding is shown to be electrostatic in origin. The results obtained for Al(+) are compared with those previously reported for Mg(+).

  11. Cellulose binding domain fusion proteins

    DOEpatents

    Shoseyov, O.; Yosef, K.; Shpiegl, I.; Goldstein, M.A.; Doi, R.H.

    1998-02-17

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  12. Cellulose binding domain fusion proteins

    DOEpatents

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  13. Positive Emotion Facilitates Audiovisual Binding

    PubMed Central

    Kitamura, Miho S.; Watanabe, Katsumi; Kitagawa, Norimichi

    2016-01-01

    It has been shown that positive emotions can facilitate integrative and associative information processing in cognitive functions. The present study examined whether emotions in observers can also enhance perceptual integrative processes. We tested 125 participants in total for revealing the effects of emotional states and traits in observers on the multisensory binding between auditory and visual signals. Participants in Experiment 1 observed two identical visual disks moving toward each other, coinciding, and moving away, presented with a brief sound. We found that for participants with lower depressive tendency, induced happy moods increased the width of the temporal binding window of the sound-induced bounce percept in the stream/bounce display, while no effect was found for the participants with higher depressive tendency. In contrast, no effect of mood was observed for a simple audiovisual simultaneity discrimination task in Experiment 2. These results provide the first empirical evidence of a dependency of multisensory binding upon emotional states and traits, revealing that positive emotions can facilitate the multisensory binding processes at a perceptual level. PMID:26834585

  14. Hebrew as a Binding Force.

    ERIC Educational Resources Information Center

    Fischler, Ben-Zion

    1990-01-01

    The role of the Hebrew language as a cohesive force and the history of modern Hebrew instruction are chronicled. It is proposed that despite the scattering of its speakers and periods of use only as a literary or business language, Hebrew has been a binding force for the Jewish people. It was with considerable struggle that Hebrew gained…

  15. Protein binding assay for hyaluronate

    SciTech Connect

    Lacy, B.E.; Underhill, C.B.

    1986-11-01

    A relatively quick and simple assay for hyaluronate was developed using the specific binding protein, hyaluronectin. The hyaluronectin was obtained by homogenizing the brains of Sprague-Dawley rats, and then centrifuging the homogenate. The resulting supernatant was used as a source of crude hyaluronectin. In the binding assay, the hyaluronectin was mixed with (/sup 3/H)hyaluronate, followed by an equal volume of saturated (NH/sub 4/)/sub 2/SO/sub 4/, which precipitated the hyaluronectin and any (/sup 3/H)hyaluronate associated with it, but left free (/sup 3/H)hyaluronate in solution. The mixture was then centrifuged, and the amount of bound (/sup 3/H)hyaluronate in the precipitate was determined. Using this assay, the authors found that hyaluronectin specifically bound hyaluronate, since other glycosaminoglycans failed to compete for the binding protein. In addition, the interaction between hyaluronectin and hyaluronate was of relatively high affinity, and the size of the hyaluronate did not appear to substantially alter the amount of binding. To determine the amount of hyaluronate in an unknown sample, they used a competition assay in which the binding of a set amount of (/sup 3/H)hyaluronate was blocked by the addition of unlabeled hyaluronate. By comparing the degree of competition of the unknown samples with that of known amounts of hyaluronate, it was possible to determine the amount of hyaluronate in the unknowns. They have found that this method is sensitive to 1 ..mu..g or less of hyaluronate, and is unaffected by the presence of proteins.

  16. The prion protein binds thiamine.

    PubMed

    Perez-Pineiro, Rolando; Bjorndahl, Trent C; Berjanskii, Mark V; Hau, David; Li, Li; Huang, Alan; Lee, Rose; Gibbs, Ebrima; Ladner, Carol; Dong, Ying Wei; Abera, Ashenafi; Cashman, Neil R; Wishart, David S

    2011-11-01

    Although highly conserved throughout evolution, the exact biological function of the prion protein is still unclear. In an effort to identify the potential biological functions of the prion protein we conducted a small-molecule screening assay using the Syrian hamster prion protein [shPrP(90-232)]. The screen was performed using a library of 149 water-soluble metabolites that are known to pass through the blood-brain barrier. Using a combination of 1D NMR, fluorescence quenching and surface plasmon resonance we identified thiamine (vitamin B1) as a specific prion ligand with a binding constant of ~60 μM. Subsequent studies showed that this interaction is evolutionarily conserved, with similar binding constants being seen for mouse, hamster and human prions. Various protein construct lengths, both with and without the unstructured N-terminal region in the presence and absence of copper, were examined. This indicates that the N-terminus has no influence on the protein's ability to interact with thiamine. In addition to thiamine, the more biologically abundant forms of vitamin B1 (thiamine monophosphate and thiamine diphosphate) were also found to bind the prion protein with similar affinity. Heteronuclear NMR experiments were used to determine thiamine's interaction site, which is located between helix 1 and the preceding loop. These data, in conjunction with computer-aided docking and molecular dynamics, were used to model the thiamine-binding pharmacophore and a comparison with other thiamine binding proteins was performed to reveal the common features of interaction. PMID:21848803

  17. SH2 domains prevent tyrosine dephosphorylation of the EGF receptor: identification of Tyr992 as the high-affinity binding site for SH2 domains of phospholipase C gamma.

    PubMed Central

    Rotin, D; Margolis, B; Mohammadi, M; Daly, R J; Daum, G; Li, N; Fischer, E H; Burgess, W H; Ullrich, A; Schlessinger, J

    1992-01-01

    Several cytoplasmic tyrosine kinases contain a conserved, non-catalytic stretch of approximately 100 amino acids called the src homology 2 (SH2) domain, and a region of approximately 50 amino acids called the SH3 domain. SH2/SH3 domains are also found in several other proteins, including phospholipase C-gamma (PLC gamma). Recent studies indicate that SH2 domains promote association between autophosphorylated growth factor receptors such as the epidermal growth factor (EGF) receptor and signal transducing molecules such as PLC gamma. Because SH2 domains bind specifically to protein sequences containing phosphotyrosine, we examined their capacity to prevent tyrosine dephosphorylation of the EGF and other receptors with tyrosine kinase activity. For this purpose, various SH2/SH3 constructs of PLC gamma were expressed in Escherichia coli as glutathione-S-transferase fusion proteins. Our results show that purified SH2 domains of PLC gamma are able to prevent tyrosine dephosphorylation of the EGF receptor and other receptors with tyrosine activity. The inhibition of tyrosine dephosphorylation paralleled the capacity of various SH2-containing constructs to bind to the EGF receptor, suggesting that the tyrosine phosphatase and the SH2 domain compete for the same tyrosine phosphorylation sites in the carboxy-terminal tail of the EGF receptor. Analysis of the phosphorylation sites protected from dephosphorylation by PLC gamma-SH2 revealed substantial inhibition of dephosphorylation of Tyr992 at 1 microM SH2. This indicates that Tyr992 and its flanking sequence is the high-affinity binding site for SH2 domains of PLC gamma.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:1537335

  18. Mg(+)-ligand binding energies

    NASA Technical Reports Server (NTRS)

    Bauschlicher, Charles W., Jr.; Partridge, Harry

    1991-01-01

    Ab initio calculations are used to optimize the structures and determine the binding energies of Mg(+) to a series of ligands. Mg(+) bonds electrostatically with benzene, acetone, H2, CO, and NH3 and a self-consistent-field treatment gives a good description of the bonding. The bonding in MgCN(+) and MgCH3(+) is largely covalent and a correlated treatment is required.

  19. Anion binding in biological systems

    NASA Astrophysics Data System (ADS)

    Feiters, Martin C.; Meyer-Klaucke, Wolfram; Kostenko, Alexander V.; Soldatov, Alexander V.; Leblanc, Catherine; Michel, Gurvan; Potin, Philippe; Küpper, Frithjof C.; Hollenstein, Kaspar; Locher, Kaspar P.; Bevers, Loes E.; Hagedoorn, Peter-Leon; Hagen, Wilfred R.

    2009-11-01

    We compare aspects of biological X-ray absorption spectroscopy (XAS) studies of cations and anions, and report on some examples of anion binding in biological systems. Brown algae such as Laminaria digitata (oarweed) are effective accumulators of I from seawater, with tissue concentrations exceeding 50 mM, and the vanadate-containing enzyme haloperoxidase is implicated in halide accumulation. We have studied the chemical state of iodine and its biological role in Laminaria at the I K edge, and bromoperoxidase from Ascophyllum nodosum (knotted wrack) at the Br K edge. Mo is essential for many forms of life; W only for certain archaea, such as Archaeoglobus fulgidus and the hyperthermophilic archaeon Pyrococcus furiosus, and some bacteria. The metals are bound and transported as their oxo-anions, molybdate and tungstate, which are similar in size. The transport protein WtpA from P. furiosus binds tungstate more strongly than molybdate, and is related in sequence to Archaeoglobus fulgidus ModA, of which a crystal structure is known. We have measured A. fulgidus ModA with tungstate at the W L3 (2p3/2) edge, and compared the results with the refined crystal structure. XAS studies of anion binding are feasible even if only weak interactions are present, are biologically relevant, and give new insights in the spectroscopy.

  20. Preclinical Profile of BI 224436, a Novel HIV-1 Non-Catalytic-Site Integrase Inhibitor

    PubMed Central

    Amad, Ma'an; Bailey, Murray D.; Bethell, Richard; Bös, Michael; Bonneau, Pierre; Cordingley, Michael; Coulombe, René; Duan, Jianmin; Edwards, Paul; Faucher, Anne-Marie; Garneau, Michel; Jakalian, Araz; Kawai, Stephen; Lamorte, Louie; LaPlante, Steven; Luo, Laibin; Mason, Steve; Poupart, Marc-André; Rioux, Nathalie; Schroeder, Patricia; Simoneau, Bruno; Tremblay, Sonia; Tsantrizos, Youla; Witvrouw, Myriam; Yoakim, Christiane

    2014-01-01

    BI 224436 is an HIV-1 integrase inhibitor with effective antiviral activity that acts through a mechanism that is distinct from that of integrase strand transfer inhibitors (INSTIs). This 3-quinolineacetic acid derivative series was identified using an enzymatic integrase long terminal repeat (LTR) DNA 3′-processing assay. A combination of medicinal chemistry, parallel synthesis, and structure-guided drug design led to the identification of BI 224436 as a candidate for preclinical profiling. It has antiviral 50% effective concentrations (EC50s) of <15 nM against different HIV-1 laboratory strains and cellular cytotoxicity of >90 μM. BI 224436 also has a low, ∼2.1-fold decrease in antiviral potency in the presence of 50% human serum and, by virtue of a steep dose-response curve slope, exhibits serum-shifted EC95 values ranging between 22 and 75 nM. Passage of virus in the presence of inhibitor selected for either A128T, A128N, or L102F primary resistance substitutions, all mapping to a conserved allosteric pocket on the catalytic core of integrase. BI 224436 also retains full antiviral activity against recombinant viruses encoding INSTI resistance substitutions N155S, Q148H, and E92Q. In drug combination studies performed in cellular antiviral assays, BI 224436 displays an additive effect in combination with most approved antiretrovirals, including INSTIs. BI 224436 has drug-like in vitro absorption, distribution, metabolism, and excretion (ADME) properties, including Caco-2 cell permeability, solubility, and low cytochrome P450 inhibition. It exhibited excellent pharmacokinetic profiles in rat (clearance as a percentage of hepatic flow [CL], 0.7%; bioavailability [F], 54%), monkey (CL, 23%; F, 82%), and dog (CL, 8%; F, 81%). Based on the excellent biological and pharmacokinetic profile, BI 224436 was advanced into phase 1 clinical trials. PMID:24663024

  1. Comparison of non-catalytic and catalytic fast pyrolysis of corncob in a fluidized bed reactor.

    PubMed

    Zhang, Huiyan; Xiao, Rui; Huang, He; Xiao, Gang

    2009-02-01

    Fast pyrolysis of corncob with and without catalyst was investigated in a fluidized bed to determine the effects of pyrolysis parameters (temperature, gas flow rate, static bed height and particle size) and a HZSM-5 zeolite catalyst on the product yields and the qualities of the liquid products. The result showed that the optimal conditions for liquid yield (56.8%) were a pyrolysis temperature of 550 degrees C, gas flow rate of 3.4 L/min, static bed height of 10 cm and particle size of 1.0-2.0mm. The presence of the catalyst increased the yields of non-condensable gas, water and coke, while decreased the liquid and char yields. The elemental analysis showed that more than 25% decrease in oxygen content of the collected liquid in the second condenser with HZSM-5 was observed compared with that without catalyst. The H/C, O/C molar ratios and the higher heating value of the oil fraction in the collected liquid with the catalyst were 1.511, 0.149 and 34.6 MJ/kg, respectively. It was indicated that the collected liquid in the second condenser had high qualities and might be used as transport oil. PMID:18829306

  2. Non-cell autonomous and non-catalytic activities of ATX in the developing brain.

    PubMed

    Greenman, Raanan; Gorelik, Anna; Sapir, Tamar; Baumgart, Jan; Zamor, Vanessa; Segal-Salto, Michal; Levin-Zaidman, Smadar; Aidinis, Vassilis; Aoki, Junken; Nitsch, Robert; Vogt, Johannes; Reiner, Orly

    2015-01-01

    The intricate formation of the cerebral cortex requires a well-coordinated series of events, which are regulated at the level of cell-autonomous and non-cell autonomous mechanisms. Whereas cell-autonomous mechanisms that regulate cortical development are well-studied, the non-cell autonomous mechanisms remain poorly understood. A non-biased screen allowed us to identify Autotaxin (ATX) as a non-cell autonomous regulator of neural stem cells. ATX (also known as ENPP2) is best known to catalyze lysophosphatidic acid (LPA) production. Our results demonstrate that ATX affects the localization and adhesion of neuronal progenitors in a cell autonomous and non-cell autonomous manner, and strikingly, this activity is independent from its catalytic activity in producing LPA. PMID:25788872

  3. Non-catalytic liquefaction of coal with bagasse, a biomass waste

    SciTech Connect

    Rafiqul, I.; Lugang, B.; Yan, Y.; Li, T.

    1999-07-01

    Liquefaction of a Chinese bituminous coal with bagasse, a kind of biomass waste, have been carried out in an autoclave of 300 ml capacity at a temperature range of 350--450 C, reaction time 15--45 min and cool hydrogen pressure 300--700 PSIG (2.04 4.76 MPa). Optimization of the co-liquefaction process was done with respect to oil yield by Factorial Experiment Design Method. Oil yield reached 48% at optimum conditions of temperature: 420 C, cool hydrogen pressure: 500 PSIG and reaction time: 40 min. A polynomial mathematical model, a second order response surface model, has been obtained for correlating the oil yield response factor as well as conversion with the major process variables. The equation derived by the authors holds good in determining the effect of process variables on response factors for any regime conditions in the range of the Planned Experimental Design. Experimental data were also correlated by a kinetic model. The model is based on coal and bagasse, undergoing thermal cracking, is first converted parallel to form preasphaltene and asphaltene and low molecular gaseous products; then consecutively oil is formed from preasphaltene and asphaltene. Activation energies for these three reactions are 32.51 KJ/mol, 75.14 KJ/mol and 44.65 KJ/mol, respectively. These values are lower than that of liquefaction of coal alone. It justifies that the addition of bagasse is effective in enhancing the process of co-liquefaction and giving higher yield of oil than for liquefaction of coal alone. Calculated values from this model fairly agree with the experimental data.

  4. TET2 Regulates Mast Cell Differentiation and Proliferation through Catalytic and Non-catalytic Activities.

    PubMed

    Montagner, Sara; Leoni, Cristina; Emming, Stefan; Della Chiara, Giulia; Balestrieri, Chiara; Barozzi, Iros; Piccolo, Viviana; Togher, Susan; Ko, Myunggon; Rao, Anjana; Natoli, Gioacchino; Monticelli, Silvia

    2016-05-17

    Dioxygenases of the TET family impact genome functions by converting 5-methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine (5hmC). Here, we identified TET2 as a crucial regulator of mast cell differentiation and proliferation. In the absence of TET2, mast cells showed disrupted gene expression and altered genome-wide 5hmC deposition, especially at enhancers and in the proximity of downregulated genes. Impaired differentiation of Tet2-ablated cells could be relieved or further exacerbated by modulating the activity of other TET family members, and mechanistically it could be linked to the dysregulated expression of C/EBP family transcription factors. Conversely, the marked increase in proliferation induced by the loss of TET2 could be rescued exclusively by re-expression of wild-type or catalytically inactive TET2. Our data indicate that, in the absence of TET2, mast cell differentiation is under the control of compensatory mechanisms mediated by other TET family members, while proliferation is strictly dependent on TET2 expression. PMID:27160912

  5. A kinetic model of municipal sludge degradation during non-catalytic wet oxidation.

    PubMed

    Prince-Pike, Arrian; Wilson, David I; Baroutian, Saeid; Andrews, John; Gapes, Daniel J

    2015-12-15

    Wet oxidation is a successful process for the treatment of municipal sludge. In addition, the resulting effluent from wet oxidation is a useful carbon source for subsequent biological nutrient removal processes in wastewater treatment. Owing to limitations with current kinetic models, this study produced a kinetic model which predicts the concentrations of key intermediate components during wet oxidation. The model was regressed from lab-scale experiments and then subsequently validated using data from a wet oxidation pilot plant. The model was shown to be accurate in predicting the concentrations of each component, and produced good results when applied to a plant 500 times larger in size. A statistical study was undertaken to investigate the validity of the regressed model parameters. Finally the usefulness of the model was demonstrated by suggesting optimum operating conditions such that volatile fatty acids were maximised. PMID:26426294

  6. Catalytic and non-catalytic wet air oxidation of sodium dodecylbenzene sulfonate: kinetics and biodegradability enhancement.

    PubMed

    Suárez-Ojeda, María Eugenia; Kim, Jungkwon; Carrera, Julián; Metcalfe, Ian S; Font, Josep

    2007-06-18

    Wet air oxidation (WAO) and catalytic wet air oxidation (CWAO) were investigated as suitable precursors for the biological treatment of industrial wastewater containing sodium dodecylbenzene sulfonate (DBS). Two hours WAO semi-batch experiments were conducted at 15 bar of oxygen partial pressure (P(O2)) and at 180, 200 and 220 degrees C. It was found that the highest temperature provides appreciable total organic carbon (TOC) and chemical oxygen demand (COD) abatement of about 42 and 47%, correspondingly. Based on the main identified intermediates (acetic acid and sulfobenzoic acid) a reaction pathway for DBS and a kinetic model in WAO were proposed. In the case of CWAO experiments, seventy-two hours tests were done in a fixed bed reactor in continuous trickle flow regime, using a commercial activated carbon (AC) as catalyst. The temperature and P(O2) were 140-160 degrees C and 2-9 bar, respectively. The influence of the operating conditions on the DBS oxidation, the occurrence of oxidative coupling reactions over the AC, and the catalytic activity (in terms of substrate removal) were established. The results show that the AC without any supported active metal behaves bi-functional as adsorbent and catalyst, giving TOC conversions up to 52% at 160 degrees C and 2 bar of P(O2), which were comparable to those obtained in WAO experiments. Respirometric tests were completed before and after CWAO and to the main intermediates identified through the WAO and CWAO oxidation route. Then, the readily biodegradable COD (COD(RB)) of the CWAO and WAO effluents were found. Taking into account these results it was possible to compare whether or not the CWAO or WAO effluents were suitable for a conventional activated sludge plant inoculated with non adapted culture. PMID:17363148

  7. Thermodynamic and kinetic study of phenol degradation by a non-catalytic wet air oxidation process.

    PubMed

    Lefèvre, Sébastien; Boutin, Olivier; Ferrasse, Jean-Henry; Malleret, Laure; Faucherand, Rémy; Viand, Alain

    2011-08-01

    This work is dedicated to an accurate evaluation of thermodynamic and kinetics aspects of phenol degradation using wet air oxidation process. Phenol is a well known polluting molecule and therefore it is important having data of its behaviour during this process. A view cell is used for the experimental study, with an internal volume of 150 mL, able to reach pressures up to 30 MPa and temperatures up to 350°C. Concerning the thermodynamic phase equilibria, experimental and modelling results are obtained for different binary systems (water/nitrogen, water/air) and ternary system (water/nitrogen/phenol). The best model is the Predictive Soave Redlich Kwong one. This information is necessary to predict the composition of the gas phase during the process. It is also important for an implementation in a process simulation. The second part is dedicated to kinetics evaluation of the degradation of phenol. Different compounds have been detected using GC coupled with a MS. A kinetic scheme is deduced, taking into account the evolution of phenol, hydroquinones, catechol, resorcinol and acetic acid. The kinetic parameters are calculated for this scheme. These data are important to evaluate the evolution of the concentration of the different polluting molecules during the process. A simplified kinetic scheme, which can be easily implemented in a process simulation, is also determined for the direct degradation of phenol into H(2)O and CO(2). The Arrhenius law data obtained for the phenol disappearance are the following: k=1.8×10(6)±3.9×10(5)M(-1)s(-1) (pre-exponential factor) and E(a)=77±8 kJ mol(-1) (activation energy). PMID:21700312

  8. Biodiesel production from Jatropha oil by catalytic and non-catalytic approaches: an overview.

    PubMed

    Juan, Joon Ching; Kartika, Damayani Agung; Wu, Ta Yeong; Hin, Taufiq-Yap Yun

    2011-01-01

    Biodiesel (fatty acids alkyl esters) is a promising alternative fuel to replace petroleum-based diesel that is obtained from renewable sources such as vegetable oil, animal fat and waste cooking oil. Vegetable oils are more suitable source for biodiesel production compared to animal fats and waste cooking since they are renewable in nature. However, there is a concern that biodiesel production from vegetable oil would disturb the food market. Oil from Jatropha curcas is an acceptable choice for biodiesel production because it is non-edible and can be easily grown in a harsh environment. Moreover, alkyl esters of jatropha oil meet the standard of biodiesel in many countries. Thus, the present paper provides a review on the transesterification methods for biodiesel production using jatropha oil as feedstock. PMID:21094045

  9. DNA replication: polymerase epsilon as a non-catalytic converter of the helicase.

    PubMed

    Zegerman, Philip

    2013-04-01

    In eukaryotes DNA polymerase epsilon (ε) synthesises the leading DNA strand during replication. A new study provides insight into how this polymerase also functions independently of its enzyme activity to assemble and activate the replicative helicase. PMID:23578873

  10. Non-cell autonomous and non-catalytic activities of ATX in the developing brain

    PubMed Central

    Greenman, Raanan; Gorelik, Anna; Sapir, Tamar; Baumgart, Jan; Zamor, Vanessa; Segal-Salto, Michal; Levin-Zaidman, Smadar; Aidinis, Vassilis; Aoki, Junken; Nitsch, Robert; Vogt, Johannes; Reiner, Orly

    2015-01-01

    The intricate formation of the cerebral cortex requires a well-coordinated series of events, which are regulated at the level of cell-autonomous and non-cell autonomous mechanisms. Whereas cell-autonomous mechanisms that regulate cortical development are well-studied, the non-cell autonomous mechanisms remain poorly understood. A non-biased screen allowed us to identify Autotaxin (ATX) as a non-cell autonomous regulator of neural stem cells. ATX (also known as ENPP2) is best known to catalyze lysophosphatidic acid (LPA) production. Our results demonstrate that ATX affects the localization and adhesion of neuronal progenitors in a cell autonomous and non-cell autonomous manner, and strikingly, this activity is independent from its catalytic activity in producing LPA. PMID:25788872

  11. Non-catalytic heterogeneous biodiesel production via a continuous flow system.

    PubMed

    Kwon, Eilhann E; Yi, Haakrho; Park, Jongseok; Seo, Jaegun

    2012-06-01

    This study provides a novel methodology for biodiesel (FAME) production under ambient pressure, which resolves most drawbacks of current commercialized biodiesel conversion via the transesterification reaction. This has been achieved by means of a thermo-chemical process and a true continuous flow system. This also enables combination of esterification of free fatty acids (FFAs) and transesterification of triglycerides into a single process without utilizing a catalyst, and leads to a 98-99±0.5 % conversion efficiency of FAME within 1 min in the temperature range of 350-500 °C. High FFA content in oil feedstock is not a matter of the new process, which enables the use of a broader variety of feedstocks, including all edible and inedible fats. Another feature of this novel method is that it does not produce wastewater. Thus, the new process has potential to spur a breakthrough in the lowest cost of biodiesel production. Moreover, this method also requires utilization of carbon dioxide during biodiesel production, an additional environmental benefit. PMID:22520224

  12. Erythropoietin binding protein from mammalian serum

    DOEpatents

    Clemons, G.K.

    1997-04-29

    Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described. 11 figs.

  13. Erythropoietin binding protein from mammalian serum

    DOEpatents

    Clemons, Gisela K.

    1997-01-01

    Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described.

  14. Feature-Based Binding and Phase Theory

    ERIC Educational Resources Information Center

    Antonenko, Andrei

    2012-01-01

    Current theories of binding cannot provide a uniform account for many facts associated with the distribution of anaphors, such as long-distance binding effects and the subject-orientation of monomorphemic anaphors. Further, traditional binding theory is incompatible with minimalist assumptions. In this dissertation I propose an analysis of…

  15. Evolution of Protein-binding DNA Sequences through Competitive Binding

    NASA Astrophysics Data System (ADS)

    Peng, Weiqun; Gerland, Ulrich; Hwa, Terence; Levine, Herbert

    2002-03-01

    The dynamics of in vitro DNA evolution controlled via competitive binding of DNA sequences to proteins has been explored in a recent serial transfer experiment footnote B. Dubertret, S.Liu, Q. Ouyang, A. Libchaber, Phys. Rev. Lett. 86, 6022 (2001).. Motivated by the experiment, we investigate a continuum model for this evolution process in various parameter regimes. We establish a self-consistent mean-field evolution equation, determine its dynamical properties and finite population size corrections. In addition, we discuss the experimental implications of our results.

  16. Localization of the chaperone binding site

    NASA Technical Reports Server (NTRS)

    Boyle, D.; Gopalakrishnan, S.; Takemoto, L.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    The hypothesis derived from models of the multi-oligomeric chaperone complex suggests that partially denatured proteins bind in a central cavity in the aggregate. To test this hypothesis, the molecular chaperone, alpha crystallin, was bound to partially denatured forms of gamma crystallin, and the binding site was visualized by immunogold localization. In an alternative approach, gold particles were directly complexed with gamma crystallin, followed by binding to the alpha crystallin aggregate. In both cases, binding was localized to the central region of the aggregate, confirming for the first time that partially denatured proteins do indeed bind to a central region of the molecular chaperone aggregate.

  17. Synthetic heparin-binding factor analogs

    DOEpatents

    Pena, Louis A.; Zamora, Paul O.; Lin, Xinhua; Glass, John D.

    2010-04-20

    The invention provides synthetic heparin-binding growth factor analogs having at least one peptide chain, and preferably two peptide chains branched from a dipeptide branch moiety composed of two trifunctional amino acid residues, which peptide chain or chains bind a heparin-binding growth factor receptor and are covalently bound to a non-signaling peptide that includes a heparin-binding domain, preferably by a linker, which may be a hydrophobic linker. The synthetic heparin-binding growth factor analogs are useful as pharmaceutical agents, soluble biologics or as surface coatings for medical devices.

  18. Unraveling determinants of transcription factor binding outside the core binding site.

    PubMed

    Levo, Michal; Zalckvar, Einat; Sharon, Eilon; Dantas Machado, Ana Carolina; Kalma, Yael; Lotam-Pompan, Maya; Weinberger, Adina; Yakhini, Zohar; Rohs, Remo; Segal, Eran

    2015-07-01

    Binding of transcription factors (TFs) to regulatory sequences is a pivotal step in the control of gene expression. Despite many advances in the characterization of sequence motifs recognized by TFs, our ability to quantitatively predict TF binding to different regulatory sequences is still limited. Here, we present a novel experimental assay termed BunDLE-seq that provides quantitative measurements of TF binding to thousands of fully designed sequences of 200 bp in length within a single experiment. Applying this binding assay to two yeast TFs, we demonstrate that sequences outside the core TF binding site profoundly affect TF binding. We show that TF-specific models based on the sequence or DNA shape of the regions flanking the core binding site are highly predictive of the measured differential TF binding. We further characterize the dependence of TF binding, accounting for measurements of single and co-occurring binding events, on the number and location of binding sites and on the TF concentration. Finally, by coupling our in vitro TF binding measurements, and another application of our method probing nucleosome formation, to in vivo expression measurements carried out with the same template sequences serving as promoters, we offer insights into mechanisms that may determine the different expression outcomes observed. Our assay thus paves the way to a more comprehensive understanding of TF binding to regulatory sequences and allows the characterization of TF binding determinants within and outside of core binding sites. PMID:25762553

  19. Infinite sets and double binds.

    PubMed

    Arden, M

    1984-01-01

    There have been many attempts to bring psychoanalytical theory up to date. This paper approaches the problem by discussing the work of Gregory Bateson and Ignacio Matte-Blanco, with particular reference to the use made by these authors of Russell's theory of logical types. Bateson's theory of the double bind and Matte-Blanco's bilogic are both based on concepts of logical typing. It is argued that the two theories can be linked by the idea that neurotic symptoms are based on category errors in thinking. Clinical material is presented from the analysis of a middle-aged woman. The intention is to demonstrate that the process of making interpretations can be thought of as revealing errors in thinking. Changes in the patient's inner world are then seen to be the result of clarifying childhood experiences based on category errors. Matte-Blanco's theory of bilogic and infinite experiences is a re-evaluation of the place of the primary process in mental life. It is suggested that a combination of bilogic and double bind theory provides a possibility of reformulating psychoanalytical theory. PMID:6544755

  20. Comparing binding site information to binding affinity reveals that Crp/DNA complexes have several distinct binding conformers

    PubMed Central

    Holmquist, Peter C.; Holmquist, Gerald P.; Summers, Michael L.

    2011-01-01

    We show that the cAMP receptor protein (Crp) binds to DNA as several different conformers. This situation has precluded discovering a high correlation between any sequence property and binding affinity for proteins that bend DNA. Experimentally quantified affinities of Synechocystis sp. PCC 6803 cAMP receptor protein (SyCrp1), the Escherichia coli Crp (EcCrp, also CAP) and DNA were analyzed to mathematically describe, and make human-readable, the relationship of DNA sequence and binding affinity in a given system. Here, sequence logos and weight matrices were built to model SyCrp1 binding sequences. Comparing the weight matrix model to binding affinity revealed several distinct binding conformations. These Crp/DNA conformations were asymmetrical (non-palindromic). PMID:21586590

  1. Receptor-binding sites: bioinformatic approaches.

    PubMed

    Flower, Darren R

    2006-01-01

    It is increasingly clear that both transient and long-lasting interactions between biomacromolecules and their molecular partners are the most fundamental of all biological mechanisms and lie at the conceptual heart of protein function. In particular, the protein-binding site is the most fascinating and important mechanistic arbiter of protein function. In this review, I examine the nature of protein-binding sites found in both ligand-binding receptors and substrate-binding enzymes. I highlight two important concepts underlying the identification and analysis of binding sites. The first is based on knowledge: when one knows the location of a binding site in one protein, one can "inherit" the site from one protein to another. The second approach involves the a priori prediction of a binding site from a sequence or a structure. The full and complete analysis of binding sites will necessarily involve the full range of informatic techniques ranging from sequence-based bioinformatic analysis through structural bioinformatics to computational chemistry and molecular physics. Integration of both diverse experimental and diverse theoretical approaches is thus a mandatory requirement in the evaluation of binding sites and the binding events that occur within them. PMID:16671408

  2. Nucleotide-binding mechanisms in pseudokinases

    PubMed Central

    Hammarén, Henrik M.; Virtanen, Anniina T.; Silvennoinen, Olli

    2015-01-01

    Pseudokinases are classified by the lack of one or several of the highly conserved motifs involved in nucleotide (nt) binding or catalytic activity of protein kinases (PKs). Pseudokinases represent ∼10% of the human kinome and they are found in all evolutionary classes of kinases. It has become evident that pseudokinases, which were initially considered somewhat peculiar dead kinases, are important components in several signalling cascades. Furthermore, several pseudokinases have been linked to human diseases, particularly cancer, which is raising interest for therapeutic approaches towards these proteins. The ATP-binding pocket is a well-established drug target and elucidation of the mechanism and properties of nt binding in pseudokinases is of significant interest and importance. Recent studies have demonstrated that members of the pseudokinase family are very diverse in structure as well as in their ability and mechanism to bind nts or perform phosphoryl transfer reactions. This diversity also precludes prediction of pseudokinase function, or the importance of nt binding for said function, based on primary sequence alone. Currently available data indicate that ∼40% of pseudokinases are able to bind nts, whereas only few are able to catalyse occasional phosphoryl transfer. Pseudokinases employ diverse mechanisms to bind nts, which usually occurs at low, but physiological, affinity. ATP binding serves often a structural role but in most cases the functional roles are not precisely known. In the present review, we discuss the various mechanisms that pseudokinases employ for nt binding and how this often low-affinity binding can be accurately analysed. PMID:26589967

  3. Synthetic LPS-Binding Polymer Nanoparticles

    NASA Astrophysics Data System (ADS)

    Jiang, Tian

    Lipopolysaccharide (LPS), one of the principal components of most gram-negative bacteria's outer membrane, is a type of contaminant that can be frequently found in recombinant DNA products. Because of its strong and even lethal biological effects, selective LPS removal from bioproducts solution is of particular importance in the pharmaceutical and health care industries. In this thesis, for the first time, a proof-of-concept study on preparing LPS-binding hydrogel-like NPs through facile one-step free-radical polymerization was presented. With the incorporation of various hydrophobic (TBAm), cationic (APM, GUA) monomers and cross-linkers (BIS, PEG), a small library of NPs was constructed. Their FITC-LPS binding behaviors were investigated and compared with those of commercially available LPS-binding products. Moreover, the LPS binding selectivity of the NPs was also explored by studying the NPs-BSA interactions. The results showed that all NPs obtained generally presented higher FITC-LPS binding capacity in lower ionic strength buffer than higher ionic strength. However, unlike commercial poly-lysine cellulose and polymyxin B agarose beads' nearly linear increase of FITC-LPS binding with particle concentration, NPs exhibited serious aggregation and the binding quickly saturated or even decreased at high particle concentration. Among various types of NPs, higher FITC-LPS binding capacity was observed for those containing more hydrophobic monomers (TBAm). However, surprisingly, more cationic NPs with higher content of APM exhibited decreased FITC-LPS binding in high ionic strength conditions. Additionally, when new cationic monomer and cross-linker, GUA and PEG, were applied to replace APM and BIS, the obtained NPs showed improved FITC-LPS binding capacity at low NP concentration. But compared with APM- and BIS-containing NPs, the FITC-LPS binding capacity of GUA- and PEG-containing NPs saturated earlier. To investigate the NPs' binding to proteins, we tested the NPs

  4. Hemoglobin binding to deglycosylated haptoglobin.

    PubMed

    Kaartinen, V; Mononen, I

    1988-04-14

    The carbohydrate portion of polymeric haptoglobin was gradually removed by exoglycosidases in order to investigate its role in complex formation between haptoglobin and hemoglobin. Total removal of sialic acid diminished the haptoglobin-hemoglobin complex formation 15%. Removal of about 25% of the galactose residues from asialohaptoglobin, i.e., about 40% of the total weight of the carbohydrate moiety, totally inhibited the ability of haptoglobin to form complex with hemoglobin and react with haptoglobin-specific antibodies. Liberation of further galactose residues resulted in slow precipitation of the protein. Removal of a similar part of the carbohydrate moiety from haptoglobin-hemoglobin complex did not liberate hemoglobin from it, and the complex reacted with haptoglobin antibodies. The combined data indicate that the carbohydrate portion is essential for the functionally active form of polymeric haptoglobin to complex with hemoglobin, but it hardly has any direct role in the binding event, and other factors are responsible for the stability of the complex. PMID:3128331

  5. DNA Binding Hydroxyl Radical Probes

    PubMed Central

    Tang, Vicky J; Konigsfeld, Katie M; Aguilera, Joe A; Milligan, Jamie R

    2011-01-01

    The hydroxyl radical is the primary mediator of DNA damage by the indirect effect of ionizing radiation. It is a powerful oxidizing agent produced by the radiolysis of water and is responsible for a significant fraction of the DNA damage associated with ionizing radiation. There is therefore an interest in the development of sensitive assays for its detection. The hydroxylation of aromatic groups to produce fluorescent products has been used for this purpose. We have examined four different chromophores which produce fluorescent products when hydroxylated. Of these, the coumarin system suffers from the fewest disadvantages. We have therefore examined its behavior when linked to a cationic peptide ligand designed to bind strongly to DNA. PMID:22125376

  6. An RNA motif that binds ATP

    NASA Technical Reports Server (NTRS)

    Sassanfar, M.; Szostak, J. W.

    1993-01-01

    RNAs that contain specific high-affinity binding sites for small molecule ligands immobilized on a solid support are present at a frequency of roughly one in 10(10)-10(11) in pools of random sequence RNA molecules. Here we describe a new in vitro selection procedure designed to ensure the isolation of RNAs that bind the ligand of interest in solution as well as on a solid support. We have used this method to isolate a remarkably small RNA motif that binds ATP, a substrate in numerous biological reactions and the universal biological high-energy intermediate. The selected ATP-binding RNAs contain a consensus sequence, embedded in a common secondary structure. The binding properties of ATP analogues and modified RNAs show that the binding interaction is characterized by a large number of close contacts between the ATP and RNA, and by a change in the conformation of the RNA.

  7. Secretin: specific binding to rat brain membranes

    SciTech Connect

    Fremeau, R.T. Jr.; Jensen, R.T.; Charlton, C.G.; Miller, R.L.; O'Donohue, T.L.; Moody, T.W.

    1983-08-01

    The binding of (/sup 125/I)secretin to rat brain membranes was investigated. Radiolabeled secretin bound with high affinity (KD . 0.2 nM) to a single class of noninteracting sites. Binding was specific, saturable, and reversible. Regional distribution studies indicated that the specific binding was greatest in the cerebellum, intermediate in the cortex, thalamus, striatum, hippocampus, and hypothalamus, and lowest in the midbrain and medulla/pons. Pharmacological studies indicated that only secretin, but not other peptides, inhibits binding of (/sup 125/I)secretin with high affinity. Also, certain guanine nucleotides inhibited high affinity binding. These data indicate that rat brain membranes possess high affinity binding sites specific for secretin and that with the use of (/sup 125/I) secretin the kinetics, stoichiometry, specificity, and distribution of secretin receptors can be directly investigated.

  8. Binding of TH-iloprost to rat gastric mucosa: a pitfall in performing radioligand binding assays

    SciTech Connect

    Beinborn, M.; Kromer, W.; Staar, U.; Sewing, K.F.

    1985-09-01

    Binding of TH-iloprost was studied in a 20,000 x g sediment of the rat gastric mucosa. When pH in both test tubes for total and non-specific binding was kept identical, no displaceable binding of iloprost could be detected. When no care was taken to keep the pH identical in corresponding test tubes of the binding assay, changes in pH simulated specific and displaceable binding of iloprost. Therefore it is concluded that - in contrast to earlier reports - it is not possible to demonstrate specific iloprost binding using the given method.

  9. Calcium-binding proteins and development

    NASA Technical Reports Server (NTRS)

    Beckingham, K.; Lu, A. Q.; Andruss, B. F.; McIntire, L. V. (Principal Investigator)

    1998-01-01

    The known roles for calcium-binding proteins in developmental signaling pathways are reviewed. Current information on the calcium-binding characteristics of three classes of cell-surface developmental signaling proteins (EGF-domain proteins, cadherins and integrins) is presented together with an overview of the intracellular pathways downstream of these surface receptors. The developmental roles delineated to date for the universal intracellular calcium sensor, calmodulin, and its targets, and for calcium-binding regulators of the cytoskeleton are also reviewed.

  10. Binding Efficiency of Protein-Protein Complexes

    PubMed Central

    Day, Eric S.; Cote, Shaun M.; Whitty, Adrian

    2012-01-01

    We examine the relationship between binding affinity and interface size for reversible protein-protein interactions (PPI), using cytokines from the tumor necrosis factor (TNF) superfamily and their receptors as a test case. Using surface plasmon resonance, we measured single-site binding affinities for the large receptor TNFR1 binding to its ligands TNFα (KD = 1.4 ± 0.4 nM) and lymphotoxin-α (KD = 50 ± 10 nM), and also for the small receptor Fn14 binding to TWEAK (KD = 70 ± 10 nM). We additionally assembled data for all other TNF/TNFR family complexes for which reliable single site binding affinities have been reported. We used these values to calculate the binding efficiency – defined as binding energy per Å2 of surface area buried at the contact interface – for the nine of these complexes for which co-crystal structures are available, and compared the results to those for a set of 144 protein-protein complexes with published affinity values. The results show that the most efficient PPI complexes generate ~20 cal.mol−1/Å2 of binding energy. A minimum contact area of ~500 Å2 is required for a stable complex, required to generate sufficient interaction energy to pay the entropic cost of co-localizing two proteins from 1 M solution. The most compact and efficient TNF/TNFR complex was BAFF/BR3, which achieved ~80% of the maximum achievable binding efficiency. Other small receptors also gave high binding efficiencies, while the larger receptors generated only 44-49% of this limit despite interacting primarily through just a single small domain. The results provide new insight into how much binding energy can be generated by a PPI interface of a given size, and establish a quantitative method to predict how large a natural or engineered contact interface must be to achieve a given level of binding affinity. PMID:23088250

  11. Mutated Leguminous Lectin Containing a Heparin-Binding like Motif in a Carbohydrate-Binding Loop Specifically Binds to Heparin

    PubMed Central

    Abo, Hirohito; Soga, Keisuke; Tanaka, Atsuhiro; Tateno, Hiroaki; Hirabayashi, Jun; Yamamoto, Kazuo

    2015-01-01

    We previously introduced random mutations in the sugar-binding loops of a leguminous lectin and screened the resulting mutated lectins for novel specificities using cell surface display. Screening of a mutated peanut agglutinin (PNA), revealed a mutated PNA with a distinct preference for heparin. Glycan microarray analyses using the mutated lectin fused to the Fc region of human immunoglobulin, revealed that a particular sulfated glycosaminoglycan (GAG), heparin, had the highest binding affinity for mutated PNA among 97 glycans tested, although wild-type PNA showed affinity towards Galβ1-3GalNAc and similar galactosylated glycans. Further analyses of binding specificity using an enzyme-linked immunoadsorbent assay demonstrated that the mutated PNA specifically binds to heparin, and weakly to de-2-O-sulfated heparin, but not to other GAG chains including de-6-O-sulfated and de-N-sulfated heparins. The mutated PNA had six amino acid substitutions within the eight amino acid-long sugar-binding loop. In this loop, the heparin-binding like motif comprised three arginine residues at positions 124, 128, and 129, and a histidine at position 125 was present. Substitution of each arginine or histidine residue to alanine reduced heparin-binding ability, indicating that all of these basic amino acid residues contributed to heparin binding. Inhibition assay demonstrated that heparin and dextran sulfate strongly inhibited mutated PNA binding to heparin in dose-dependent manner. The mutated PNA could distinguish between CHO cells and proteoglycan-deficient mutant cells. This is the first report establishing a novel leguminous lectin that preferentially binds to highly sulfated heparin and may provide novel GAG-binding probes to distinguish between heterogeneous GAG repeating units. PMID:26714191

  12. Evolution of Protein Binding Modes in Homooligomers

    PubMed Central

    Dayhoff, Judith E.; Shoemaker, Benjamin A.; Bryant, Stephen H.; Panchenko, Anna R.

    2009-01-01

    The evolution of protein interactions cannot be deciphered without a detailed analysis of interaction interfaces and binding modes. We performed a large-scale study of protein homooligomers in terms of their symmetry, interface sizes, and conservation of binding modes. We also focused specifically on the evolution of protein binding modes from nine families of homooligomers and mapped 60 different binding modes and oligomerization states onto the phylogenetic trees of these families. We observed a significant tendency for the same binding modes to be clustered together and conserved within clades on phylogenetic trees; this trend is especially pronounced for close homologs with 70% sequence identity or higher. Some binding modes are conserved among very distant homologs, pointing to their ancient evolutionary origin, while others are very specific for a certain phylogenetic group. Moreover, we found that the most ancient binding modes have a tendency to involve symmetrical (isologous) homodimer binding arrangements with larger interfaces, while recently evolved binding modes more often exhibit asymmetrical arrangements and smaller interfaces. PMID:19879880

  13. Radiation abolishes inducer binding to lactose repressor.

    PubMed

    Gillard, Nathalie; Spotheim-Maurizot, Mélanie; Charlier, Michel

    2005-04-01

    The lactose operon functions under the control of the repressor-operator system. Binding of the repressor to the operator prevents the expression of the structural genes. This interaction can be destroyed by the binding of an inducer to the repressor. If ionizing radiations damage the partners, a dramatic dysfunction of the regulation system may be expected. We showed previously that gamma irradiation hinders repressor-operator binding through protein damage. Here we show that irradiation of the repressor abolishes the binding of the gratuitous inducer isopropyl-1-beta-D-thiogalactoside (IPTG) to the repressor. The observed lack of release of the repressor from the complex results from the loss of the ability of the inducer to bind to the repressor due to the destruction of the IPTG binding site. Fluorescence measurements show that both tryptophan residues located in or near the IPTG binding site are damaged. Since tryptophan damage is strongly correlated with the loss of IPTG binding ability, we conclude that it plays a critical role in the effect. A model was built that takes into account the kinetic analysis of damage production and the observed protection of its binding site by IPTG. This model satisfactorily accounts for the experimental results and allows us to understand the radiation-induced effects. PMID:15799700

  14. Fundamental considerations in ski binding analysis.

    PubMed

    Mote, C D; Hull, M L

    1976-01-01

    1. The static adjustment of a ski binding by hand or by available machines is only an adjustment and is neither a static nor a dynamic evaluation of the binding design. Bindings of different design with identical static adjustments will perform differently in environments in which the forces are static or dynamic. 2. The concept of binding release force is a useful measure of binding adjustment, but it is inappropriate as a criterion for binding evaluation. First, it does not direct attention toward the injury causing mechanism, strain, or displacement in the leg. Second, it is only part of the evaluation in dynamic problems. 3. The binding release decision in present bindings is displacement controlled. The relative displacement of the boot and ski is the system variable. For any specified relative displacement the binding force can be any of an infinite number of possibilities determined by the loading path. 4. The response of the leg-ski system to external impulses applied to the ski is independent of the boot-ski relative motion as long as the boot recenters quickly in the binding. Response is dependent upon the external impulse plus system inertia, damping and stiffness. 5. When tested under half sinusoidal forces applied to a test ski, all bindings will demonstrate static and impulse loading regions. In the static region the force drives the binding to a relative release displacement. In the impulse region the initial velocity of the ski drives the binding to a release displacement. 6. The transition between the static and impulse loading regions is determined by the binding's capacity to store and dissipate energy along the principal loading path. Increased energy capacity necessitates larger external impulses to produce release. 7. In all bindings examined to date, the transmitted leg displacement or strain at release under static loading exceeds leg strain under dynamic or impact loading. Because static loading is responsible for many injuries, a skier

  15. Lack of [3H]quinuclidinyl benzylate binding to biologically relevant binding sites on mononuclear cells.

    PubMed

    Adams, E M; Lubrano, T M; Gordon, J; Fields, J Z

    1992-09-01

    We analyzed the binding characteristics of [3H]quinuclidinyl benzylate ([3H]QNB), a muscarinic cholinergic ligand, to rat and human mononuclear cells (MNC). Under various assay conditions, atropine-sensitive, saturable binding occurred with an apparent Kd of 10 nM. Conditions which disrupted the MNC membrane reduced total binding and eliminated specific binding. Muscarinic agonists were unable to inhibit [3H]QNB binding to MNC at concentrations up to 10(-2) M. Stereoisomers dexetimide and levetimide were equipotent inhibitors of binding (IC50 2 x 10(-5) M). We conclude that, although atropine-sensitive binding of [3H]QNB to MNC occurs, the binding is not consistent with the presence of a biologically relevant muscarinic cholinergic receptor. PMID:1392105

  16. New DNA-binding radioprotectors

    NASA Astrophysics Data System (ADS)

    Martin, Roger

    The normal tissue damage associated with cancer radiotherapy has motivated the development at Peter Mac of a new class of DNA-binding radioprotecting drugs that could be applied top-ically to normal tissues at risk. Methylproamine (MP), the lead compound, reduces radiation induced cell kill at low concentrations. For example, experiments comparing the clonogenic survival of transformed human keratinocytes treated with 30 micromolar MP before and dur-ing various doses of ionising radiation, with the radiation dose response for untreated cells, indicate a dose reduction factor (DRF) of 2. Similar survival curve experiments using various concentrations of MP, with parallel measurements of uptake of MP into cell nuclei, have en-abled the relationship between drug uptake and extent of radioprotection to be established. Radioprotection has also been demonstrated after systemic administration to mice, for three different endpoints, namely lung, jejunum and bone marrow (survival at 30 days post-TBI). The results of pulse radiolysis studies indicated that the drugs act by reduction of transient radiation-induced oxidative species on DNA. This hypothesis was substantiated by the results of experiments in which MP radioprotection of radiation-induced DNA double-strand breaks, assessed as -H2AX foci, in the human keratinocyte cell line. For both endpoints, the extent of radioprotection increased with MP concentration up to a maximal value. These results are consistent with the hypothesis that radioprotection by MP is mediated by attenuation of the extent of initial DNA damage. However, although MP is a potent radioprotector, it becomes cytotoxic at higher concentrations. This limitation has been addressed in an extensive program of lead optimisation and some promising analogues have emerged from which the next lead will be selected. Given the clinical potential of topical radioprotection, the new analogues are being assessed in terms of delivery to mouse oral mucosa. This is

  17. Methods for Improving Aptamer Binding Affinity.

    PubMed

    Hasegawa, Hijiri; Savory, Nasa; Abe, Koichi; Ikebukuro, Kazunori

    2016-01-01

    Aptamers are single stranded oligonucleotides that bind a wide range of biological targets. Although aptamers can be isolated from pools of random sequence oligonucleotides using affinity-based selection, aptamers with high affinities are not always obtained. Therefore, further refinement of aptamers is required to achieve desired binding affinities. The optimization of primary sequences and stabilization of aptamer conformations are the main approaches to refining the binding properties of aptamers. In particular, sequence optimization using combined in silico sequence recombinations and in vitro functional evaluations is effective for the improvement of binding affinities, however, the binding affinities of aptamers are limited by the low hydrophobicity of nucleic acids. Accordingly, introduction of hydrophobic moieties into aptamers expands the diversity of interactions between aptamers and targets. Moreover, construction of multivalent aptamers by connecting aptamers that recognize distinct epitopes is an attractive approach to substantial increases in binding affinity. In addition, binding affinities can be tuned by optimizing the scaffolds of multivalent constructs. In this review, we summarize the various techniques for improving the binding affinities of aptamers. PMID:27043498

  18. Multiple instance learning of Calmodulin binding sites

    PubMed Central

    Minhas, Fayyaz ul Amir Afsar; Ben-Hur, Asa

    2012-01-01

    Motivation: Calmodulin (CaM) is a ubiquitously conserved protein that acts as a calcium sensor, and interacts with a large number of proteins. Detection of CaM binding proteins and their interaction sites experimentally requires a significant effort, so accurate methods for their prediction are important. Results: We present a novel algorithm (MI-1 SVM) for binding site prediction and evaluate its performance on a set of CaM-binding proteins extracted from the Calmodulin Target Database. Our approach directly models the problem of binding site prediction as a large-margin classification problem, and is able to take into account uncertainty in binding site location. We show that the proposed algorithm performs better than the standard SVM formulation, and illustrate its ability to recover known CaM binding motifs. A highly accurate cascaded classification approach using the proposed binding site prediction method to predict CaM binding proteins in Arabidopsis thaliana is also presented. Availability: Matlab code for training MI-1 SVM and the cascaded classification approach is available on request. Contact: fayyazafsar@gmail.com or asa@cs.colostate.edu PMID:22962461

  19. Backbone Dynamics Of Intracellular Lipid Binding Proteins

    NASA Astrophysics Data System (ADS)

    Gutiérrez-González, Luis H.

    2005-04-01

    The family of intracellular lipid binding proteins (iLBPs) comprises a group of homologous 14-15 kDa proteins that specifically bind and facilitate the transport of fatty acids, bile acids, retinoids or eicosanoids. Members of this family include several types of fatty acid binding proteins (FABPs), ileal lipid binding protein, cellular retinoic acid binding proteins and cellular retinoid binding proteins. As a contribution to understanding the structure-function relationship in this protein family, the solution structure and backbone dynamics of human epidermal-type FABP (E-FABP) determined by NMR spectroscopy are reported. Moreover, hydrogen/deuterium exchange experiments indicated a direct correlation between the stability of the hydrogen-bonding network in the β-sheet structure and the conformational exchange in the millisecond-to-microsecond time range. The features of E-FABP backbone dynamics discussed in the present study are compared with those obtained for other phylogenetically related proteins. A strong interdependence with the overall protein stability and possibly also with the ligand-binding affinity for members of the lipid-binding protein family is shown.

  20. Biodiscovery of aluminum binding peptides

    NASA Astrophysics Data System (ADS)

    Adams, Bryn L.; Sarkes, Deborah A.; Finch, Amethist S.; Hurley, Margaret M.; Stratis-Cullum, Dimitra

    2013-05-01

    Cell surface peptide display systems are large and diverse libraries of peptides (7-15 amino acids) which are presented by a display scaffold hosted by a phage (virus), bacteria, or yeast cell. This allows the selfsustaining peptide libraries to be rapidly screened for high affinity binders to a given target of interest, and those binders quickly identified. Peptide display systems have traditionally been utilized in conjunction with organic-based targets, such as protein toxins or carbon nanotubes. However, this technology has been expanded for use with inorganic targets, such as metals, for biofabrication, hybrid material assembly and corrosion prevention. While most current peptide display systems employ viruses to host the display scaffold, we have recently shown that a bacterial host, Escherichia coli, displaying peptides in the ubiquitous, membrane protein scaffold eCPX can also provide specific peptide binders to an organic target. We have, for the first time, extended the use of this bacterial peptide display system for the biodiscovery of aluminum binding 15mer peptides. We will present the process of biopanning with macroscopic inorganic targets, binder enrichment, and binder isolation and discovery.

  1. Haptenation: Chemical Reactivity and Protein Binding

    PubMed Central

    Chipinda, Itai; Hettick, Justin M.; Siegel, Paul D.

    2011-01-01

    Low molecular weight chemical (LMW) allergens are commonly referred to as haptens. Haptens must complex with proteins to be recognized by the immune system. The majority of occupationally related haptens are reactive, electrophilic chemicals, or are metabolized to reactive metabolites that form covalent bonds with nucleophilic centers on proteins. Nonelectrophilic protein binding may occur through disulfide exchange, coordinate covalent binding onto metal ions on metalloproteins or of metal allergens, themselves, to the major histocompatibility complex. Recent chemical reactivity kinetic studies suggest that the rate of protein binding is a major determinant of allergenic potency; however, electrophilic strength does not seem to predict the ability of a hapten to skew the response between Th1 and Th2. Modern proteomic mass spectrometry methods that allow detailed delineation of potential differences in protein binding sites may be valuable in predicting if a chemical will stimulate an immediate or delayed hypersensitivity. Chemical aspects related to both reactivity and protein-specific binding are discussed. PMID:21785613

  2. Nonphysiological binding of ethylene by plants.

    PubMed

    Abeles, F B

    1984-03-01

    Ethylene binding to seedling tissue of Vicia faba, Phaseolus vulgaris, Glycine max, and Triticum aestivum was demonstrated by determining transit time required for ethylene to move through a glass tube filled with seedling tissue. Transit time for ethylene was greater than that for methane indicating that these tissues had an affinity for ethylene. However, the following observations suggest that the binding was not physiological. Inhibitors of ethylene action such as Ag(+) ions and CO(2) did not decrease binding. Mushrooms which have no known sites of ethylene action also demonstrated ethylene binding. The binding of acetylene, propylene, ethylene, propane, and ethane more closely followed their solubility in water than any known physiological activity. PMID:16663455

  3. Nonphysiological Binding of Ethylene by Plants

    PubMed Central

    Abeles, Fred B.

    1984-01-01

    Ethylene binding to seedling tissue of Vicia faba, Phaseolus vulgaris, Glycine max, and Triticum aestivum was demonstrated by determining transit time required for ethylene to move through a glass tube filled with seedling tissue. Transit time for ethylene was greater than that for methane indicating that these tissues had an affinity for ethylene. However, the following observations suggest that the binding was not physiological. Inhibitors of ethylene action such as Ag+ ions and CO2 did not decrease binding. Mushrooms which have no known sites of ethylene action also demonstrated ethylene binding. The binding of acetylene, propylene, ethylene, propane, and ethane more closely followed their solubility in water than any known physiological activity. PMID:16663455

  4. Ethylene binding site affinity in ripening apples

    SciTech Connect

    Blankenship, S.M. . Dept. of Horticultural Science); Sisler, E.C. )

    1993-09-01

    Scatchard plots for ethylene binding in apples (Malus domestica Borkh.), which were harvested weekly for 5 weeks to include the ethylene climacteric rise, showed C[sub 50] values (concentration of ethylene needed to occupy 50% of the ethylene binding sites) of 0.10, 0.11, 0.34, 0.40, and 0.57 [mu]l ethylene/liter[sup [minus]1], respectively, for each of the 5 weeks. Higher ethylene concentrations were required to saturate the binding sites during the climacteric rise than at other times. Diffusion of [sup 14]C-ethylene from the binding sites was curvilinear and did not show any indication of multiple binding sites. Ethylene was not metabolized by apple tissue.

  5. Follitropin receptors contain cryptic ligand binding sites.

    PubMed

    Lin, Win; Bernard, Michael P; Cao, Donghui; Myers, Rebecca V; Kerrigan, John E; Moyle, William R

    2007-01-01

    Human choriogonadotropin (hCG) and follitropin (hFSH) have been shown to contact different regions of the extracellular domains of G-protein coupled lutropin (LHR) and follitropin (FSHR) receptors. We report here that hCG and hFSH analogs interact with different regions of an FSHR/LHR chimera having only two unique LHR residues and that binds both hormones with high affinity. hCG and hFSH analogs dock with this receptor chimera in a manner similar to that in which they bind LHR and FSHR, respectively. This shows that although the FSHR does not normally bind hCG, it contains a cryptic lutropin binding site that has the potential to recognize hCG in a manner similar to the LHR. The presence of this cryptic site may explain why equine lutropins bind many mammalian FSHR and why mutations in the transmembrane domain distant from the extracellular domain enable the FSHR to bind hCG. The leucine-rich repeat domain (LRD) of the FSHR also appears to contain a cryptic FSH binding site that is obscured by other parts of the extracellular domain. This will explain why contacts seen in crystals of hFSH complexed with an LRD fragment of the human FSHR are hard to reconcile with the abilities of FSH analogs to interact with membrane G-protein coupled FSHR. We speculate that cryptic lutropin binding sites in the FSHR, which are also likely to be present in thyrotropin receptors (TSHR), permit the physiological regulation of ligand binding specificity. Cryptic FSH binding sites in the LRD may enable alternate spliced forms of the FSHR to interact with FSH. PMID:17059863

  6. Improving Binding Affinity and Selectivity of Computationally Designed Ligand-Binding Proteins Using Experiments.

    PubMed

    Tinberg, Christine E; Khare, Sagar D

    2016-01-01

    The ability to de novo design proteins that can bind small molecules has wide implications for synthetic biology and medicine. Combining computational protein design with the high-throughput screening of mutagenic libraries of computationally designed proteins is emerging as a general approach for creating binding proteins with programmable binding modes, affinities, and selectivities. The computational step enables the creation of a binding site in a protein that otherwise does not (measurably) bind the intended ligand, and targeted mutagenic screening allows for validation and refinement of the computational model as well as provides orders-of-magnitude increases in the binding affinity. Deep sequencing of mutagenic libraries can provide insights into the mutagenic binding landscape and enable further affinity improvements. Moreover, in such a combined computational-experimental approach where the binding mode is preprogrammed and iteratively refined, selectivity can be achieved (and modulated) by the placement of specified amino acid side chain groups around the ligand in defined orientations. Here, we describe the experimental aspects of a combined computational-experimental approach for designing-using the software suite Rosetta-proteins that bind a small molecule of choice and engineering, using fluorescence-activated cell sorting and high-throughput yeast surface display, high affinity and ligand selectivity. We illustrated the utility of this approach by performing the design of a selective digoxigenin (DIG)-binding protein that, after affinity maturation, binds DIG with picomolar affinity and high selectivity over structurally related steroids. PMID:27094290

  7. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    SciTech Connect

    Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya; Apicella, Michael A.; Ramaswamy, S.

    2014-07-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

  8. The TRPV5/6 calcium channels contain multiple calmodulin binding sites with differential binding properties.

    PubMed

    Kovalevskaya, Nadezda V; Bokhovchuk, Fedir M; Vuister, Geerten W

    2012-06-01

    The epithelial Ca(2+) channels TRPV5/6 (transient receptor potential vanilloid 5/6) are thoroughly regulated in order to fine-tune the amount of Ca(2+) reabsorption. Calmodulin has been shown to be involved into calcium-dependent inactivation of TRPV5/6 channels by binding directly to the distal C-terminal fragment of the channels (de Groot et al. in Mol Cell Biol 31:2845-2853, 12). Here, we investigate this binding in detail and find significant differences between TRPV5 and TRPV6. We also identify and characterize in vitro four other CaM binding fragments of TRPV5/6, which likely are also involved in TRPV5/6 channel regulation. The five CaM binding sites display diversity in binding modes, binding stoichiometries and binding affinities, which may fine-tune the response of the channels to varying Ca(2+)-concentrations. PMID:22354706

  9. Thermodynamic binding constants for gallium transferrin

    SciTech Connect

    Harris, W.R.; Pecoraro, V.L.

    1983-01-18

    Gallium-67 is widely used as an imaging agent for tumors and inflammatory abscesses. It is well stablished that Ga/sup 3 +/ travels through the circulatory system bound to the serum iron transport protein transferrin and that this protein binding is an essential step in tumor localization. However, there have been conflicting reports on the magnitude of the gallium-transferrin binding constants. Therefore, thermodynamic binding constants for gallium complexation at the two specific metal binding sites of human serum transferrin at pH 7.4 and 5 mM NaHCO/sub 3/ have been determined by UV difference spectroscopy. The conditional constants calculated for 27 mM NaHCO/sub 3/ are log K/sub 1/* = 20.3 and log K/sub 2/* = 19.3. These results are discussed in relation to the thermodynamics of transferrin binding of Fe/sup 3 +/ and to previous reports on gallium binding. The strength of transferrin complexation is also compared to that of a series of low molecular weight ligands by using calculated pM values (pM = -log (Ga(H/sub 2/O)/sub 6/)) to express the effective binding strength at pH 7.4.

  10. Copper(II) binding properties of hepcidin.

    PubMed

    Kulprachakarn, Kanokwan; Chen, Yu-Lin; Kong, Xiaole; Arno, Maria C; Hider, Robert C; Srichairatanakool, Somdet; Bansal, Sukhvinder S

    2016-06-01

    Hepcidin is a peptide hormone that regulates the homeostasis of iron metabolism. The N-terminal domain of hepcidin is conserved amongst a range of species and is capable of binding Cu(II) and Ni(II) through the amino terminal copper-nickel binding motif (ATCUN). It has been suggested that the binding of copper to hepcidin may have biological relevance. In this study we have investigated the binding of Cu(II) with model peptides containing the ATCUN motif, fluorescently labelled hepcidin and hepcidin using MALDI-TOF mass spectrometry. As with albumin, it was found that tetrapeptide models of hepcidin possessed a higher affinity for Cu(II) than that of native hepcidin. The log K 1 value of hepcidin for Cu(II) was determined as 7.7. Cu(II) binds to albumin more tightly than hepcidin (log K 1 = 12) and in view of the serum concentration difference of albumin and hepcidin, the bulk of kinetically labile Cu(II) present in blood will be bound to albumin. It is estimated that the concentration of Cu(II)-hepcidin will be less than one femtomolar in normal serum and thus the binding of copper to hepcidin is unlikely to play a role in iron homeostasis. As with albumin, small tri and tetra peptides are poor models for the metal binding properties of hepcidin. PMID:26883683

  11. Calmodulin Binding Proteins and Alzheimer's Disease.

    PubMed

    O'Day, Danton H; Eshak, Kristeen; Myre, Michael A

    2015-01-01

    The small, calcium-sensor protein, calmodulin, is ubiquitously expressed and central to cell function in all cell types. Here the literature linking calmodulin to Alzheimer's disease is reviewed. Several experimentally-verified calmodulin-binding proteins are involved in the formation of amyloid-β plaques including amyloid-β protein precursor, β-secretase, presenilin-1, and ADAM10. Many others possess potential calmodulin-binding domains that remain to be verified. Three calmodulin binding proteins are associated with the formation of neurofibrillary tangles: two kinases (CaMKII, CDK5) and one protein phosphatase (PP2B or calcineurin). Many of the genes recently identified by genome wide association studies and other studies encode proteins that contain putative calmodulin-binding domains but only a couple (e.g., APOE, BIN1) have been experimentally confirmed as calmodulin binding proteins. At least two receptors involved in calcium metabolism and linked to Alzheimer's disease (mAchR; NMDAR) have also been identified as calmodulin-binding proteins. In addition to this, many proteins that are involved in other cellular events intimately associated with Alzheimer's disease including calcium channel function, cholesterol metabolism, neuroinflammation, endocytosis, cell cycle events, and apoptosis have been tentatively or experimentally verified as calmodulin binding proteins. The use of calmodulin as a potential biomarker and as a therapeutic target is discussed. PMID:25812852

  12. Human liver aldehyde dehydrogenase: coenzyme binding

    SciTech Connect

    Kosley, L.L.; Pietruszko, R.

    1987-05-01

    The binding of (U-/sup 14/C) NAD to mitochondrial (E2) and cytoplasmin(E1) aldehyde dehydrogenase was measured by gel filtration and sedimentation techniques. The binding data for NAD and (E1) yielded linear Scatchard plots giving a dissociation constant of 25 (+/- 8) uM and the stoichiometry of 2 mol of NAD bound per mol of E1. The binding data for NAD and (E2) gave nonlinear Scatchard plots. The binding of NADH to E2 was measured via fluorescence enhancement; this could not be done with E1 because there was no signal. The dissociation constant for E2 by this technique was 0.7 (+/- 0.4) uM and stoichiometry of 1.0 was obtained. The binding of (U-/sup 14/C) NADH to (E1) and (E2) was also measured by the sedimentation technique. The binding data for (E1) and NADH gave linear Scatchard plots giving a dissociation constant of 13 (+/- 6) uM and the stoichiometry of 2.0. The binding data for NADH to (E2) gave nonlinear Scatchard plots. With (E1), the dissociation constants for both NAD and NADH are similar to those determined kinetically, but the stoichiometry is only half of that found by stopped flow technique. With (E2) the dissociation constant by fluorometric procedure was 2 orders of magnitude less than that from catalytic reaction.

  13. Transcription factor binding energy vs. biological function

    NASA Astrophysics Data System (ADS)

    Djordjevic, M.; Grotewold, E.

    2007-03-01

    Transcription factors (TFs) are proteins that bind to DNA and regulate expression of genes. Identification of transcription factor binding sites within the regulatory segments of genomic DNA is an important step towards understanding of gene regulatory networks. Recent theoretical advances that we developed [1,2], allow us to infer TF-DNA interaction parameters from in-vitro selection experiments [3]. We use more than 6000 binding sequences [3], assembled under controlled conditions, to obtain protein-DNA interaction parameters for a mammalian TF with up to now unprecedented accuracy. Can one accurately identify biologically functional TF binding sites (i.e. the binding sites that regulate gene expression), even with the best possible protein-DNA interaction parameters? To address this issue we i) compare our prediction of protein binding with gene expression data, ii) use evolutionary comparison between related mammalian genomes. Our results strongly suggest that in a genome there exists a large number of randomly occurring high energy binding sites that are not biologically functional. [1] M Djordjevic, submitted to Biomol. Eng. [2] M. Djordjevic and A. M. Sengupta, Phys. Biol. 3: 13, 2006. [3] E. Roulet et al., Nature Biotech. 20: 831, 2002.

  14. DNA Triplexes That Bind Several Cofactor Molecules.

    PubMed

    Vollmer, Sven; Richert, Clemens

    2015-12-14

    Cofactors are critical for energy-consuming processes in the cell. Harnessing such processes for practical applications requires control over the concentration of cofactors. We have recently shown that DNA triplex motifs with a designed binding site can be used to capture and release nucleotides with low micromolar dissociation constants. In order to increase the storage capacity of such triplex motifs, we have explored the limits of ligand binding through designed cavities in the oligopurine tract. Oligonucleotides with up to six non-nucleotide bridges between purines were synthesized and their ability to bind ATP, cAMP or FAD was measured. Triplex motifs with several single-nucleotide binding sites were found to bind purines more tightly than triplexes with one large binding site. The optimized triplex consists of 59 residues and four C3-bridges. It can bind up to four equivalents of ligand with apparent Kd values of 52 µM for ATP, 9 µM for FAD, and 2 µM for cAMP. An immobilized version fuels bioluminescence via release of ATP at body temperature. These results show that motifs for high-density capture, storage and release of energy-rich biomolecules can be constructed from synthetic DNA. PMID:26561335

  15. Improved flow cytometer measurement of binding assays

    DOEpatents

    Saunders, G.C.

    1984-05-30

    The invention relates to a method of measuring binding assays carried out with different size particles wherein the binding assay sample is run through a flow cytometer without separating the sample from the marking agent. The amount of a binding reactant present in a sample is determined by providing particles with a coating of binder and also a known quantity of smaller particles with a coating of binder reactant. The binding reactant is the same as the binding reactant present in the sample. The smaller particles also contain a fluorescent chemical. The particles are combined with the sample and the binding reaction is allowed to occur for a set length of time followed by combining the smaller particles with the mixture of the particles and the sample produced and allowing the binding reactions to proceed to equilibrium. The fluorescence and light scatter of the combined mixture is then measured as the combined mixture passes through a flow cytometer equipped with a laser to bring about fluorescence, and the number and strength of fluorescent events are compared. A similar method is also provided for determining the amount of antigen present in the sample by providing spheres with an antibody coating and some smaller spheres with an antigen coating. (LEW)

  16. Binding of perlecan to transthyretin in vitro.

    PubMed Central

    Smeland, S; Kolset, S O; Lyon, M; Norum, K R; Blomhoff, R

    1997-01-01

    Transthyretin is one of two specific proteins involved in the transport of thyroid hormones in plasma; it possesses two binding sites for serum retinol-binding protein. In the present study we demonstrate that transthyretin also interacts in vitro with [35S]sulphate-labelled material from the medium of HepG2 cells. By using the same strategy as for purifying serum retinol-binding protein, [35S]sulphate-labelled medium was specifically eluted from a transthyretin-affinity column. Ion-exchange chromatography showed that the material was highly polyanionic, and its size and alkali susceptibility suggested that it was a proteoglycan. Structural analyses with chondroitinase ABC lyase and nitrous acid revealed that approx. 20% was chondroitin sulphate and 80% heparan sulphate. Immunoprecipitation showed that the [35S]sulphate-labelled material contained perlecan. Further analysis by binding studies revealed specific and saturable binding of 125I-transthyretin to perlecan-enriched Matrigel. Because inhibition of sulphation by treating HepG2 cells with sodium chlorate increased the affinity of the perlecan for transthyretin, and [3H]heparin was not retained by the transthyretin affinity column, the binding is probably mediated by the core protein and is not a protein-glycosaminoglycan interaction. Because perlecan is released from transthyretin in water, the binding might be due to hydrophobic interactions. PMID:9307034

  17. Lipopolysaccharides of Actinobacillus pleuropneumoniae bind pig hemoglobin.

    PubMed Central

    Bélanger, M; Bégin, C; Jacques, M

    1995-01-01

    A previous study indicated that lipopolysaccharides (LPS) extracted from Actinobacillus pleuropneumoniae bind two low-molecular-mass proteins, of approximately 10 and 11 kDa, present in porcine respiratory tract secretions (M. Bélanger, D. Dubreuil, and M. Jacques, Infect. Immun. 62:868-873, 1994). In the present study, we determined the N-terminal amino acid sequences of these two proteins, which revealed high homology with the alpha and beta chains of pig hemoglobin. Some isolates of A. pleuropneumoniae were able to use hemoglobin from various animal species as well as other heme compounds as sole sources of iron for growth, while other isolates were unable to use them. Immunoelectron microscopy showed binding of pig hemoglobin at the surface of all A. pleuropneumoniae isolates as well as labeling of outer membrane blebs. We observed, using Western blotting (immunoblotting), that the lipid A-core region of LPS of all isolates was binding pig hemoglobin. Furthermore, lipid A obtained after acid hydrolysis of LPS extracted from A. pleuropneumoniae was able to bind pig hemoglobin and this binding was completely abolished by preincubation of lipid A with polymyxin B but was not inhibited by preincubation with glucosamines. Fatty acids constituting the lipid A of A. pleuropneumoniae, namely, dodecanoic acid, tetradecanoic acid, 3-hydroxytetradecanoic acid, hexadecanoic acid, and octadecanoic acid, were also binding pig hemoglobin. Our results indicate that LPS of all A. pleuropneumoniae isolates tested bind pig hemoglobin and that lipid A is involved in this binding. Our results also indicate that some A. pleuropneumoniae isolates are, in addition, able to use hemoglobin for growth. Binding of hemoglobin to LPS might represent an important means by which A. pleuropneumoniae acquires iron in vivo from hemoglobin released from erythrocytes lysed by the action of its hemolysins. PMID:7822035

  18. Relativistic corrections to the triton binding energy

    SciTech Connect

    Sammarruca, F.; Xu, D.P.; Machleidt, R. )

    1992-11-01

    The influence of relativity on the triton binding energy is investigated. The relativistic three-dimensional version of the Bethe-Salpeter equation proposed by Blankenbecler and Sugar (BbS) is used. Relativistic (nonseparable) one-boson-exchange potentials (constructed in the BbS framework) are employed for the two-nucleon interaction. In a 34-channel Faddeev calculation, it is found that relativistic effects increase the triton binding energy by about 0.2 MeV. Including charge dependence (besides relativity), the final triton binding energy predictions are 8.33 and 8.16 MeV for the Bonn A and B potentials, respectively.

  19. α-Enolase binds to RNA.

    PubMed

    Hernández-Pérez, Liliana; Depardón, Francisco; Fernández-Ramírez, Fernando; Sánchez-Trujillo, Alejandra; Bermúdez-Crúz, Rosa María; Dangott, Lawrence; Montañez, Cecilia

    2011-09-01

    To detect proteins binding to CUG triplet repeats, we performed magnetic bead affinity assays and North-Western analysis using a (CUG)(10) ssRNA probe and either nuclear or total extracts from rat L6 myoblasts. We report the isolation and identification by mass spectrometry and immunodetection of α-enolase, as a novel (CUG)n triplet repeat binding protein. To confirm our findings, rat recombinant α-enolase was cloned, expressed and purified; the RNA binding activity was verified by electrophoretic mobility shift assays using radiolabeled RNA probes. Enolase may play other roles in addition to its well described function in glycolysis. PMID:21621582

  20. Muscarine binding sites in bovine adrenal medulla.

    PubMed

    Barron, B A; Murrin, L C; Hexum, T D

    1986-03-18

    The presence of muscarinic binding sites in the bovine adrenal medulla was investigated using [3H]QNB and the bovine adrenal medulla. Scatchard analysis combined with computer analysis yielded data consistent with a two binding site configuration. KDs of 0.15 and 14 nM and Bmax s of 29 and 210 fmol/mg protein, respectively, were observed. Displacement of [3H]QNB by various cholinergic agents is, in order of decreasing potency: QNB, dexetimide, atropine, scopolamine, imipramine, desipramine, oxotremorine, pilocarpine, acetylcholine, methacholine and carbachol. These results demonstrate the presence of more than one muscarine binding site in the bovine adrenal gland. PMID:3709656

  1. Measuring Binding Affinity of Protein-Ligand Interaction Using Spectrophotometry: Binding of Neutral Red to Riboflavin-Binding Protein

    ERIC Educational Resources Information Center

    Chenprakhon, Pirom; Sucharitakul, Jeerus; Panijpan, Bhinyo; Chaiyen, Pimchai

    2010-01-01

    The dissociation constant, K[subscript d], of the binding of riboflavin-binding protein (RP) with neutral red (NR) can be determined by titrating RP to a fixed concentration of NR. Upon adding RP to the NR solution, the maximum absorption peak of NR shifts to 545 nm from 450 nm for the free NR. The change of the absorption can be used to determine…

  2. Two nucleotide binding sites modulate ( sup 3 H) glyburide binding to rat cortex membranes

    SciTech Connect

    Johnson, D.E.; Gopalakrishnan, M.; Triggle, D.J.; Janis, R.A. State Univ. of New York, Buffalo )

    1991-03-11

    The effects of nucleotides on the binding of the ATP-dependent K{sup +}-channel antagonist ({sup 3}H)glyburide (GLB) to rat cortex membranes were examined. Nucleotide triphosphates (NTPs) and nucleotide diphosphate (NDPs) inhibited the binding of GLB. This effect was dependent on the presence of dithiothreitol (DTT). Inhibition of binding by NTPs, with the exception of ATP{gamma}S, was dependent on the presence of Mg{sup 2+}. GLB binding showed a biphasic response to ADP: up to 3 mM, ADP inhibited binding, and above this concentration GLB binding increased rapidly, and was restored to normal levels by 10 mM ADP. In the presence of Mg{sup 2+}, ADP did not stimulate binding. Saturation analysis in the presence of Mg{sup 2+} and increasing concentrations of ADP showed that ADP results primarily in a change of the B{sub max} for GLB binding. The differential effects of NTPS and NDPs indicate that two nucleotide binding sites regulate GLB binding.

  3. Ligand Binding to Macromolecules: Allosteric and Sequential Models of Cooperativity.

    ERIC Educational Resources Information Center

    Hess, V. L.; Szabo, Attila

    1979-01-01

    A simple model is described for the binding of ligands to macromolecules. The model is applied to the cooperative binding by hemoglobin and aspartate transcarbamylase. The sequential and allosteric models of cooperative binding are considered. (BB)

  4. Lamin-Binding Proteins in Caenorhabditis elegans.

    PubMed

    Dobrzynska, Agnieszka; Askjaer, Peter; Gruenbaum, Yosef

    2016-01-01

    The nuclear lamina, composed of lamins and numerous lamin-associated proteins, is required for mechanical stability, mechanosensing, chromatin organization, developmental gene regulation, mRNA transcription, DNA replication, nuclear assembly, and nuclear positioning. Mutations in lamins or lamin-binding proteins cause at least 18 distinct human diseases that affect specific tissues such as muscle, adipose, bone, nerve, or skin, and range from muscular dystrophies to lipodystrophy, peripheral neuropathy, or accelerated aging. Caenorhabditis elegans has unique advantages in studying lamin-binding proteins. These advantages include the low complexity of genes encoding lamin and lamin-binding proteins, advanced transgenic techniques, simple application of RNA interference, sophisticated genetic strategies, and a large collection of mutant lines. This chapter provides detailed and comprehensive protocols for the genetic and phenotypic analysis of lamin-binding proteins in C. elegans. PMID:26778571

  5. Binding of ATP to the progesterone receptor.

    PubMed Central

    Moudgil, V K; Toft, D O

    1975-01-01

    The possible interaction of progesterone--receptor complexes with nucleotides was tested by affinity chromatography. The cytosol progesterone receptor from hen oviduct was partially purified by ammonium sulfate precipitation before use. When progesterone was bound to the receptor, the resulting complex could be selectively adsorbed onto columns of ATP-Sepharose. This interaction was reversible and of an ionic nature since it could be disrupted by high-salt conditions. A competitive binding assay was used to test the specificity of receptor binding to several other nucleotides, including ADP, AMP, and cAMP. A clear specificity for binding ATP was evident from these studies. When ATP was added to receptor preparations, the nucleotide did not affect the sedimentation properties or hormone binding characteristics of the receptor. Although the function of ATP remains unknown, these studies indicate a role of this nucleotide in some aspect of hormone receptor activity. PMID:165493

  6. Universal binding energy relations in metallic adhesion

    NASA Technical Reports Server (NTRS)

    Ferrante, J.; Smith, J. R.; Rose, J. H.

    1981-01-01

    Scaling relations which map metallic adhesive binding energy onto a single universal binding energy curve are discussed in relation to adhesion, friction, and wear in metals. The scaling involved normalizing the energy to the maximum binding energy and normalizing distances by a suitable combination of Thomas-Fermi screening lengths. The universal curve was found to be accurately represented by E*(A*)= -(1+beta A) exp (-Beta A*) where E* is the normalized binding energy, A* is the normalized separation, and beta is the normalized decay constant. The calculated cohesive energies of potassium, barium, copper, molybdenum, and samarium were also found to scale by similar relations, suggesting that the universal relation may be more general than for the simple free electron metals.

  7. Structure and Function of Lipopolysaccharide Binding Protein

    NASA Astrophysics Data System (ADS)

    Schumann, Ralf R.; Leong, Steven R.; Flaggs, Gail W.; Gray, Patrick W.; Wright, Samuel D.; Mathison, John C.; Tobias, Peter S.; Ulevitch, Richard J.

    1990-09-01

    The primary structure of lipopolysaccharide binding protein (LBP), a trace plasma protein that binds to the lipid A moiety of bacterial lipopolysaccharides (LPSs), was deduced by sequencing cloned complementary DNA. LBP shares sequence identity with another LPS binding protein found in granulocytes, bactericidal/permeability-increasing protein, and with cholesterol ester transport protein of the plasma. LBP may control the response to LPS under physiologic conditions by forming high-affinity complexes with LPS that bind to monocytes and macrophages, which then secrete tumor necrosis factor. The identification of this pathway for LPS-induced monocyte stimulation may aid in the development of treatments for diseases in which Gram-negative sepsis or endotoxemia are involved.

  8. Human Frataxin: Iron And Ferrochelatase Binding Surface

    SciTech Connect

    Bencze, K.Z.; Yoon, T.; Millan-Pacheco, C.; Bradley, P.B.; Pastor, N.; Cowan, J.A.; Stemmler, T.L.

    2009-06-02

    The coordinated iron structure and ferrochelatase binding surface of human frataxin have been characterized to provide insight into the protein's ability to serve as the iron chaperone during heme biosynthesis.

  9. Hardware device binding and mutual authentication

    SciTech Connect

    Hamlet, Jason R; Pierson, Lyndon G

    2014-03-04

    Detection and deterrence of device tampering and subversion by substitution may be achieved by including a cryptographic unit within a computing device for binding multiple hardware devices and mutually authenticating the devices. The cryptographic unit includes a physically unclonable function ("PUF") circuit disposed in or on the hardware device, which generates a binding PUF value. The cryptographic unit uses the binding PUF value during an enrollment phase and subsequent authentication phases. During a subsequent authentication phase, the cryptographic unit uses the binding PUF values of the multiple hardware devices to generate a challenge to send to the other device, and to verify a challenge received from the other device to mutually authenticate the hardware devices.

  10. Exciton Binding Energy of Monolayer WS2

    PubMed Central

    Zhu, Bairen; Chen, Xi; Cui, Xiaodong

    2015-01-01

    The optical properties of monolayer transition metal dichalcogenides (TMDC) feature prominent excitonic natures. Here we report an experimental approach to measuring the exciton binding energy of monolayer WS2 with linear differential transmission spectroscopy and two-photon photoluminescence excitation spectroscopy (TP-PLE). TP-PLE measurements show the exciton binding energy of 0.71 ± 0.01 eV around K valley in the Brillouin zone. PMID:25783023

  11. Bilirubin Binding Capacity in the Preterm Neonate.

    PubMed

    Amin, Sanjiv B

    2016-06-01

    Total serum/plasma bilirubin (TB), the biochemical measure currently used to evaluate and manage hyperbilirubinemia, is not a useful predictor of bilirubin-induced neurotoxicity in premature infants. Altered bilirubin-albumin binding in premature infants limits the usefulness of TB in premature infants. In this article, bilirubin-albumin binding, a modifying factor for bilirubin-induced neurotoxicity, in premature infants is reviewed. PMID:27235205

  12. DNA-aptamers binding aminoglycoside antibiotics.

    PubMed

    Nikolaus, Nadia; Strehlitz, Beate

    2014-01-01

    Aptamers are short, single stranded DNA or RNA oligonucleotides that are able to bind specifically and with high affinity to their non-nucleic acid target molecules. This binding reaction enables their application as biorecognition elements in biosensors and assays. As antibiotic residues pose a problem contributing to the emergence of antibiotic-resistant pathogens and thereby reducing the effectiveness of the drug to fight human infections, we selected aptamers targeted against the aminoglycoside antibiotic kanamycin A with the aim of constructing a robust and functional assay that can be used for water analysis. With this work we show that aptamers that were derived from a Capture-SELEX procedure targeting against kanamycin A also display binding to related aminoglycoside antibiotics. The binding patterns differ among all tested aptamers so that there are highly substance specific aptamers and more group specific aptamers binding to a different variety of aminoglycoside antibiotics. Also the region of the aminoglycoside antibiotics responsible for aptamer binding can be estimated. Affinities of the different aptamers for their target substance, kanamycin A, are measured with different approaches and are in the micromolar range. Finally, the proof of principle of an assay for detection of kanamycin A in a real water sample is given. PMID:24566637

  13. Molecular design of substrate binding sites

    SciTech Connect

    Shelnutt, J.A.; Hobbs, J.D.

    1991-12-31

    Computer-aided molecular design methods were used to tailor binding sites for small substrate molecules, including CO{sub 2} and methane. The goal is to design a cavity, adjacent to a catalytic metal center, into which the substrate will selectively bind through only non-bonding interactions with the groups lining the binding pocket. Porphyrins are used as a basic molecular structure, with various substituents added to construct the binding pocket. The conformations of these highly-substituted porphyrins are predicted using molecular mechanics calculations with a force field that gives accurate predictions for metalloporhyrins. Dynamics and energy-minimization calculations of substrate molecules bound to the cavity indicate high substrate binding affinity. The size, shape and charge-distribution of groups surrounding the cavity provide molecular selectivity. Specifically, calculated binding energies of methane, benzene, dichloromethane, CO{sub 2} and chloroform vary by about 10 kcal/mol for metal octaethyl-tetraphenylporphyrins (OETPPs) with chloroform, dichloromethane, and CO{sub 2} having the lowest. Significantly, a solvent molecule is found in the cavity in the X-ray structures of Co- and CuOETPP crystals obtained from dichloromethane. 5 refs., 3 figs., 3 tabs.

  14. Molecular design of substrate binding sites

    SciTech Connect

    Shelnutt, J.A.; Hobbs, J.D.

    1991-01-01

    Computer-aided molecular design methods were used to tailor binding sites for small substrate molecules, including CO{sub 2} and methane. The goal is to design a cavity, adjacent to a catalytic metal center, into which the substrate will selectively bind through only non-bonding interactions with the groups lining the binding pocket. Porphyrins are used as a basic molecular structure, with various substituents added to construct the binding pocket. The conformations of these highly-substituted porphyrins are predicted using molecular mechanics calculations with a force field that gives accurate predictions for metalloporhyrins. Dynamics and energy-minimization calculations of substrate molecules bound to the cavity indicate high substrate binding affinity. The size, shape and charge-distribution of groups surrounding the cavity provide molecular selectivity. Specifically, calculated binding energies of methane, benzene, dichloromethane, CO{sub 2} and chloroform vary by about 10 kcal/mol for metal octaethyl-tetraphenylporphyrins (OETPPs) with chloroform, dichloromethane, and CO{sub 2} having the lowest. Significantly, a solvent molecule is found in the cavity in the X-ray structures of Co- and CuOETPP crystals obtained from dichloromethane. 5 refs., 3 figs., 3 tabs.

  15. Anion binding to the ubiquitin molecule.

    PubMed Central

    Makhatadze, G. I.; Lopez, M. M.; Richardson, J. M.; Thomas, S. T.

    1998-01-01

    Effects of different salts (NaCl, MgCl2, CaCl2, GdmCl, NaBr, NaClO4, NaH2PO4, Na2SO4) on the stability of the ubiquitin molecule at pH 2.0 have been studied by differential scanning calorimetry, circular dichroism, and Tyr fluorescence spectroscopies. It is shown that all of the salts studied significantly increase the thermostability of the ubiquitin molecule, and that this stabilization can be interpreted in terms of anion binding. Estimated thermodynamic parameters of binding for Cl- show that this binding is relatively weak (Kd = 0.15 M) and is characterized by a negative enthalpy of -15 kJ/mol per site. Particularly surprising was the observed stabilizing effect of GdmCl through the entire concentration range studied (0.01-2 M), however, to a lesser extent than stabilization by NaCl. This stabilizing effect of GdmCl appears to arise from the binding of Cl- ions. Analysis of the observed changes in the stability of the ubiquitin molecule in the presence of GdmCl can be adequately described by combining the thermodynamic model of denaturant binding with Cl- binding effects. PMID:9541401

  16. Folding and binding energy of a calmodulin-binding cell antiproliferative peptide.

    PubMed

    Almudallal, Ahmad M; Saika-Voivod, Ivan; Stewart, John M

    2015-09-01

    We carry out a computational study of a calmodulin-binding peptide shown to be effective in reducing cell proliferation. We find several folded states for two short variants of different length of the peptide and determine the location of the binding site on calmodulin, the binding free energy for the different conformers and structural details that play a role in optimal binding. Binding to a hydrophobic pocket in calmodulin occurs via an anchoring phenylalanine residue of the natively disordered peptide, and is enhanced when a neighbouring hydrophobic residue acts as a co-anchor. The shorter sequence possesses better binding to calmodulin, which is encouraging in terms of the development of non-peptide analogues as therapeutic agents. PMID:26310499

  17. Theoretical studies of binding of mannose-binding protein to monosaccharides

    NASA Astrophysics Data System (ADS)

    Aida-Hyugaji, Sachiko; Takano, Keiko; Takada, Toshikazu; Hosoya, Haruo; Kojima, Naoya; Mizuochi, Tsuguo; Inoue, Yasushi

    2004-11-01

    Binding properties of mannose-binding protein (MBP) to monosaccharides are discussed based on ab initio molecular orbital calculations for cluster models constructed. The calculated binding energies indicate that MBP has an affinity for N-acetyl- D-glucosamine, D-mannose, L-fucose, and D-glucose rather than D-galactose and N-acetyl- D-galactosamine, which is consistent with the biochemical experimental results. Electrostatic potential surfaces at the binding site of four monosaccharides having binding properties matched well with that of MBP. A vacant frontier orbital was found to be localized around the binding site of MBP, suggesting that MBP-monosaccharide interaction may occur through electrostatic and orbital interactions.

  18. Fucose-binding Lotus tetragonolobus lectin binds to human polymorphonuclear leukocytes and induces a chemotactic response.

    PubMed

    VanEpps, D E; Tung, K S

    1977-09-01

    Fucose-binding L. tetragonolobus lectin to the surface of human polymorphonuclear leukocytes (PMN) and induces a chemotactic response. Both surface binding and chemotaxis are inhibited by free fucose but not by fructose, mannose, or galactose. The lectin-binding sites on PMN are unrelated to the A, B, or O blood group antigen. Utilization of this lectin should be a useful tool in isolating PMN membrane components and in analyzing the mechanism of neutrophil chemotaxis. PMID:330752

  19. Leukocyte Protease Binding to Nucleic Acids Promotes Nuclear Localization and Cleavage of Nucleic Acid Binding Proteins

    PubMed Central

    Thomas, Marshall P.; Whangbo, Jennifer; McCrossan, Geoffrey; Deutsch, Aaron; Martinod, Kimberly; Walch, Michael; Lieberman, Judy

    2014-01-01

    Killer lymphocyte granzyme (Gzm) serine proteases induce apoptosis of pathogen-infected cells and tumor cells. Many known Gzm substrates are nucleic acid binding proteins, and the Gzms accumulate in the target cell nucleus by an unknown mechanism. Here we show that human Gzms bind to DNA and RNA with nanomolar affinity. Gzms cleave their substrates most efficiently when both are bound to nucleic acids. RNase treatment of cell lysates reduces Gzm cleavage of RNA binding protein (RBP) targets, while adding RNA to recombinant RBP substrates increases in vitro cleavage. Binding to nucleic acids also influences Gzm trafficking within target cells. Pre-incubation with competitor DNA and DNase treatment both reduce Gzm nuclear localization. The Gzms are closely related to neutrophil proteases, including neutrophil elastase (NE) and cathepsin G (CATG). During neutrophil activation, NE translocates to the nucleus to initiate DNA extrusion into neutrophil extracellular traps (NETs), which bind NE and CATG. These myeloid cell proteases, but not digestive serine proteases, also bind DNA strongly and localize to nuclei and NETs in a DNA-dependent manner. Thus, high affinity nucleic acid binding is a conserved and functionally important property specific to leukocyte serine proteases. Furthermore, nucleic acid binding provides an elegant and simple mechanism to confer specificity of these proteases for cleavage of nucleic acid binding protein substrates that play essential roles in cellular gene expression and cell proliferation. PMID:24771851

  20. Radiation inactivation reveals discrete cation binding sites that modulate dihydropyridine binding sites

    SciTech Connect

    Bolger, G.T.; Skolnick, P.; Kempner, E.S. )

    1989-08-01

    In low ionic strength buffer (5 mM Tris.HCl), the binding of (3H) nitrendipine to dihydropyridine calcium antagonist binding sites of mouse forebrain membranes is increased by both Na{sup +} and Ca{sup 2+}. Radiation inactivation was used to determine the target size of ({sup 3}H)nitrendipine binding sites in 5 mM Tris.HCl buffer, in the presence and absence of these cations. After irradiation, ({sup 3}H) nitrendipine binding in buffer with or without Na+ was diminished, due to a loss of binding sites and also to an increase in Kd. After accounting for radiation effects on the dissociation constant, the target size for the nitrendipine binding site in buffer was 160-170 kDa and was 170-180 kDa in the presence of sodium. In the presence of calcium ions, ({sup 3}H)nitrendipine binding showed no radiation effects on Kd and yielded a target size of 150-170 kDa. These findings suggest, as in the case of opioid receptors, the presence of high molecular weight membrane components that modulate cation-induced alterations in radioligand binding to dihydropyridine binding sites.

  1. RNA binding protein and binding site useful for expression of recombinant molecules

    DOEpatents

    Mayfield, Stephen

    2000-01-01

    The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

  2. RNA binding protein and binding site useful for expression of recombinant molecules

    DOEpatents

    Mayfield, Stephen P.

    2006-10-17

    The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

  3. Crystal Structure of the Botulinum Neurotoxin Type G Binding Domain: Insight into Cell Surface Binding

    SciTech Connect

    Stenmark, Pål; Dong, Min; Dupuy, Jérôme; Chapman, Edwin R.; Stevens, Raymond C.

    2011-11-02

    Botulinum neurotoxins (BoNTs) typically bind the neuronal cell surface via dual interactions with both protein receptors and gangliosides. We present here the 1.9-{angstrom} X-ray structure of the BoNT serotype G (BoNT/G) receptor binding domain (residues 868-1297) and a detailed view of protein receptor and ganglioside binding regions. The ganglioside binding motif (SxWY) has a conserved structure compared to the corresponding regions in BoNT serotype A and BoNT serotype B (BoNT/B), but several features of interactions with the hydrophilic face of the ganglioside are absent at the opposite side of the motif in the BoNT/G ganglioside binding cleft. This may significantly reduce the affinity between BoNT/G and gangliosides. BoNT/G and BoNT/B share the protein receptor synaptotagmin (Syt) I/II. The Syt binding site has a conserved hydrophobic plateau located centrally in the proposed protein receptor binding interface (Tyr1189, Phe1202, Ala1204, Pro1205, and Phe1212). Interestingly, only 5 of 14 residues that are important for binding between Syt-II and BoNT/B are conserved in BoNT/G, suggesting that the means by which BoNT/G and BoNT/B bind Syt diverges more than previously appreciated. Indeed, substitution of Syt-II Phe47 and Phe55 with alanine residues had little effect on the binding of BoNT/G, but strongly reduced the binding of BoNT/B. Furthermore, an extended solvent-exposed hydrophobic loop, located between the Syt binding site and the ganglioside binding cleft, may serve as a third membrane association and binding element to contribute to high-affinity binding to the neuronal membrane. While BoNT/G and BoNT/B are homologous to each other and both utilize Syt-I/Syt-II as their protein receptor, the precise means by which these two toxin serotypes bind to Syt appears surprisingly divergent.

  4. Recent improvements to Binding MOAD: a resource for protein-ligand binding affinities and structures.

    PubMed

    Ahmed, Aqeel; Smith, Richard D; Clark, Jordan J; Dunbar, James B; Carlson, Heather A

    2015-01-01

    For over 10 years, Binding MOAD (Mother of All Databases; http://www.BindingMOAD.org) has been one of the largest resources for high-quality protein-ligand complexes and associated binding affinity data. Binding MOAD has grown at the rate of 1994 complexes per year, on average. Currently, it contains 23,269 complexes and 8156 binding affinities. Our annual updates curate the data using a semi-automated literature search of the references cited within the PDB file, and we have recently upgraded our website and added new features and functionalities to better serve Binding MOAD users. In order to eliminate the legacy application server of the old platform and to accommodate new changes, the website has been completely rewritten in the LAMP (Linux, Apache, MySQL and PHP) environment. The improved user interface incorporates current third-party plugins for better visualization of protein and ligand molecules, and it provides features like sorting, filtering and filtered downloads. In addition to the field-based searching, Binding MOAD now can be searched by structural queries based on the ligand. In order to remove redundancy, Binding MOAD records are clustered in different families based on 90% sequence identity. The new Binding MOAD, with the upgraded platform, features and functionalities, is now equipped to better serve its users. PMID:25378330

  5. Folding funnels, binding funnels, and protein function.

    PubMed Central

    Tsai, C. J.; Kumar, S.; Ma, B.; Nussinov, R.

    1999-01-01

    Folding funnels have been the focus of considerable attention during the last few years. These have mostly been discussed in the general context of the theory of protein folding. Here we extend the utility of the concept of folding funnels, relating them to biological mechanisms and function. In particular, here we describe the shape of the funnels in light of protein synthesis and folding; flexibility, conformational diversity, and binding mechanisms; and the associated binding funnels, illustrating the multiple routes and the range of complexed conformers. Specifically, the walls of the folding funnels, their crevices, and bumps are related to the complexity of protein folding, and hence to sequential vs. nonsequential folding. Whereas the former is more frequently observed in eukaryotic proteins, where the rate of protein synthesis is slower, the latter is more frequent in prokaryotes, with faster translation rates. The bottoms of the funnels reflect the extent of the flexibility of the proteins. Rugged floors imply a range of conformational isomers, which may be close on the energy landscape. Rather than undergoing an induced fit binding mechanism, the conformational ensembles around the rugged bottoms argue that the conformers, which are most complementary to the ligand, will bind to it with the equilibrium shifting in their favor. Furthermore, depending on the extent of the ruggedness, or of the smoothness with only a few minima, we may infer nonspecific, broad range vs. specific binding. In particular, folding and binding are similar processes, with similar underlying principles. Hence, the shape of the folding funnel of the monomer enables making reasonable guesses regarding the shape of the corresponding binding funnel. Proteins having a broad range of binding, such as proteolytic enzymes or relatively nonspecific endonucleases, may be expected to have not only rugged floors in their folding funnels, but their binding funnels will also behave similarly

  6. Predicting Ca(2+)-binding sites in proteins.

    PubMed Central

    Nayal, M; Di Cera, E

    1994-01-01

    The coordination shell of Ca2+ ions in proteins contains almost exclusively oxygen atoms supported by an outer shell of carbon atoms. The bond-strength contribution of each ligating oxygen in the inner shell can be evaluated by using an empirical expression successfully applied in the analysis of crystals of metal oxides. The sum of such contributions closely approximates the valence of the bound cation. When a protein is embedded in a very fine grid of points and an algorithm is used to calculate the valence of each point representing a potential Ca(2+)-binding site, a typical distribution of valence values peaked around 0.4 is obtained. In 32 documented Ca(2+)-binding proteins, containing a total of 62 Ca(2+)-binding sites, a very small fraction of points in the distribution has a valence close to that of Ca2+. Only 0.06% of the points have a valence > or = 1.4. These points share the remarkable tendency to cluster around documented Ca2+ ions. A high enough value of the valence is both necessary (58 out of 62 Ca(2+)-binding sites have a valence > or = 1.4) and sufficient (87% of the grid points with a valence > or = 1.4 are within 1.0 A from a documented Ca2+ ion) to predict the location of bound Ca2+ ions. The algorithm can also be used for the analysis of other cations and predicts the location of Mg(2+)- and Na(+)-binding sites in a number of proteins. The valence is, therefore, a tool of pinpoint accuracy for locating cation-binding sites, which can also be exploited in engineering high-affinity binding sites and characterizing the linkage between structural components and functional energetics for molecular recognition of metal ions by proteins. Images Fig. 4 PMID:8290605

  7. Mannose-binding geometry of pradimicin A.

    PubMed

    Nakagawa, Yu; Doi, Takashi; Taketani, Takara; Takegoshi, K; Igarashi, Yasuhiro; Ito, Yukishige

    2013-08-01

    Pradimicins (PRMs) and benanomicins are the only family of non-peptidic natural products with lectin-like properties, that is, they recognize D-mannopyranoside (Man) in the presence of Ca(2+) ions. Coupled with their unique Man binding ability, they exhibit antifungal and anti-HIV activities through binding to Man-containing glycans of pathogens. Notwithstanding the great potential of PRMs as the lectin mimics and therapeutic leads, their molecular basis of Man recognition has yet to be established. Their aggregate-forming propensity has impeded conventional interaction analysis in solution, and the analytical difficulty is exacerbated by the existence of two Man binding sites in PRMs. In this work, we investigated the geometry of the primary Man binding of PRM-A, an original member of PRMs, by the recently developed analytical strategy using the solid aggregate composed of the 1:1 complex of PRM-A and Man. Evaluation of intermolecular distances by solid-state NMR spectroscopy revealed that the C2-C4 region of Man is in close contact with the primary binding site of PRM-A, while the C1 and C6 positions of Man are relatively distant. The binding geometry was further validated by co-precipitation experiments using deoxy-Man derivatives, leading to the proposal that PRM-A binds not only to terminal Man residues at the non-reducing end of glycans, but also to internal 6-substituted Man residues. The present study provides new insights into the molecular basis of Man recognition and glycan specificity of PRM-A. PMID:23832850

  8. Evidence for chemoreceptors with bimodular ligand-binding regions harboring two signal-binding sites

    PubMed Central

    Pineda-Molina, Estela; Reyes-Darias, José-Antonio; Lacal, Jesús; Ramos, Juan L.; García-Ruiz, Juan Manuel; Gavira, Jose A.; Krell, Tino

    2012-01-01

    Chemoreceptor-based signaling is a central mechanism in bacterial signal transduction. Receptors are classified according to the size of their ligand-binding region. The well-studied cluster I proteins have a 100- to 150-residue ligand-binding region that contains a single site for chemoattractant recognition. Cluster II receptors, which contain a 220- to 300-residue ligand-binding region and which are almost as abundant as cluster I receptors, remain largely uncharacterized. Here, we report high-resolution structures of the ligand-binding region of the cluster II McpS chemotaxis receptor (McpS-LBR) of Pseudomonas putida KT2440 in complex with different chemoattractants. The structure of McpS-LBR represents a small-molecule binding domain composed of two modules, each able to bind different signal molecules. Malate and succinate were found to bind to the membrane-proximal module, whereas acetate binds to the membrane-distal module. A structural alignment of the two modules revealed that the ligand-binding sites could be superimposed and that amino acids involved in ligand recognition are conserved in both binding sites. Ligand binding to both modules was shown to trigger chemotactic responses. Further analysis showed that McpS-like receptors were found in different classes of proteobacteria, indicating that this mode of response to different carbon sources may be universally distributed. The physiological relevance of the McpS architecture may lie in its capacity to respond with high sensitivity to the preferred carbon sources malate and succinate and, at the same time, mediate lower sensitivity responses to the less preferred but very abundant carbon source acetate. PMID:23112148

  9. Molecular interactions and metal binding in the theophylline-binding core of an RNA aptamer.

    PubMed Central

    Zimmermann, G R; Wick, C L; Shields, T P; Jenison, R D; Pardi, A

    2000-01-01

    An RNA aptamer containing a 15-nt binding site shows high affinity and specificity for the bronchodilator theophylline. A variety of base modifications or 2' deoxyribose substitutions in binding-site residues were tested for theophyllinebinding affinity and the results were compared with the previously determined three-dimensional structure of the RNA-theophylline complex. The RNA-theophylline complex contains a U6-A28-U23 base triple, and disruption of this A28-U23 Hoogsteen-pair by a 7-deaza, 2'-deoxy A28 mutant reduces theophylline binding >45-fold at 25 degrees C. U24 is part of a U-turn in the core of the RNA, and disruption of this U-turn motif by a 2'-deoxy substitution of U24 also reduces theophylline binding by >90-fold. Several mutations outside the "conserved core" of the RNA aptamer showed reduced binding affinity, and these effects could be rationalized by comparison with the three-dimensional structure of the complex. Divalent ions are absolutely required for high-affinity theophylline binding. High-affinity binding was observed with 5 mM Mg2+, Mn2+, or Co2+ ions, whereas little or no significant binding was observed for other divalent or lanthanide ions. A metal-binding site in the core of the complex was revealed by paramagnetic Mn2+-induced broadening of specific RNA resonances in the NMR spectra. When caffeine is added to the aptamer in tenfold excess, the NMR spectra show no evidence for binding in the conserved core and instead the drug stacks on the terminal helix. The lack of interaction between caffeine and the theophylline-binding site emphasizes the extreme molecular discrimination of this RNA aptamer. PMID:10836787

  10. Promoter-distal RNA polymerase II binding discriminates active from inactive CCAAT/ enhancer-binding protein beta binding sites

    PubMed Central

    Savic, Daniel; Roberts, Brian S.; Carleton, Julia B.; Partridge, E. Christopher; White, Michael A.; Cohen, Barak A.; Cooper, Gregory M.; Gertz, Jason; Myers, Richard M.

    2015-01-01

    Transcription factors (TFs) bind to thousands of DNA sequences in mammalian genomes, but most of these binding events appear to have no direct effect on gene expression. It is unclear why only a subset of TF bound sites are actively involved in transcriptional regulation. Moreover, the key genomic features that accurately discriminate between active and inactive TF binding events remain ambiguous. Recent studies have identified promoter-distal RNA polymerase II (RNAP2) binding at enhancer elements, suggesting that these interactions may serve as a marker for active regulatory sequences. Despite these correlative analyses, a thorough functional validation of these genomic co-occupancies is still lacking. To characterize the gene regulatory activity of DNA sequences underlying promoter-distal TF binding events that co-occur with RNAP2 and TF sites devoid of RNAP2 occupancy using a functional reporter assay, we performed cis-regulatory element sequencing (CRE-seq). We tested more than 1000 promoter-distal CCAAT/enhancer-binding protein beta (CEBPB)-bound sites in HepG2 and K562 cells, and found that CEBPB-bound sites co-occurring with RNAP2 were more likely to exhibit enhancer activity. CEBPB-bound sites further maintained substantial cell-type specificity, indicating that local DNA sequence can accurately convey cell-type–specific regulatory information. By comparing our CRE-seq results to a comprehensive set of genome annotations, we identified a variety of genomic features that are strong predictors of regulatory element activity and cell-type–specific activity. Collectively, our functional assay results indicate that RNAP2 occupancy can be used as a key genomic marker that can distinguish active from inactive TF bound sites. PMID:26486725

  11. Ag(I)-binding to phytochelatins.

    PubMed

    Mehra, R K; Tran, K; Scott, G W; Mulchandani, P; Saini, S S

    1996-02-01

    Phytochelatins (PCs) are glutathione-derived peptides with the general structure (gamma-Glu-Cys)nGly, where n varies from 2 to 11. A variety of metal ions such as Cu(II), Cd(II), Pb(II), Zn(II), and Ag(I) induce PC synthesis in plants and some yeasts. It has generally been assumed that the inducer metals also bind PCs. However, very little information is available on the binding of metals other than Cu(I) and Cd(II) to PCs. In this paper, we describe the Ag(I)-binding characteristics of PCs with the structure (gamma-Glu-Cys)2Gly, (gamma-Glu-Cys)3Gly, and (gamma-Glu-Cys)4Gly. The Ag(I)-binding stoichiometries of these three peptides were determined by (i) UV/VIS spectrophotometry, (ii) luminescence spectroscopy at 77 K, and (iii) reverse-phase HPLC. The three techniques yielded similar results. ApoPCs exhibit featureless absorption in the 220-340 nm range. The binding of Ag(I) to PCs induced the appearance of specific absorption shoulders. The titration end point was indicated by the flattening of the characteristic absorption shoulders. Similarly, luminescence at 77 K due to Ag(I)-thiolate clusters increased with the addition of graded Ag(I) equivalents. The luminescence declined when Ag(I) equivalents in excess of the saturating amounts were added to the peptides. At neutral pH, (gamma-Glu-Cys)2Gly, (gamma-Glu-Cys)3Gly, and (gamma-Glu-Cys)4Gly bind 1.0, 1.5, and 4.0 equivalents of Ag(I), respectively. The Ag(I)-binding capacity of (gamma-Glu-Cys)2Gly and (gamma-Glu-Cys)3Gly was increased at pH 5.0 and below so that Ag(I)/-SH ratio approached 1.0. A similar pH-dependent binding of Ag(I) to glutathione was also observed. The increased Ag(I)-binding to PCs at lower pH is of physiological significance as these peptides accumulate in acidic vacuoles. We also report lifetime data on Ag(I)-PCs. The relatively long decay-times (approximately 0.1-0.3 msec) accompanied with a large Stokes shift in the emission band are indicative of spin-forbidden phosphorescence. PMID

  12. Conformational heterogeneity of the calmodulin binding interface

    NASA Astrophysics Data System (ADS)

    Shukla, Diwakar; Peck, Ariana; Pande, Vijay S.

    2016-04-01

    Calmodulin (CaM) is a ubiquitous Ca2+ sensor and a crucial signalling hub in many pathways aberrantly activated in disease. However, the mechanistic basis of its ability to bind diverse signalling molecules including G-protein-coupled receptors, ion channels and kinases remains poorly understood. Here we harness the high resolution of molecular dynamics simulations and the analytical power of Markov state models to dissect the molecular underpinnings of CaM binding diversity. Our computational model indicates that in the absence of Ca2+, sub-states in the folded ensemble of CaM's C-terminal domain present chemically and sterically distinct topologies that may facilitate conformational selection. Furthermore, we find that local unfolding is off-pathway for the exchange process relevant for peptide binding, in contrast to prior hypotheses that unfolding might account for binding diversity. Finally, our model predicts a novel binding interface that is well-populated in the Ca2+-bound regime and, thus, a candidate for pharmacological intervention.

  13. C60 fullerene binding to DNA

    NASA Astrophysics Data System (ADS)

    Alshehri, Mansoor H.; Cox, Barry J.; Hill, James M.

    2014-09-01

    Fullerenes have attracted considerable attention in various areas of science and technology. Owing to their exceptional physical, chemical, and biological properties, they have many applications, particularly in cosmetic and medical products. Using the Lennard-Jones 6-12 potential function and the continuum approximation, which assumes that intermolecular interactions can be approximated by average atomic surface densities, we determine the binding energies of a C60 fullerene with respect to both single-strand and double-strand DNA molecules. We assume that all configurations are in a vacuum and that the C60 fullerene is initially at rest. Double integrals are performed to determine the interaction energy of the system. We find that the C60 fullerene binds to the double-strand DNA molecule, at either the major or minor grooves, with binding energies of -4.7 eV or -2.3 eV, respectively, and that the C60 molecule binds to the single-strand DNA molecule with a binding energy of -1.6 eV. Our results suggest that the C60 molecule is most likely to be linked to the major groove of the dsDNA molecule.

  14. Conformational heterogeneity of the calmodulin binding interface

    PubMed Central

    Shukla, Diwakar; Peck, Ariana; Pande, Vijay S.

    2016-01-01

    Calmodulin (CaM) is a ubiquitous Ca2+ sensor and a crucial signalling hub in many pathways aberrantly activated in disease. However, the mechanistic basis of its ability to bind diverse signalling molecules including G-protein-coupled receptors, ion channels and kinases remains poorly understood. Here we harness the high resolution of molecular dynamics simulations and the analytical power of Markov state models to dissect the molecular underpinnings of CaM binding diversity. Our computational model indicates that in the absence of Ca2+, sub-states in the folded ensemble of CaM's C-terminal domain present chemically and sterically distinct topologies that may facilitate conformational selection. Furthermore, we find that local unfolding is off-pathway for the exchange process relevant for peptide binding, in contrast to prior hypotheses that unfolding might account for binding diversity. Finally, our model predicts a novel binding interface that is well-populated in the Ca2+-bound regime and, thus, a candidate for pharmacological intervention. PMID:27040077

  15. Improved flow cytometer measurement of binding assays

    NASA Astrophysics Data System (ADS)

    Saunders, G. C.

    1984-05-01

    A method of measuring binding assays is carried out with different size particles wherein the binding assay sample is run through a flow cytometer without separating the sample from the marking agent. The amount of a binding reactant present in a sample is determined by providing particles with a coating of binder and also known quantity of smaller particles with a coating of binder reactant. The smaller particles also contain a fluorescent chemical. The particles are combined with the sample and the binding reaction is allowed to occur for a set length of time followed by combining the smaller particles with the mixture of the particles and the sample produced and allowing the binding reactions to proceed to equilibrium. The fluorescence and light scatter of the combined mixture is then measured as the combined mixture passes through a flow cytometer equipped with a laser to bring about fluorescence, and the number of fluorescent events are compared. A similar method is also provided for determining the amount of antigen present in the sample by providing spheres with an antibody coating and some smaller spheres with an antigen coating.

  16. Stretching DNA to quantify nonspecific protein binding

    NASA Astrophysics Data System (ADS)

    Goyal, Sachin; Fountain, Chandler; Dunlap, David; Family, Fereydoon; Finzi, Laura

    2012-07-01

    Nonspecific binding of regulatory proteins to DNA can be an important mechanism for target search and storage. This seems to be the case for the lambda repressor protein (CI), which maintains lysogeny after infection of E. coli. CI binds specifically at two distant regions along the viral genome and induces the formation of a repressive DNA loop. However, single-molecule imaging as well as thermodynamic and kinetic measurements of CI-mediated looping show that CI also binds to DNA nonspecifically and that this mode of binding may play an important role in maintaining lysogeny. This paper presents a robust phenomenological approach using a recently developed method based on the partition function, which allows calculation of the number of proteins bound nonspecific to DNA from measurements of the DNA extension as a function of applied force. This approach was used to analyze several cycles of extension and relaxation of λ DNA performed at several CI concentrations to measure the dissociation constant for nonspecific binding of CI (˜100 nM), and to obtain a measurement of the induced DNA compaction (˜10%) by CI.

  17. DNA Binding to the Silica Surface.

    PubMed

    Shi, Bobo; Shin, Yun Kyung; Hassanali, Ali A; Singer, Sherwin J

    2015-08-27

    We investigate the DNA-silica binding mechanism using molecular dynamics simulations. This system is of technological importance, and also of interest to explore how negatively charged DNA can bind to a silica surface, which is also negatively charged at pH values above its isoelectric point near pH 3. We find that the two major binding mechanisms are attractive interactions between DNA phosphate and surface silanol groups and hydrophobic bonding between DNA base and silica hydrophobic region. Umbrella sampling and the weighted histogram analysis method (WHAM) are used to calculate the free energy surface for detachment of DNA from a binding configuration to a location far from the silica surface. Several factors explain why single-stranded DNA (ssDNA) has been observed to be more strongly attracted to silica than double-stranded (dsDNA): (1) ssDNA is more flexible and therefore able to maximize the number of binding interactions. (2) ssDNA has free unpaired bases to form hydrophobic attachment to silica while dsDNA has to break hydrogen bonds with base partners to get free bases. (3) The linear charge density of dsDNA is twice that of ssDNA. We devise a procedure to approximate the atomic forces between biomolecules and amorphous silica to enable large-scale biomolecule-silica simulations as reported here. PMID:25966319

  18. Peptide binding at the GLP-1 receptor.

    PubMed

    Mann, R; Nasr, N; Hadden, D; Sinfield, J; Abidi, F; Al-Sabah, S; de Maturana, R López; Treece-Birch, J; Willshaw, A; Donnelly, D

    2007-08-01

    The receptor for GLP-1 [glucagon-like peptide-1-(7-36)-amide] is a member of the 'Family B' of GPCRs (G-protein-coupled receptors) comprising an extracellular N-terminal domain containing six conserved cysteine residues (the N-domain) and a core domain (or J-domain) comprising the seven transmembrane helices and interconnecting loop regions. According to the two-domain model for peptide binding, the N-domain is primarily responsible for providing most of the peptide binding energy, whereas the core domain is responsible for binding the N-terminal region of the peptide agonists and transmitting the signal to the intracellular G-protein. Two interesting differences between the binding properties of two GLP-1 receptor agonists, GLP-1 and EX-4 (exendin-4), can be observed. First, while GLP-1 requires its full length to maintain high affinity, the eight N-terminal residues of EX-4 can be removed with little reduction in affinity. Secondly, EX-4 (but not GLP-1) can bind to the fully isolated N-domain of the receptor with an affinity matching that of the full-length receptor. In order to better understand these differences, we have studied the interaction between combinations of full-length or truncated ligands with full-length or truncated receptors. PMID:17635131

  19. Binding of Dissolved Strontium by Micrococcus luteus

    PubMed Central

    Faison, Brendlyn D.; Cancel, Carmen A.; Lewis, Susan N.; Adler, Howard I.

    1990-01-01

    Resting cells of Micrococcus luteus have been shown to remove strontium (Sr) from dilute aqueous solutions of SrCl2 at pH 7. Loadings of 25 mg of Sr per g of cell dry weight were achieved by cells exposed to a solution containing 50 ppm (mg/liter) of Sr. Sr binding occurred in the absence of nutrients and did not require metabolic activity. Initial binding was quite rapid (<0.5 h), although a slow, spontaneous release of Sr was observed over time. Sr binding was inhibited in the presence of polyvalent cations but not monovalent cations. Ca and Sr were bound preferentially over all other cations tested. Sr-binding activity was localized on the cell envelope and was sensitive to various chemical and physical pretreatments. Bound Sr was displaced by divalent ions or by H+. Other monovalent ions were less effective. Bound Sr was also removed by various chelating agents. It was concluded that Sr binding by M. luteus is a reversible equilibrium process. Both ion exchange mediated by acidic cell surface components and intracellular uptake may be involved in this activity. PMID:16348370

  20. Predicting tissue specific transcription factor binding sites

    PubMed Central

    2013-01-01

    Background Studies of gene regulation often utilize genome-wide predictions of transcription factor (TF) binding sites. Most existing prediction methods are based on sequence information alone, ignoring biological contexts such as developmental stages and tissue types. Experimental methods to study in vivo binding, including ChIP-chip and ChIP-seq, can only study one transcription factor in a single cell type and under a specific condition in each experiment, and therefore cannot scale to determine the full set of regulatory interactions in mammalian transcriptional regulatory networks. Results We developed a new computational approach, PIPES, for predicting tissue-specific TF binding. PIPES integrates in vitro protein binding microarrays (PBMs), sequence conservation and tissue-specific epigenetic (DNase I hypersensitivity) information. We demonstrate that PIPES improves over existing methods on distinguishing between in vivo bound and unbound sequences using ChIP-seq data for 11 mouse TFs. In addition, our predictions are in good agreement with current knowledge of tissue-specific TF regulation. Conclusions We provide a systematic map of computationally predicted tissue-specific binding targets for 284 mouse TFs across 55 tissue/cell types. Such comprehensive resource is useful for researchers studying gene regulation. PMID:24238150

  1. Aminoglycoside binding to Oxytricha Nova Telomeric DNA

    PubMed Central

    Ranjan, Nihar; Andreasen, Katrine F.; Kumar, Sunil; Hyde-volpe, David; Arya, Dev P.

    2012-01-01

    Telomeric DNA sequences have been at the center stage of drug design for cancer treatment in recent years. The ability of these DNA structures to form four stranded nucleic acid structures, called G-quadruplexes, has been perceived as target for inhibiting telomerase activity vital for the longevity of cancer cells. Being highly diverse in structural forms, these G-quadruplexes are subjects of detailed studies of ligand–DNA interactions of different classes, which will pave the way for logical design of more potent ligands in future. The binding of aminoglycosides were investigated with Oxytricha Nova quadruplex forming DNA sequence (GGGGTTTTGGGG)2. Isothermal Titration calorimetry (ITC) determined ligand to quadruplex binding ratio shows 1:1 neomycin:quadruplex binding with association constants (Ka ) ~ 105M−1 while paromomycin was found to have a two-fold weaker affinity than neomycin. The CD titration experiments with neomycin resulted in minimal changes in the CD signal. FID assays, performed to determine the minimum concentration required to displace half of the fluorescent probe bound, showed neomycin as the best of the all aminoglycosides studied for quadruplex binding. Initial NMR footprint suggests that ligand-DNA interactions occur in the wide groove of the quadruplex. Computational docking studies also indicate that aminoglycosides bind in the wide groove of the quadruplex. PMID:20886815

  2. Binding of dissolved strontium by Micrococcus luteus

    SciTech Connect

    Faison, B.D.; Cancel, C.A.; Lewis, S.N.; Adler, H.I. )

    1990-12-01

    Resting cells of Micrococcus luteus have been shown to remove strontium (Sr) from dilute aqueous solutions of SrCl{sub 2} at pH 7. Loadings of 25 mg of Sr per g of cell dry weight were achieved by cells exposed to a solution containing 50 ppm (mg/liter) of Sr. Sr binding occurred in the absence of nutrients and did not require metabolic activity. Initial binding was quite rapid (<0.5 h), although a slow, spontaneous release of Sr was observed over time. Sr binding was inhibited in the presence of polyvalent cations but not monovalent cations. Ca and Sr were bound preferentially over all other cations tested. Sr-binding activity was localized on the cell envelope and was sensitive to various chemical and physical pretreatments. Bound Sr was displaced by divalent ions or by H{sup +}. Other monovalent ions were less effective. Bound Sr was also removed by various chelating agents. It was concluded that Sr binding by M. luteus is a reversible equilibrium process. Both ion exchange mediated by acidic cell surface components and intracellular uptake may be involved in this activity.

  3. Ancestral Protein Reconstruction Yields Insights into Adaptive Evolution of Binding Specificity in Solute-Binding Proteins.

    PubMed

    Clifton, Ben E; Jackson, Colin J

    2016-02-18

    The promiscuous functions of proteins are an important reservoir of functional novelty in protein evolution, but the molecular basis for binding promiscuity remains elusive. We used ancestral protein reconstruction to experimentally characterize evolutionary intermediates in the functional expansion of the polar amino acid-binding protein family, which has evolved to bind a variety of amino acids with high affinity and specificity. High-resolution crystal structures of an ancestral arginine-binding protein in complex with l-arginine and l-glutamine show that the promiscuous binding of l-glutamine is enabled by multi-scale conformational plasticity, water-mediated interactions, and selection of an alternative conformational substate productive for l-glutamine binding. Evolution of specialized glutamine-binding proteins from this ancestral protein was achieved by displacement of water molecules from the protein-ligand interface, reducing the entropic penalty associated with the promiscuous interaction. These results provide a structural and thermodynamic basis for the co-option of a promiscuous interaction in the evolution of binding specificity. PMID:26853627

  4. Quantification of Cooperativity in Heterodimer-DNA Binding Improves the Accuracy of Binding Specificity Models.

    PubMed

    Isakova, Alina; Berset, Yves; Hatzimanikatis, Vassily; Deplancke, Bart

    2016-05-01

    Many transcription factors (TFs) have the ability to cooperate on DNA elements as heterodimers. Despite the significance of TF heterodimerization for gene regulation, a quantitative understanding of cooperativity between various TF dimer partners and its impact on heterodimer DNA binding specificity models is still lacking. Here, we used a novel integrative approach, combining microfluidics-steered measurements of dimer-DNA assembly with mechanistic modeling of the implicated protein-protein-DNA interactions to quantitatively interrogate the cooperative DNA binding behavior of the adipogenic peroxisome proliferator-activated receptor γ (PPARγ):retinoid X receptor α (RXRα) heterodimer. Using the high throughput MITOMI (mechanically induced trapping of molecular interactions) platform, we derived equilibrium DNA binding data for PPARγ, RXRα, as well as the PPARγ:RXRα heterodimer to more than 300 target DNA sites and variants thereof. We then quantified cooperativity underlying heterodimer-DNA binding and derived an integrative heterodimer DNA binding constant. Using this cooperativity-inclusive constant, we were able to build a heterodimer-DNA binding specificity model that has superior predictive power than the one based on a regular one-site equilibrium. Our data further revealed that individual nucleotide substitutions within the target site affect the extent of cooperativity in PPARγ:RXRα-DNA binding. Our study therefore emphasizes the importance of assessing cooperativity when generating DNA binding specificity models for heterodimers. PMID:26912662

  5. Binding of the Ah receptor to receptor binding factors in chromatin.

    PubMed

    Dunn, R T; Ruh, T S; Ruh, M F

    1993-03-01

    Dioxin induces biological responses through interaction with a specific intracellular receptor, the Ah receptor, and the subsequent interaction of the Ah receptor with chromatin. We report the binding of the Ah receptor, partially purified from rabbit liver, to receptor binding factors in chromatin. Rabbit liver chromatin proteins (CP) were isolated by adsorption of chromatin to hydroxylapatite followed by sequential extraction with 1-8 M GdnHCl. To assay for receptor binding a portion of each CP fraction was reconstituted to rabbit double-stranded DNA using a reverse gradient dialysis of 7.5 to 0 M GdnHCl. These reconstituted nucleoacidic proteins were then examined for binding to [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin ([3H]TCDD)-receptor complexes by the streptomycin filter assay. Prior to the binding assay, [3H]TCDD-receptor complexes were partially purified by step elution from DEAE-cellulose columns. CP fractions 2, 5, and 7 were found to bind to the Ah receptor with high affinity. Scatchard analysis yielded Kd values in the nanomolar range. Competition with 2-fold excess unlabeled TCDD-receptor complexes was demonstrated, and binding was reduced markedly when the receptor was prepared in the presence of 10 mM molybdate. Such chromatin receptor binding factors (RBFs) may participate in the interaction of receptor with specific DNA sequences resulting in modulation of specific gene expression. PMID:8384852

  6. Selective polyamine-binding proteins. Spermine binding by an androgen-sensitive phosphoprotein.

    PubMed

    Liang, T; Mezzetti, G; Chen, C; Liao, S

    1978-09-01

    Rat ventral prostate contains an acidic protein which can bind spermine selectively. The relative binding affinities of various aliphatic amines for the protein are, in decreasing order, spermine greater than thermine greater than greater than putrecine greater than 1,10-diaminodecane, cadaverine and 1,12-diaminododecane. The binding protein has an isoelectric point at pH 4.3 and a sedimentation coefficient of 3 S. Its molecular weight is approx. 30 000. Histones and nuclear chromatin preparations of the prostate can interact with the binding protein. The spermine-binding activity of the purified prostate protein can be inactivated by treatment with intestinal alkaline phosphatases. The phosphatase treated preparation can then be reactivated by beef heart protein kinase in the presence of cyclic AMP and ATP. The spermine-binding activity of the prostate cytosol protein fraction decreases after castration, but increases very rapidly after the castrated rats are injected with 5alpha-dihydrotestosterone. This finding raises the possibility that, in the postate, certain androgen actions may be dependent on the androgen-induced increase in the acidic protein binding of polyamines and their translocation to a functional cellular site such as nuclear chromatin. In the prostate cytosol, spermine also binds to 4-S tRNAs and to a unique RNA which has a sedimentation coefficient of 1.5 S. PMID:28786

  7. Binding dynamics and energetic insight into the molecular forces driving nucleotide binding by guanylate kinase.

    PubMed

    Kandeel, Mahmoud; Kitade, Yukio

    2011-01-01

    Plasmodium deoxyguanylate pathways are an attractive area of investigation for future metabolic and drug discovery studies due to their unique substrate specificities. We investigated the energetic contribution to guanylate kinase substrate binding and the forces underlying ligand recognition. In the range from 20 to 35°C, the thermodynamic profiles displayed marked decrease in binding enthalpy, while the free energy of binding showed little changes. GMP produced a large binding heat capacity change of -356 cal mol(-1) K(-1), indicating considerable conformational changes upon ligand binding. Interestingly, the calculated ΔCp was -32 cal mol(-1) K(-1), indicating that the accessible surface area is not the central change in substrate binding, and that other entropic forces, including conformational changes, are more predominant. The thermodynamic signature for GMP is inconsistent with rigid-body association, while dGMP showed more or less rigid-body association. These binding profiles explain the poor catalytic efficiency and low affinity for dGMP compared with GMP. At low temperature, the ligands bind to the receptor site under the effect of hydrophobic forces. Interestingly, by increasing the temperature, the entropic forces gradually vanish and proceed to a nonfavorable contribution, and the interaction occurs mainly through bonding, electrostatic forces, and van der Waals interactions. PMID:21360614

  8. Estrophilin immunoreactivity versus estrogen receptor binding activity in meningiomas: evidence for multiple estrogen binding sites

    SciTech Connect

    Lesch, K.P.; Schott, W.; Gross, S.

    1987-09-01

    The existence of estrogen receptors in human meningiomas has long been a controversial issue. This may be explained, in part, by apparent heterogeneity of estrogen binding sites in meningioma tissue. In this study, estrogen receptors were determined in 58 meningiomas with an enzyme immunoassay using monoclonal antibodies against human estrogen receptor protein (estrophilin) and with a sensitive radioligand binding assay using /sup 125/I-labeled estradiol (/sup 125/I-estradiol) as radioligand. Low levels of estrophilin immunoreactivity were found in tumors from 62% of patients, whereas radioligand binding activity was demonstrated in about 46% of the meningiomas examined. In eight (14%) tissue samples multiple binding sites for estradiol were observed. The immunoreactive binding sites correspond to the classical, high affinity estrogen receptors: the Kd for /sup 125/I-estradiol binding to the receptor was approximately 0.2 nM and the binding was specific for estrogens. The second, low affinity class of binding sites considerably influenced measurement of the classical receptor even at low ligand concentrations. The epidemiological and clinical data from patients with meningiomas, and the existence of specific estrogen receptors confirmed by immunochemical detection, may be important factors in a theory of oncogenesis.

  9. Natural ligand binding and transfer from liver fatty acid binding protein (LFABP) to membranes.

    PubMed

    De Gerónimo, Eduardo; Hagan, Robert M; Wilton, David C; Córsico, Betina

    2010-09-01

    Liver fatty acid-binding protein (LFABP) is distinctive among fatty acid-binding proteins because it binds more than one molecule of long-chain fatty acid and a variety of diverse ligands. Also, the transfer of fluorescent fatty acid analogues to model membranes under physiological ionic strength follows a different mechanism compared to most of the members of this family of intracellular lipid binding proteins. Tryptophan insertion mutants sensitive to ligand binding have allowed us to directly measure the binding affinity, ligand partitioning and transfer to model membranes of natural ligands. Binding of fatty acids shows a cooperative mechanism, while acyl-CoAs binding presents a hyperbolic behavior. Saturated fatty acids seem to have a stronger partition to protein vs. membranes, compared to unsaturated fatty acids. Natural ligand transfer rates are more than 200-fold higher compared to fluorescently-labeled analogues. Interestingly, oleoyl-CoA presents a markedly different transfer behavior compared to the rest of the ligands tested, probably indicating the possibility of specific targeting of ligands to different metabolic fates. PMID:20541621

  10. Quantification of Cooperativity in Heterodimer-DNA Binding Improves the Accuracy of Binding Specificity Models*

    PubMed Central

    Isakova, Alina; Berset, Yves; Hatzimanikatis, Vassily; Deplancke, Bart

    2016-01-01

    Many transcription factors (TFs) have the ability to cooperate on DNA elements as heterodimers. Despite the significance of TF heterodimerization for gene regulation, a quantitative understanding of cooperativity between various TF dimer partners and its impact on heterodimer DNA binding specificity models is still lacking. Here, we used a novel integrative approach, combining microfluidics-steered measurements of dimer-DNA assembly with mechanistic modeling of the implicated protein-protein-DNA interactions to quantitatively interrogate the cooperative DNA binding behavior of the adipogenic peroxisome proliferator-activated receptor γ (PPARγ):retinoid X receptor α (RXRα) heterodimer. Using the high throughput MITOMI (mechanically induced trapping of molecular interactions) platform, we derived equilibrium DNA binding data for PPARγ, RXRα, as well as the PPARγ:RXRα heterodimer to more than 300 target DNA sites and variants thereof. We then quantified cooperativity underlying heterodimer-DNA binding and derived an integrative heterodimer DNA binding constant. Using this cooperativity-inclusive constant, we were able to build a heterodimer-DNA binding specificity model that has superior predictive power than the one based on a regular one-site equilibrium. Our data further revealed that individual nucleotide substitutions within the target site affect the extent of cooperativity in PPARγ:RXRα-DNA binding. Our study therefore emphasizes the importance of assessing cooperativity when generating DNA binding specificity models for heterodimers. PMID:26912662