WorldWideScience

Sample records for active site conformation

  1. Active site loop conformation regulates promiscuous activity in a lactonase from Geobacillus kaustophilus HTA426.

    Yu Zhang

    Full Text Available Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL from Geobacillus kaustophilus HTA426 (GkaP exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a "hot spot" in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (kcat/Km toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity.

  2. Fragment-based identification of determinants of conformational and spectroscopic change at the ricin active site

    Soares Alexei S

    2007-11-01

    Full Text Available Abstract Background Ricin is a potent toxin and known bioterrorism threat with no available antidote. The ricin A-chain (RTA acts enzymatically to cleave a specific adenine base from ribosomal RNA, thereby blocking translation. To understand better the relationship between ligand binding and RTA active site conformational change, we used a fragment-based approach to find a minimal set of bonding interactions able to induce rearrangements in critical side-chain positions. Results We found that the smallest ligand stabilizing an open conformer of the RTA active site pocket was an amide group, bound weakly by only a few hydrogen bonds to the protein. Complexes with small amide-containing molecules also revealed a switch in geometry from a parallel towards a splayed arrangement of an arginine-tryptophan cation-pi interaction that was associated with an increase and red-shift in tryptophan fluorescence upon ligand binding. Using the observed fluorescence signal, we determined the thermodynamic changes of adenine binding to the RTA active site, as well as the site-specific binding of urea. Urea binding had a favorable enthalpy change and unfavorable entropy change, with a ΔH of -13 ± 2 kJ/mol and a ΔS of -0.04 ± 0.01 kJ/(K*mol. The side-chain position of residue Tyr80 in a complex with adenine was found not to involve as large an overlap of rings with the purine as previously considered, suggesting a smaller role for aromatic stacking at the RTA active site. Conclusion We found that amide ligands can bind weakly but specifically to the ricin active site, producing significant shifts in positions of the critical active site residues Arg180 and Tyr80. These results indicate that fragment-based drug discovery methods are capable of identifying minimal bonding determinants of active-site side-chain rearrangements and the mechanistic origins of spectroscopic shifts. Our results suggest that tryptophan fluorescence provides a sensitive probe for the

  3. Rate of hydrolysis in ATP synthase is fine-tuned by  -subunit motif controlling active site conformation

    Beke-Somfai, T.

    2013-01-23

    Computer-designed artificial enzymes will require precise understanding of how conformation of active sites may control barrier heights of key transition states, including dependence on structure and dynamics at larger molecular scale. F(o)F(1) ATP synthase is interesting as a model system: a delicate molecular machine synthesizing or hydrolyzing ATP using a rotary motor. Isolated F(1) performs hydrolysis with a rate very sensitive to ATP concentration. Experimental and theoretical results show that, at low ATP concentrations, ATP is slowly hydrolyzed in the so-called tight binding site, whereas at higher concentrations, the binding of additional ATP molecules induces rotation of the central γ-subunit, thereby forcing the site to transform through subtle conformational changes into a loose binding site in which hydrolysis occurs faster. How the 1-Å-scale rearrangements are controlled is not yet fully understood. By a combination of theoretical approaches, we address how large macromolecular rearrangements may manipulate the active site and how the reaction rate changes with active site conformation. Simulations reveal that, in response to γ-subunit position, the active site conformation is fine-tuned mainly by small α-subunit changes. Quantum mechanics-based results confirm that the sub-Ångström gradual changes between tight and loose binding site structures dramatically alter the hydrolysis rate.

  4. Active site conformational changes of prostasin provide a new mechanism of protease regulation by divalent cations

    Prostasin or human channel-activating protease 1 has been reported to play a critical role in the regulation of extracellular sodium ion transport via its activation of the epithelial cell sodium channel. Here, the structure of the extracellular portion of the membrane associated serine protease has been solved to high resolution in complex with a nonselective d-FFR chloromethyl ketone inhibitor, in an apo form, in a form where the apo crystal has been soaked with the covalent inhibitor camostat and in complex with the protein inhibitor aprotinin. It was also crystallized in the presence of the divalent cation Ca+2. Comparison of the structures with each other and with other members of the trypsin-like serine protease family reveals unique structural features of prostasin and a large degree of conformational variation within specificity determining loops. Of particular interest is the S1 subsite loop which opens and closes in response to basic residues or divalent ions, directly binding Ca+2 cations. This induced fit active site provides a new possible mode of regulation of trypsin-like proteases adapted in particular to extracellular regions with variable ionic concentrations such as the outer membrane layer of the epithelial cell.

  5. Conformational Change in the Active Site of Streptococcal Unsaturated Glucuronyl Hydrolase Through Site-Directed Mutagenesis at Asp-115.

    Nakamichi, Yusuke; Oiki, Sayoko; Mikami, Bunzo; Murata, Kousaku; Hashimoto, Wataru

    2016-08-01

    Bacterial unsaturated glucuronyl hydrolase (UGL) degrades unsaturated disaccharides generated from mammalian extracellular matrices, glycosaminoglycans, by polysaccharide lyases. Two Asp residues, Asp-115 and Asp-175 of Streptococcus agalactiae UGL (SagUGL), are completely conserved in other bacterial UGLs, one of which (Asp-175 of SagUGL) acts as a general acid and base catalyst. The other Asp (Asp-115 of SagUGL) also affects the enzyme activity, although its role in the enzyme reaction has not been well understood. Here, we show substitution of Asp-115 in SagUGL with Asn caused a conformational change in the active site. Tertiary structures of SagUGL mutants D115N and D115N/K370S with negligible enzyme activity were determined at 2.00 and 1.79 Å resolution, respectively, by X-ray crystallography. The side chain of Asn-115 is drastically shifted in both mutants owing to the interaction with several residues, including Asp-175, by formation of hydrogen bonds. This interaction between Asn-115 and Asp-175 probably prevents the mutants from triggering the enzyme reaction using Asp-175 as an acid catalyst. PMID:27402448

  6. Kinetic and conformational effects of lysine substitutions for arginines 35 and 87 in the active site of Staphylococcal nuclease

    The high-resolution X-ray crystal structure of staphylococcal nuclease (SNase) suggests that the guanidinium groups of Arg 35 and Arg 87 participate as electrophilic catalysts in the attack of water on the substrate phosphodiester. Both arginine residues have been replaced with conservative lysine residues so that both the importance of these residues in catalysis and the effect of changes in electrostatic interactions on active site conformation can be assessed. The catalytic efficiencies of R35K and R87K are decreased by factors of 104 and 105 relative to wild-type SNase, with R87K showing a very significant reduction in its affinity for both DNA substrate and the competitive inhibitor thymidine 3',5'-bisphosphate (pdTp). The thermal denaturation behavior of both mutant enzymes differs from that of wild type both in the absence and in the presence of the active site ligands Ca2+ and pdTp. Both the 1H NMR chemical shifts and interresidue nuclear Overhauser effects (NOEs) of residues previously assigned to be in the hydrophobic core of SNase are altered in R35K and R87K. These observations suggest that lysine substitutions are not conservative in SNase and disrupt the conformation of the active site. Thus, the kinetic properties of R35K and R87K cannot be used to describe the roles of Arg 35 and Arg 87 in catalysis by the wild-type enzyme. Furthermore, on the basis of the effects of these conservative substitutions for Arg 35 and Arg 87. The authors predict that other substitutions for these residues, including neutral substitutions, are also likely to cause conformational alterations in the active site

  7. Effects of protonation state of Asp181 and position of active site water molecules on the conformation of PTP1B.

    Ozcan, Ahmet; Olmez, Elif Ozkirimli; Alakent, Burak

    2013-05-01

    In protein tyrosine phosphatase 1B (PTP1B), the flexible WPD loop adopts a closed conformation (WPDclosed ) in the active state of PTP1B, bringing the catalytic Asp181 close to the active site pocket, while WPD loop is in an open conformation (WPDopen ) in the inactive state. Previous studies showed that Asp181 may be protonated at physiological pH, and ordered water molecules exist in the active site. In the current study, molecular dynamics simulations are employed at different Asp181 protonation states and initial positions of active site water molecules, and compared with the existing crystallographic data of PTP1B. In WPDclosed conformation, the active site is found to maintain its conformation only in the protonated state of Asp181 in both free and liganded states, while Asp181 is likely to be deprotonated in WPDopen conformation. When the active site water molecule network that is a part of the free WPDclosed crystal structure is disrupted, intermediate WPD loop conformations, similar to that in the PTPRR crystal structure, are sampled in the MD simulations. In liganded PTP1B, one active site water molecule is found to be important for facilitating the orientation of Cys215 and the phosphate ion, thus may play a role in the reaction. In conclusion, conformational stability of WPD loop, and possibly catalytic activity of PTP1B, is significantly affected by the protonation state of Asp181 and position of active site water molecules, showing that these aspects should be taken into consideration both in MD simulations and inhibitor design. PMID:23239271

  8. Magnesium-Dependent Active-Site Conformational Selection in the Diels-Alderase Ribozyme

    Berezniak, Tomasz [University of Heidelberg; Zahran, Mai [ORNL; Imhof, Petra [University of Heidelberg; Jaeschke, Andres [Free University of Berlin; Smith, Jeremy C [ORNL

    2010-10-01

    The Diels-Alderase ribozyme, an in vitro-evolved ribonucleic acid enzyme, accelerates the formation of carbon-carbon bonds between an anthracene diene and a maleimide dienophile in a [4 + 2] cycloaddition, a reaction with broad application in organic chemistry. Here, the Diels-Alderase ribozyme is examined via molecular dynamics (MD) simulations in both crystalline and aqueous solution environments. The simulations indicate that the catalytic pocket is highly dynamic. At low Mg(2+) ion concentrations, inactive states with the catalytic pocket closed dominate. Stabilization of the enzymatically active, open state of the catalytic pocket requires a high concentration of Mg(2+) ions (e.g., 54 mM), with cations binding to specific phosphate sites on the backbone of the residues bridging the opposite strands of the pocket. The free energy profile for pocket opening at high Mg(2+) cation concentration exhibits a double minimum, with a barrier to opening of approximately 5.5 kJ/mol and the closed state approximately 3 kJ/mol lower than the open state. Selection of the open state on substrate binding leads to the catalytic activity of the ribozyme. The simulation results explain structurally the experimental observation that full catalytic activity depends on the Mg(2+) ion concentration

  9. Two-step mechanism involving active-site conformational changes regulates human telomerase DNA binding.

    Tomlinson, Christopher G; Moye, Aaron L; Holien, Jessica K; Parker, Michael W; Cohen, Scott B; Bryan, Tracy M

    2015-01-15

    The ribonucleoprotein enzyme telomerase maintains telomeres and is essential for cellular immortality in most cancers. Insight into the telomerase mechanism can be gained from syndromes such as dyskeratosis congenita, in which mutation of telomerase components manifests in telomere dysfunction. We carried out detailed kinetic and thermodynamic analyses of wild-type telomerase and two disease-associated mutations in the reverse transcriptase domain. Differences in dissociation rates between primers with different 3' ends were independent of DNA affinities, revealing that initial binding of telomerase to telomeric DNA occurs through a previously undescribed two-step mechanism involving enzyme conformational changes. Both mutations affected DNA binding, but through different mechanisms: P704S specifically affected protein conformational changes during DNA binding, whereas R865H showed defects in binding to the 3' region of the DNA. To gain further insight at the structural level, we generated the first homology model of the human telomerase reverse transcriptase domain; the positions of P704S and R865H corroborate their observed mechanistic defects, providing validation for the structural model. Our data reveal the importance of protein interactions with the 3' end of telomeric DNA and the role of protein conformational change in telomerase DNA binding, and highlight naturally occurring disease mutations as a rich source of mechanistic insight. PMID:25365545

  10. A remote palm domain residue of RB69 DNA polymerase is critical for enzyme activity and influences the conformation of the active site.

    Agata Jacewicz

    Full Text Available Non-conserved amino acids that are far removed from the active site can sometimes have an unexpected effect on enzyme catalysis. We have investigated the effects of alanine replacement of residues distant from the active site of the replicative RB69 DNA polymerase, and identified a substitution in a weakly conserved palm residue (D714A, that renders the enzyme incapable of sustaining phage replication in vivo. D714, located several angstroms away from the active site, does not contact the DNA or the incoming dNTP, and our apoenzyme and ternary crystal structures of the Pol(D714A mutant demonstrate that D714A does not affect the overall structure of the protein. The structures reveal a conformational change of several amino acid side chains, which cascade out from the site of the substitution towards the catalytic center, substantially perturbing the geometry of the active site. Consistent with these structural observations, the mutant has a significantly reduced k pol for correct incorporation. We propose that the observed structural changes underlie the severe polymerization defect and thus D714 is a remote, non-catalytic residue that is nevertheless critical for maintaining an optimal active site conformation. This represents a striking example of an action-at-a-distance interaction.

  11. Conformational Lability in Serine Protease Active Sites: Structures of Hepatocyte Growth Factor Activator (HGFA) Alone and with the Inhibitory Domain from HGFA Inhibitor-1B

    Hepatocyte growth factor activator (HGFA) is a serine protease that converts hepatocyte growth factor (HGF) into its active form. When activated HGF binds its cognate receptor Met, cellular signals lead to cell growth, differentiation, and migration, activities which promote tissue regeneration in liver, kidney and skin. Intervention in the conversion of HGF to its active form has the potential to provide therapeutic benefit where HGF/Met activity is associated with tumorigenesis. To help identify ways to moderate HGF/Met effects, we have determined the molecular structure of the protease domain of HGFA. The structure we determined, at 2.7 (angstrom) resolution, with no pseudo-substrate or inhibitor bound is characterized by an unconventional conformation of key residues in the enzyme active site. In order to find whether this apparently non-enzymatically competent arrangement would persist in the presence of a strongly-interacting inhibitor, we also have determined, at 2.6 (angstrom) resolution, the X-ray structure of HGFA complexed with the first Kunitz domain (KD1) from the physiological inhibitor hepatocyte growth factor activator inhibitor 1B (HAI-1B). In this complex we observe a rearranged substrate binding cleft that closely mirrors the cleft of other serine proteases, suggesting an extreme conformational dynamism. We also characterize the inhibition of 16 serine proteases by KD1, finding that the previously reported enzyme specificity of the intact extracellular region of HAI-1B resides in KD1 alone. We find that HGFA, matriptase, hepsin, plasma kallikrein and trypsin are potently inhibited, and use the complex structure to rationalize the structural basis of these results.

  12. The Role of Active Site Flexible Loops in Catalysis and of Zinc in Conformational Stability of Bacillus cereus 569/H/9 β-Lactamase.

    Montagner, Caroline; Nigen, Michaël; Jacquin, Olivier; Willet, Nicolas; Dumoulin, Mireille; Karsisiotis, Andreas Ioannis; Roberts, Gordon C K; Damblon, Christian; Redfield, Christina; Matagne, André

    2016-07-29

    Metallo-β-lactamases catalyze the hydrolysis of most β-lactam antibiotics and hence represent a major clinical concern. The development of inhibitors for these enzymes is complicated by the diversity and flexibility of their substrate-binding sites, motivating research into their structure and function. In this study, we examined the conformational properties of the Bacillus cereus β-lactamase II in the presence of chemical denaturants using a variety of biochemical and biophysical techniques. The apoenzyme was found to unfold cooperatively, with a Gibbs free energy of stabilization (ΔG(0)) of 32 ± 2 kJ·mol(-1) For holoBcII, a first non-cooperative transition leads to multiple interconverting native-like states, in which both zinc atoms remain bound in an apparently unaltered active site, and the protein displays a well organized compact hydrophobic core with structural changes confined to the enzyme surface, but with no catalytic activity. Two-dimensional NMR data revealed that the loss of activity occurs concomitantly with perturbations in two loops that border the enzyme active site. A second cooperative transition, corresponding to global unfolding, is observed at higher denaturant concentrations, with ΔG(0) value of 65 ± 1.4 kJ·mol(-1) These combined data highlight the importance of the two zinc ions in maintaining structure as well as a relatively well defined conformation for both active site loops to maintain enzymatic activity. PMID:27235401

  13. Ubiquitin vinyl methyl ester binding orients the misaligned active site of the ubiquitin hydrolase UCHL1 into productive conformation

    Boudreaux, David A.; Maiti, Tushar K.; Davies, Christopher W.; Das, Chittaranjan (Purdue)

    2010-07-06

    Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a Parkinson disease-associated, putative cysteine protease found abundantly and selectively expressed in neurons. The crystal structure of apo UCHL1 showed that the active-site residues are not aligned in a canonical form, with the nucleophilic cysteine being 7.7 {angstrom} from the general base histidine, an arrangement consistent with an inactive form of the enzyme. Here we report the crystal structures of the wild type and two Parkinson disease-associated variants of the enzyme, S18Y and I93M, bound to a ubiquitin-based suicide substrate, ubiquitin vinyl methyl ester. These structures reveal that ubiquitin vinyl methyl ester binds primarily at two sites on the enzyme, with its carboxy terminus at the active site and with its amino-terminal {beta}-hairpin at the distal site - a surface-exposed hydrophobic crevice 17 {angstrom} away from the active site. Binding at the distal site initiates a cascade of side-chain movements in the enzyme that starts at a highly conserved, surface-exposed phenylalanine and is relayed to the active site resulting in the reorientation and proximal placement of the general base within 4 {angstrom} of the catalytic cysteine, an arrangement found in productive cysteine proteases. Mutation of the distal-site, surface-exposed phenylalanine to alanine reduces ubiquitin binding and severely impairs the catalytic activity of the enzyme. These results suggest that the activity of UCHL1 may be regulated by its own substrate.

  14. Crystallographic evidence of drastic conformational changes in the active site of a flavin-dependent N-hydroxylase.

    Setser, Jeremy W; Heemstra, John R; Walsh, Christopher T; Drennan, Catherine L

    2014-09-30

    The soil actinomycete Kutzneria sp. 744 produces a class of highly decorated hexadepsipeptides, which represent a new chemical scaffold that has both antimicrobial and antifungal properties. These natural products, known as kutznerides, are created via nonribosomal peptide synthesis using various derivatized amino acids. The piperazic acid moiety contained in the kutzneride scaffold, which is vital for its antibiotic activity, has been shown to derive from the hydroxylated product of l-ornithine, l-N(5)-hydroxyornithine. The production of this hydroxylated species is catalyzed by the action of an FAD- and NAD(P)H-dependent N-hydroxylase known as KtzI. We have been able to structurally characterize KtzI in several states along its catalytic trajectory, and by pairing these snapshots with the biochemical and structural data already available for this enzyme class, we propose a structurally based reaction mechanism that includes novel conformational changes of both the protein backbone and the flavin cofactor. Further, we were able to recapitulate these conformational changes in the protein crystal, displaying their chemical competence. Our series of structures, with corroborating biochemical and spectroscopic data collected by us and others, affords mechanistic insight into this relatively new class of flavin-dependent hydroxylases and adds another layer to the complexity of flavoenzymes. PMID:25184411

  15. Conformational Flexibility of a Short Loop near the Active Site of the SARS-3CLpro is Essential to Maintain Catalytic Activity

    Li, Chunmei; Teng, Xin; Qi, Yifei; Tang, Bo; Shi, Hailing; Ma, Xiaomin; Lai, Luhua

    2016-02-01

    The SARS 3C-like proteinase (SARS-3CLpro), which is the main proteinase of the SARS coronavirus, is essential to the virus life cycle. This enzyme has been shown to be active as a dimer in which only one protomer is active. However, it remains unknown how the dimer structure maintains an active monomer conformation. It has been observed that the Ser139-Leu141 loop forms a short 310-helix that disrupts the catalytic machinery in the inactive monomer structure. We have tried to disrupt this helical conformation by mutating L141 to T in the stable inactive monomer G11A/R298A/Q299A. The resulting tetra-mutant G11A/L141T/R298A/Q299A is indeed enzymatically active as a monomer. Molecular dynamics simulations revealed that the L141T mutation disrupts the 310-helix and helps to stabilize the active conformation. The coil-310-helix conformational transition of the Ser139-Leu141 loop serves as an enzyme activity switch. Our study therefore indicates that the dimer structure can stabilize the active conformation but is not a required structure in the evolution of the active enzyme, which can also arise through simple mutations.

  16. Structure of Mycobacterium tuberculosis phosphopantetheine adenylyltransferase in complex with the feedback inhibitor CoA reveals only one active-site conformation

    The X-ray structure of phosphopantetheine adenylyltransferase (PPAT) from M. tuberculosis in complex with its feedback regulator coenzyme A (CoA) was determined to 2.11 Å resolution. Unlike previous X-ray structures of PPAT–CoA complexes from other bacteria, which showed two distinct conformations of bound CoA, only one conformation of bound CoA is observed in the M. tuberculosis PPAT–CoA complex. Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in the coenzyme A (CoA) biosynthetic pathway, reversibly transferring an adenylyl group from ATP to 4′-phosphopantetheine to form dephosphocoenzyme A (dPCoA). To complement recent biochemical and structural studies on Mycobacterium tuberculosis PPAT (MtPPAT) and to provide further insight into the feedback regulation of MtPPAT by CoA, the X-ray crystal structure of the MtPPAT enzyme in complex with CoA was determined to 2.11 Å resolution. Unlike previous X-ray crystal structures of PPAT–CoA complexes from other bacteria, which showed two distinct CoA conformations bound to the active site, only one conformation of CoA is observed in the MtPPAT–CoA complex

  17. Structure of Mycobacterium tuberculosis phosphopantetheine adenylyltransferase in complex with the feedback inhibitor CoA reveals only one active-site conformation

    Wubben, T.; Mesecar, A.D. (Purdue); (UIC)

    2014-10-02

    Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in the coenzyme A (CoA) biosynthetic pathway, reversibly transferring an adenylyl group from ATP to 4'-phosphopantetheine to form dephosphocoenzyme A (dPCoA). To complement recent biochemical and structural studies on Mycobacterium tuberculosis PPAT (MtPPAT) and to provide further insight into the feedback regulation of MtPPAT by CoA, the X-ray crystal structure of the MtPPAT enzyme in complex with CoA was determined to 2.11 {angstrom} resolution. Unlike previous X-ray crystal structures of PPAT-CoA complexes from other bacteria, which showed two distinct CoA conformations bound to the active site, only one conformation of CoA is observed in the MtPPAT-CoA complex.

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

    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

  19. Active site conformational dynamics are coupled to catalysis in the mRNA decapping enzyme Dcp2

    Aglietti, Robin A.; Floor, Stephen N.; McClendon, Chris L.; Matthew P Jacobson; Gross, John D.

    2013-01-01

    Removal of the 5′ cap structure by Dcp2 is a major step in several 5′–3′ mRNA decay pathways. The activity of Dcp2 is enhanced by Dcp1 and bound coactivators, yet the details of how these interactions are linked to chemistry are poorly understood. Here we report three crystal structures of the catalytic Nudix hydrolase domain of Dcp2 that demonstrate binding of a catalytically essential metal ion, and enzyme kinetics are used to identify several key active site residues involved in acid/base ...

  20. The structure of putative N-acetyl glutamate kinase from Thermus thermophilus reveals an intermediate active site conformation of the enzyme.

    Sundaresan, Ramya; Ragunathan, Preethi; Kuramitsu, Seiki; Yokoyama, Shigeyuki; Kumarevel, Thirumananseri; Ponnuraj, Karthe

    2012-04-13

    The de novo biosynthesis of arginine in microorganisms and plants is accomplished via several enzymatic steps. The enzyme N-acetyl glutamate kinase (NAGK) catalyzes the phosphorylation of the γ-COO(-) group of N-acetyl-L-glutamate (NAG) by adenosine triphosphate (ATP) which is the second rate limiting step in arginine biosynthesis pathway. Here we report the crystal structure of putative N-acetyl glutamate kinase (NAGK) from Thermus thermophilus HB8 (TtNAGK) determined at 1.92Å resolution. The structural analysis of TtNAGK suggests that the dimeric quaternary state of the enzyme and arginine insensitive nature are similar to mesophilic Escherichia coli NAGK. These features are significantly different from its thermophilic homolog Thermatoga maritima NAGK which is hexameric and arginine-sensitive. TtNAGK is devoid of its substrates but contains two sulfates at the active site. Very interestingly the active site of the enzyme adopts a conformation which is not completely open or closed and likely represents an intermediate stage in the catalytic cycle unlike its structural homologs, which all exist either in the open or closed conformation. Engineering arginine biosynthesis pathway enzymes for the production of l-arginine is an important industrial application. The structural comparison of TtNAGK with EcNAGK revealed the structural basis of thermostability of TtNAGK and this information could be very useful to generate mutants of NAGK with increased overall stability. PMID:22452987

  1. A selective, slow binding inhibitor of factor VIIa binds to a nonstandard active site conformation and attenuates thrombus formation in vivo.

    Olivero, Alan G; Eigenbrot, Charles; Goldsmith, Richard; Robarge, Kirk; Artis, Dean R; Flygare, John; Rawson, Thomas; Sutherlin, Daniel P; Kadkhodayan, Saloumeh; Beresini, Maureen; Elliott, Linda O; DeGuzman, Geralyn G; Banner, David W; Ultsch, Mark; Marzec, Ulla; Hanson, Stephen R; Refino, Canio; Bunting, Stuart; Kirchhofer, Daniel

    2005-03-11

    The serine protease factor VIIa (FVIIa) in complex with its cellular cofactor tissue factor (TF) initiates the blood coagulation reactions. TF.FVIIa is also implicated in thrombosis-related disorders and constitutes an appealing therapeutic target for treatment of cardiovascular diseases. To this end, we generated the FVIIa active site inhibitor G17905, which displayed great potency toward TF.FVIIa (Ki = 0.35 +/- 0.11 nM). G17905 did not appreciably inhibit 12 of the 14 examined trypsin-like serine proteases, consistent with its TF.FVIIa-specific activity in clotting assays. The crystal structure of the FVIIa.G17905 complex provides insight into the molecular basis of the high selectivity. It shows that, compared with other serine proteases, FVIIa is uniquely equipped to accommodate conformational disturbances in the Gln217-Gly219 region caused by the ortho-hydroxy group of the inhibitor's aminobenzamidine moiety located in the S1 recognition pocket. Moreover, the structure revealed a novel, nonstandard conformation of FVIIa active site in the region of the oxyanion hole, a "flipped" Lys192-Gly193 peptide bond. Macromolecular substrate activation assays demonstrated that G17905 is a noncompetitive, slow-binding inhibitor. Nevertheless, G17905 effectively inhibited thrombus formation in a baboon arterio-venous shunt model, reducing platelet and fibrin deposition by approximately 70% at 0.4 mg/kg + 0.1 mg/kg/min infusion. Therefore, the in vitro potency of G17905, characterized by slow binding kinetics, correlated with efficacious antithrombotic activity in vivo. PMID:15632123

  2. Evidence from molecular dynamics simulations of conformational preorganization in the ribonuclease H active site [v2; ref status: indexed, http://f1000r.es/3pc

    Kate A. Stafford

    2014-06-01

    Full Text Available Ribonuclease H1 (RNase H enzymes are well-conserved endonucleases that are present in all domains of life and are particularly important in the life cycle of retroviruses as domains within reverse transcriptase. Despite extensive study, especially of the E. coli homolog, the interaction of the highly negatively charged active site with catalytically required magnesium ions remains poorly understood. In this work, we describe molecular dynamics simulations of the E. coli homolog in complex with magnesium ions, as well as simulations of other homologs in their apo states. Collectively, these results suggest that the active site is highly rigid in the apo state of all homologs studied and is conformationally preorganized to favor the binding of a magnesium ion. Notably, representatives of bacterial, eukaryotic, and retroviral RNases H all exhibit similar active-site rigidity, suggesting that this dynamic feature is only subtly modulated by amino acid sequence and may primarily be imposed by the distinctive RNase H protein fold.

  3. Crystallographic Evidence of Drastic Conformational Changes in the Active Site of a Flavin-Dependent N-Hydroxylase

    Setser, Jeremy W.; Heemstra, John R.; Walsh, Christopher T.; Drennan, Catherine L.

    2014-01-01

    The soil actinomycete Kutzneria sp. 744 produces a class of highly decorated hexadepsipeptides, which represent a new chemical scaffold that has both antimicrobial and antifungal properties. These natural products, known as kutznerides, are created via nonribosomal peptide synthesis using various derivatized amino acids. The piperazic acid moiety contained in the kutzneride scaffold, which is vital for its antibiotic activity, has been shown to derive from the hydroxylated product of l-ornith...

  4. SLITHER: a web server for generating contiguous conformations of substrate molecules entering into deep active sites of proteins or migrating through channels in membrane transporters.

    Lee, Po-Hsien; Kuo, Kuei-Ling; Chu, Pei-Ying; Liu, Eric M; Lin, Jung-Hsin

    2009-07-01

    Many proteins use a long channel to guide the substrate or ligand molecules into the well-defined active sites for catalytic reactions or for switching molecular states. In addition, substrates of membrane transporters can migrate to another side of cellular compartment by means of certain selective mechanisms. SLITHER (http://bioinfo.mc.ntu.edu.tw/slither/or http://slither.rcas.sinica.edu.tw/) is a web server that can generate contiguous conformations of a molecule along a curved tunnel inside a protein, and the binding free energy profile along the predicted channel pathway. SLITHER adopts an iterative docking scheme, which combines with a puddle-skimming procedure, i.e. repeatedly elevating the potential energies of the identified global minima, thereby determines the contiguous binding modes of substrates inside the protein. In contrast to some programs that are widely used to determine the geometric dimensions in the ion channels, SLITHER can be applied to predict whether a substrate molecule can crawl through an inner channel or a half-channel of proteins across surmountable energy barriers. Besides, SLITHER also provides the list of the pore-facing residues, which can be directly compared with many genetic diseases. Finally, the adjacent binding poses determined by SLITHER can also be used for fragment-based drug design. PMID:19433508

  5. A conformable active matrix LED display

    Tripathi, Ashutosh; Smits, Edsger; van der Steen, Jan-Laurens; Cauwe, Maarten; Verplancke, Rik; Myny, Kris; Maas, Joris; Kusters, Roel; Sabik, Sami; Murata, Mitsuhiro; Tomita, Yoshihiro; Ohmae, Hideki; van den Brand, Jeroen; Gelinck, Gerwin

    2015-01-01

    Conformable and stretchable displays can be integrated on complex surfaces. Such a display can assume the shape of a conformed surface by simultaneous multi-dimensional stretching and bending. Such technology provides new opportunities in the field of display applications, for example wearable displays integrated or embedded in a textile or onto complex surfaces in automotive interiors. In this work we present a conformable active matrix display using LEDs mounted on an amorphous Indium-Galli...

  6. Conformational flexibility related to enzyme activity: evidence for a dynamic active-site gatekeeper function of Tyr(215) in Aerococcus viridans lactate oxidase.

    Stoisser, Thomas; Brunsteiner, Michael; Wilson, David K; Nidetzky, Bernd

    2016-01-01

    L-Lactate oxidase (LOX) belongs to a large family of flavoenzymes that catalyze oxidation of α-hydroxy acids. How in these enzymes the protein structure controls reactivity presents an important but elusive problem. LOX contains a prominent tyrosine in the substrate binding pocket (Tyr(215) in Aerococcus viridans LOX) that is partially responsible for securing a flexible loop which sequesters the active site. To characterize the role of Tyr(215), effects of substitutions of the tyrosine (Y215F, Y215H) were analyzed kinetically, crystallographically and by molecular dynamics simulations. Enzyme variants showed slowed flavin reduction and oxidation by up to 33-fold. Pyruvate release was also decelerated and in Y215F, it was the slowest step overall. A 2.6-Å crystal structure of Y215F in complex with pyruvate shows the hydrogen bond between the phenolic hydroxyl and the keto oxygen in pyruvate is replaced with a potentially stronger hydrophobic interaction between the phenylalanine and the methyl group of pyruvate. Residues 200 through 215 or 216 appear to be disordered in two of the eight monomers in the asymmetric unit suggesting that they function as a lid controlling substrate entry and product exit from the active site. Substitutions of Tyr(215) can thus lead to a kinetic bottleneck in product release. PMID:27302031

  7. Conformational flexibility related to enzyme activity: evidence for a dynamic active-site gatekeeper function of Tyr215 in Aerococcus viridans lactate oxidase

    Stoisser, Thomas; Brunsteiner, Michael; Wilson, David K.; Nidetzky, Bernd

    2016-01-01

    L-Lactate oxidase (LOX) belongs to a large family of flavoenzymes that catalyze oxidation of α-hydroxy acids. How in these enzymes the protein structure controls reactivity presents an important but elusive problem. LOX contains a prominent tyrosine in the substrate binding pocket (Tyr215 in Aerococcus viridans LOX) that is partially responsible for securing a flexible loop which sequesters the active site. To characterize the role of Tyr215, effects of substitutions of the tyrosine (Y215F, Y215H) were analyzed kinetically, crystallographically and by molecular dynamics simulations. Enzyme variants showed slowed flavin reduction and oxidation by up to 33-fold. Pyruvate release was also decelerated and in Y215F, it was the slowest step overall. A 2.6-Å crystal structure of Y215F in complex with pyruvate shows the hydrogen bond between the phenolic hydroxyl and the keto oxygen in pyruvate is replaced with a potentially stronger hydrophobic interaction between the phenylalanine and the methyl group of pyruvate. Residues 200 through 215 or 216 appear to be disordered in two of the eight monomers in the asymmetric unit suggesting that they function as a lid controlling substrate entry and product exit from the active site. Substitutions of Tyr215 can thus lead to a kinetic bottleneck in product release. PMID:27302031

  8. Evidence from molecular dynamics simulations of conformational preorganization in the ribonuclease H active site [v1; ref status: indexed, http://f1000r.es/2z7

    Kate A. Stafford

    2014-03-01

    Full Text Available Ribonuclease H1 (RNase H enzymes are well-conserved endonucleases that are present in all domains of life and are particularly important in the life cycle of retroviruses as domains within reverse transcriptase. Despite extensive study, especially of the E. coli homolog, the interaction of the highly negatively charged active site with catalytically required magnesium ions remains poorly understood. In this work, we describe molecular dynamics simulations of the E. coli homolog in complex with magnesium ions, as well as simulations of other homologs in their apo states. Collectively, these results suggest that the active site is highly rigid in the apo state of all homologs studied and is conformationally preorganized to favor the binding of a magnesium ion. Notably, representatives of bacterial, eukaryotic, and retroviral RNases H all exhibit similar active-site rigidity, suggesting that this dynamic feature is only subtly modulated by amino acid sequence and is primarily imposed by the distinctive RNase H protein fold.

  9. Glu311 and Arg337 Stabilize a Closed Active-site Conformation and Provide a Critical Catalytic Base and Countercation for Green Bioluminescence in Beetle Luciferases.

    Viviani, V R; Simões, A; Bevilaqua, V R; Gabriel, G V M; Arnoldi, F G C; Hirano, T

    2016-08-30

    Beetle luciferases elicit the emission of different bioluminescence colors from green to red. Whereas firefly luciferases emit yellow-green light and are pH-sensitive, undergoing a typical red-shift at acidic pH and higher temperatures and in the presence of divalent heavy metals, click beetle and railroadworm luciferases emit a wider range of colors from green to red but are pH-independent. Despite many decades of study, the structural determinants and mechanisms of bioluminescence colors and pH sensitivity remain enigmatic. Here, through modeling studies, site-directed mutagenesis, and spectral and kinetic studies using recombinant luciferases from the three main families of bioluminescent beetles that emit different colors of light (Macrolampis sp2 firefly, Phrixotrix hirtus railroadworm, and Pyrearinus termitilluminans click beetle), we investigated the role of E311 and R337 in bioluminescence color determination. All mutations of these residues in firefly luciferase produced red mutants, indicating that the preservation of opposite charges and the lengths of the side chains of E311 and R337 are essential for keeping a salt bridge that stabilizes a closed hydrophobic conformation favorable for green light emission. Kinetic studies indicate that residue R337 is important for binding luciferin and creating a positively charged environment around excited oxyluciferin phenolate. In Pyrearinus green-emitting luciferase, the R334A mutation causes a 27 nm red-shift, whereas in Phrixotrix red-emitting luciferase, the L334R mutation causes a blue-shift that is no longer affected by guanidine. These results provide compelling evidence that the presence of arginine at position 334 is essential for blue-shifting the emission spectra of most beetle luciferases. Therefore, residues E311 and R337 play both structural and catalytic roles in bioluminescence color determination, by stabilizing a closed hydrophobic conformation favorable for green light emission, and also

  10. Conformational Sampling and Binding Site Assessment of Suppression of Tumorigenicity 2 Ectodomain.

    Chao-Yie Yang

    Full Text Available Suppression of Tumorigenicity 2 (ST2, a member of the interleukin-1 receptor (IL-1R family, activates type 2 immune responses to pathogens and tissue damage via binding to IL-33. Dysregulated responses contribute to asthma, graft-versus-host and autoinflammatory diseases and disorders. To study ST2 structure for inhibitor development, we performed the principal component (PC analysis on the crystal structures of IL1-1R1, IL1-1R2, ST2 and the refined ST2 ectodomain (ST2ECD models, constructed from previously reported small-angle X-ray scattering data. The analysis facilitates mapping of the ST2ECD conformations to PC subspace for characterizing structural changes. Extensive coverage of ST2ECD conformations was then obtained using the accelerated molecular dynamics simulations started with the IL-33 bound ST2ECD structure as instructed by their projected locations on the PC subspace. Cluster analysis of all conformations further determined representative conformations of ST2ECD ensemble in solution. Alignment of the representative conformations with the ST2/IL-33 structure showed that the D3 domain of ST2ECD (containing D1-D3 domains in most conformations exhibits no clashes with IL-33 in the crystal structure. Our experimental binding data informed that the D1-D2 domain of ST2ECD contributes predominantly to the interaction between ST2ECD and IL-33 underscoring the importance of the D1-D2 domain in binding. Computational binding site assessment revealed one third of the total detected binding sites in the representative conformations may be suitable for binding to potent small molecules. Locations of these sites include the D1-D2 domain ST2ECD and modulation sites conformed to ST2ECD conformations. Our study provides structural models and analyses of ST2ECD that could be useful for inhibitor discovery.

  11. Effect of Urea on Activity and Conformation of a Glycoprotein

    WEI Xiang; WANG Xiaoyun; ZHOU Bo; ZHOU Haimeng

    2006-01-01

    The changes of the activity and conformation of Aspergillus niger phytase in urea were detected by farultraviolet circular dichroism (CD) spectra, fluorescence spectra, and enzyme activity assays. The results show that no enzyme activity can be detected after phytase is incubated for 10 h in 3.0 mol/L urea, even though at this urea concentration, less than 20% of the tertiary and secondary structures in the native enzyme changed. The inactivation reaction kinetics is found to be a monophasic first-order reaction, but the unfolding is a biphasic process consisting of two first-order reactions. The inactivation rates of the free enzyme and the substrate-enzyme complex are much faster than the conformational changes during urea denaturation. All of the results indicate that, as a glycoprotein, phytase's activity is strongly dependent on its conformational integrity. The phytase active sites seem to be located in a limited region in the molecule and display more conformational fragility and flexibility to denaturants than enzyme molecular structure as a whole.

  12. Combined Use of Residual Dipolar Couplings and Solution X-ray Scattering To Rapidly Probe Rigid-Body Conformational Transitions in a Non-phosphorylatable Active-Site Mutant of the 128 kDa Enzyme I Dimer

    Takayama, Yuki; Schwieters, Charles D.; Grishaev, Alexander; Ghirlando, Rodolfo; Clore, G. Marius (NIH)

    2012-10-23

    The first component of the bacterial phosphotransferase system, enzyme I (EI), is a multidomain 128 kDa dimer that undergoes large rigid-body conformational transitions during the course of its catalytic cycle. Here we investigate the solution structure of a non-phosphorylatable active-site mutant in which the active-site histidine is substituted by glutamine. We show that perturbations in the relative orientations and positions of the domains and subdomains can be rapidly and reliably determined by conjoined rigid-body/torsion angle/Cartesian simulated annealing calculations driven by orientational restraints from residual dipolar couplings and shape and translation information afforded by small- and wide-angle X-ray scattering. Although histidine and glutamine are isosteric, the conformational space available to a Gln side chain is larger than that for the imidazole ring of His. An additional hydrogen bond between the side chain of Gln189 located on the EIN{sup {alpha}/{beta}} subdomain and an aspartate (Asp129) on the EIN{sup {alpha}} subdomain results in a small ({approx}9{sup o}) reorientation of the EIN{sup {alpha}} and EIN{sup {alpha}/{beta}} subdomains that is in turn propagated to a larger reorientation ({approx}26{sup o}) of the EIN domain relative to the EIC dimerization domain, illustrating the positional sensitivity of the EIN domain and its constituent subdomains to small structural perturbations.

  13. Transglutaminase 2 undergoes a large conformational change upon activation.

    Daniel M Pinkas

    2007-12-01

    Full Text Available Human transglutaminase 2 (TG2, a member of a large family of enzymes that catalyze protein crosslinking, plays an important role in the extracellular matrix biology of many tissues and is implicated in the gluten-induced pathogenesis of celiac sprue. Although vertebrate transglutaminases have been studied extensively, thus far all structurally characterized members of this family have been crystallized in conformations with inaccessible active sites. We have trapped human TG2 in complex with an inhibitor that mimics inflammatory gluten peptide substrates and have solved, at 2-A resolution, its x-ray crystal structure. The inhibitor stabilizes TG2 in an extended conformation that is dramatically different from earlier transglutaminase structures. The active site is exposed, revealing that catalysis takes place in a tunnel, bridged by two tryptophan residues that separate acyl-donor from acyl-acceptor and stabilize the tetrahedral reaction intermediates. Site-directed mutagenesis was used to investigate the acyl-acceptor side of the tunnel, yielding mutants with a marked increase in preference for hydrolysis over transamidation. By providing the ability to visualize this activated conformer, our results create a foundation for understanding the catalytic as well as the non-catalytic roles of TG2 in biology, and for dissecting the process by which the autoantibody response to TG2 is induced in celiac sprue patients.

  14. The insulin receptor activation process involves localized conformational changes.

    Baron, V; Kaliman, P; Gautier, N; Van Obberghen, E

    1992-11-15

    The molecular process by which insulin binding to the receptor alpha-subunit induces activation of the receptor beta-subunit with ensuing substrate phosphorylation remains unclear. In this study, we aimed at approaching this molecular mechanism of signal transduction and at delineating the cytoplasmic domains implied in this process. To do this, we used antipeptide antibodies to the following sequences of the receptor beta-subunit: (i) positions 962-972 in the juxtamembrane domain, (ii) positions 1247-1261 at the end of the kinase domain, and (iii) positions 1294-1317 and (iv) positions 1309-1326, both in the receptor C terminus. We have previously shown that insulin binding to its receptor induces a conformational change in the beta-subunit C terminus. Here, we demonstrate that receptor autophosphorylation induces an additional conformational change. This process appears to be distinct from the one produced by ligand binding and can be detected in at least three different beta-subunit regions: the juxtamembrane domain, the kinase domain, and the C terminus. Hence, the cytoplasmic part of the receptor beta-subunit appears to undergo an extended conformational change upon autophosphorylation. By contrast, the insulin-induced change does not affect the juxtamembrane domain 962-972 nor the kinase domain 1247-1261 and may be limited to the receptor C terminus. Further, we show that the hormone-dependent conformational change is maintained in a kinase-deficient receptor due to a mutation at lysine 1018. Therefore, during receptor activation, the ligand-induced change could precede ATP binding and receptor autophosphorylation. We propose that insulin binding leads to a transient receptor form that may allow ATP binding and, subsequently, autophosphorylation. The second conformational change could unmask substrate-binding sites and stabilize the receptor in an active conformation. PMID:1331080

  15. Ligand Binding Reduces Conformational Flexibility in the Active Site of Tyrosine Phosphatase Related to Biofilm Formation A (TpbA) from Pseudomonas aeruginosa

    Koveal, Dorothy; Clarkson, Michael W.; Wood, Thomas K.; Page, Rebecca; Peti, Wolfgang

    2013-01-01

    TpbA is a periplasmic dual specificity phosphatase (DUSP) that controls biofilm formation in the pathogenic bacterium, Pseudomonas aeruginosa. While DUSPs are known to regulate important cellular functions in both prokaryotes and eukaryotes, very few structures of bacterial DUSPs are available. Here, we present the solution structure of TpbA in the ligand-free open conformation, along with an analysis of the structural and dynamic changes that accompany ligand/phosphate binding. While TpbA ad...

  16. Conformational toggling controls target site choice for the heteromeric transposase element Tn7.

    Shi, Qiaojuan; Straus, Marco R; Caron, Jeremy J; Wang, Huasheng; Chung, Yu Seon; Guarné, Alba; Peters, Joseph E

    2015-12-15

    The bacterial transposon Tn7 facilitates horizontal transfer by directing transposition into actively replicating DNA with the element-encoded protein TnsE. Structural analysis of the C-terminal domain of wild-type TnsE identified a novel protein fold including a central V-shaped loop that toggles between two distinct conformations. The structure of a robust TnsE gain-of-activity variant has this loop locked in a single conformation, suggesting that conformational flexibility regulates TnsE activity. Structure-based analysis of a series of TnsE mutants relates transposition activity to DNA binding stability. Wild-type TnsE appears to naturally form an unstable complex with a target DNA, whereas mutant combinations required for large changes in transposition frequency and targeting stabilized this interaction. Collectively, our work unveils a unique structural proofreading mechanism where toggling between two conformations regulates target commitment by limiting the stability of target DNA engagement until an appropriate insertion site is identified. PMID:26384427

  17. Engineering a hyper-catalytic enzyme by photo-activated conformation modulation

    Agarwal, Pratul K [ORNL

    2012-01-01

    Enzyme engineering for improved catalysis has wide implications. We describe a novel chemical modification of Candida antarctica lipase B that allows modulation of the enzyme conformation to promote catalysis. Computational modeling was used to identify dynamical enzyme regions that impact the catalytic mechanism. Surface loop regions located distal to active site but showing dynamical coupling to the reaction were connected by a chemical bridge between Lys136 and Pro192, containing a derivative of azobenzene. The conformational modulation of the enzyme was achieved using two sources of light that alternated the azobenzene moiety in cis and trans conformations. Computational model predicted that mechanical energy from the conformational fluctuations facilitate the reaction in the active-site. The results were consistent with predictions as the activity of the engineered enzyme was found to be enhanced with photoactivation. Preliminary estimations indicate that the engineered enzyme achieved 8-52 fold better catalytic activity than the unmodulated enzyme.

  18. Photocatalytically Active Oligomeric Graphitic Carbon Nitride: Conformational Flexibility, Electronic Levels, Carrier Localization

    Blum, Volker; Lau, Vincent; Botari, Tiago; Huhn, William; Lotsch, Bettina V.

    2015-03-01

    Polymers consisting of bridged heptazine units (often called ``graphitic carbon nitride'' or ``g-C3N4'') show considerable promise as photocatalysts for solar hydrogen evolution. Recent experimental evidence suggests that oligomeric rather than fully polymerized ``g-C3N4'' exhibits increased intrinsic photocatalytic activity. Using density-functional theory (DFT; van der Waals corrected PBE functional for conformers, hybrid DFT and GW for electronic levels), we show that considerable conformational flexibility exists for the heptazine trimers and tetramers. Analysis of HOMO and LUMO locations as well as trends in photocatalytic activity among heptazine oligomers and polymers reveals the NH2 groups of the oligomers as potential charge-transfer sites. We show that conformational variations of the oligomers can lead to significant, electrostatically motivated carrier localization effects. We suggest that NH2 side groups and the intrinsic conformational variations of the oligomeric species lead to the observed enhanced catalytic activity.

  19. New binding site conformations of the dengue virus NS3 protease accessed by molecular dynamics simulation.

    Hugo de Almeida

    Full Text Available Dengue fever is caused by four distinct serotypes of the dengue virus (DENV1-4, and is estimated to affect over 500 million people every year. Presently, there are no vaccines or antiviral treatments for this disease. Among the possible targets to fight dengue fever is the viral NS3 protease (NS3PRO, which is in part responsible for viral processing and replication. It is now widely recognized that virtual screening campaigns should consider the flexibility of target protein by using multiple active conformational states. The flexibility of the DENV NS3PRO could explain the relatively low success of previous virtual screening studies. In this first work, we explore the DENV NS3PRO conformational states obtained from molecular dynamics (MD simulations to take into account protease flexibility during the virtual screening/docking process. To do so, we built a full NS3PRO model by multiple template homology modeling. The model comprised the NS2B cofactor (essential to the NS3PRO activation, a glycine flexible link and the proteolytic domain. MD simulations had the purpose to sample, as closely as possible, the ligand binding site conformational landscape prior to inhibitor binding. The obtained conformational MD sample was clustered into four families that, together with principal component analysis of the trajectory, demonstrated protein flexibility. These results allowed the description of multiple binding modes for the Bz-Nle-Lys-Arg-Arg-H inhibitor, as verified by binding plots and pair interaction analysis. This study allowed us to tackle protein flexibility in our virtual screening campaign against the dengue virus NS3 protease.

  20. Crystallographic B factor of critical residues at enzyme active site

    张海龙; 宋时英; 林政炯

    1999-01-01

    Thirty-seven sets of crystallographic enzyme data were selected from Protein Data Bank (PDB, 1995). The average temperature factors (B) of the critical residues at the active site and the whole molecule of those enzymes were calculated respectively. The statistical results showed that the critical residues at the active site of most of the enzymes had lower B factors than did the whole molecules, indicating that in the crystalline state the critical residues at the active site of the natural enzymes possess more stable conformation than do the whole molecules. The flexibility of the active site during the unfolding by denaturing was also discussed.

  1. DOE site performance assessment activities

    Information on performance assessment capabilities and activities was collected from eight DOE sites. All eight sites either currently dispose of low-level radioactive waste (LLW) or plan to dispose of LLW in the near future. A survey questionnaire was developed and sent to key individuals involved in DOE Order 5820.2A performance assessment activities at each site. The sites surveyed included: Hanford Site (Hanford), Idaho National Engineering Laboratory (INEL), Los Alamos National Laboratory (LANL), Nevada Test Site (NTS), Oak Ridge National Laboratory (ORNL), Paducah Gaseous Diffusion Plant (Paducah), Portsmouth Gaseous Diffusion Plant (Portsmouth), and Savannah River Site (SRS). The questionnaire addressed all aspects of the performance assessment process; from waste source term to dose conversion factors. This report presents the information developed from the site questionnaire and provides a comparison of site-specific performance assessment approaches, data needs, and ongoing and planned activities. All sites are engaged in completing the radioactive waste disposal facility performance assessment required by DOE Order 5820.2A. Each site has achieved various degrees of progress and have identified a set of critical needs. Within several areas, however, the sites identified common needs and questions

  2. Human insulin analogues modified at the B26 site reveal a hormone conformation that is undetected in the receptor complex

    Žáková, Lenka; Kletvíková, Emília; Lepšík, Martin; Collinsová, Michaela [Academy of Sciences of the Czech Republic, v.v.i., Flemingovo nám. 2, 166 10 Prague 6 (Czech Republic); Watson, Christopher J.; Turkenburg, Johan P. [The University of York, Heslington, York YO10 5DD (United Kingdom); Jiráček, Jiří [Academy of Sciences of the Czech Republic, v.v.i., Flemingovo nám. 2, 166 10 Prague 6 (Czech Republic); Brzozowski, Andrzej M., E-mail: marek.brzozowski@york.ac.uk [The University of York, Heslington, York YO10 5DD (United Kingdom); Academy of Sciences of the Czech Republic, v.v.i., Flemingovo nám. 2, 166 10 Prague 6 (Czech Republic)

    2014-10-01

    [AsnB26]- and [GlyB26]-insulin mutants attain a B26-turn like fold without assistance of chemical modifications. Their structures match the insulin receptor interface and expand the spectrum of insulin conformations. The structural characterization of the insulin–insulin receptor (IR) interaction still lacks the conformation of the crucial B21–B30 insulin region, which must be different from that in its storage forms to ensure effective receptor binding. Here, it is shown that insulin analogues modified by natural amino acids at the TyrB26 site can represent an active form of this hormone. In particular, [AsnB26]-insulin and [GlyB26]-insulin attain a B26-turn-like conformation that differs from that in all known structures of the native hormone. It also matches the receptor interface, avoiding substantial steric clashes. This indicates that insulin may attain a B26-turn-like conformation upon IR binding. Moreover, there is an unexpected, but significant, binding specificity of the AsnB26 mutant for predominantly the metabolic B isoform of the receptor. As it is correlated with the B26 bend of the B-chain of the hormone, the structures of AsnB26 analogues may provide the first structural insight into the structural origins of differential insulin signalling through insulin receptor A and B isoforms.

  3. Human insulin analogues modified at the B26 site reveal a hormone conformation that is undetected in the receptor complex

    [AsnB26]- and [GlyB26]-insulin mutants attain a B26-turn like fold without assistance of chemical modifications. Their structures match the insulin receptor interface and expand the spectrum of insulin conformations. The structural characterization of the insulin–insulin receptor (IR) interaction still lacks the conformation of the crucial B21–B30 insulin region, which must be different from that in its storage forms to ensure effective receptor binding. Here, it is shown that insulin analogues modified by natural amino acids at the TyrB26 site can represent an active form of this hormone. In particular, [AsnB26]-insulin and [GlyB26]-insulin attain a B26-turn-like conformation that differs from that in all known structures of the native hormone. It also matches the receptor interface, avoiding substantial steric clashes. This indicates that insulin may attain a B26-turn-like conformation upon IR binding. Moreover, there is an unexpected, but significant, binding specificity of the AsnB26 mutant for predominantly the metabolic B isoform of the receptor. As it is correlated with the B26 bend of the B-chain of the hormone, the structures of AsnB26 analogues may provide the first structural insight into the structural origins of differential insulin signalling through insulin receptor A and B isoforms

  4. Human insulin analogues modified at the B26 site reveal a hormone conformation that is undetected in the receptor complex.

    Záková, Lenka; Kletvíková, Emília; Lepšík, Martin; Collinsová, Michaela; Watson, Christopher J; Turkenburg, Johan P; Jiráček, Jiří; Brzozowski, Andrzej M

    2014-10-01

    The structural characterization of the insulin-insulin receptor (IR) interaction still lacks the conformation of the crucial B21-B30 insulin region, which must be different from that in its storage forms to ensure effective receptor binding. Here, it is shown that insulin analogues modified by natural amino acids at the TyrB26 site can represent an active form of this hormone. In particular, [AsnB26]-insulin and [GlyB26]-insulin attain a B26-turn-like conformation that differs from that in all known structures of the native hormone. It also matches the receptor interface, avoiding substantial steric clashes. This indicates that insulin may attain a B26-turn-like conformation upon IR binding. Moreover, there is an unexpected, but significant, binding specificity of the AsnB26 mutant for predominantly the metabolic B isoform of the receptor. As it is correlated with the B26 bend of the B-chain of the hormone, the structures of AsnB26 analogues may provide the first structural insight into the structural origins of differential insulin signalling through insulin receptor A and B isoforms. PMID:25286859

  5. A coevolutionary residue network at the site of a functionally important conformational change in a phosphohexomutase enzyme family.

    Yingying Lee

    Full Text Available Coevolution analyses identify residues that co-vary with each other during evolution, revealing sequence relationships unobservable from traditional multiple sequence alignments. Here we describe a coevolutionary analysis of phosphomannomutase/phosphoglucomutase (PMM/PGM, a widespread and diverse enzyme family involved in carbohydrate biosynthesis. Mutual information and graph theory were utilized to identify a network of highly connected residues with high significance. An examination of the most tightly connected regions of the coevolutionary network reveals that most of the involved residues are localized near an interdomain interface of this enzyme, known to be the site of a functionally important conformational change. The roles of four interface residues found in this network were examined via site-directed mutagenesis and kinetic characterization. For three of these residues, mutation to alanine reduces enzyme specificity to ~10% or less of wild-type, while the other has ~45% activity of wild-type enzyme. An additional mutant of an interface residue that is not densely connected in the coevolutionary network was also characterized, and shows no change in activity relative to wild-type enzyme. The results of these studies are interpreted in the context of structural and functional data on PMM/PGM. Together, they demonstrate that a network of coevolving residues links the highly conserved active site with the interdomain conformational change necessary for the multi-step catalytic reaction. This work adds to our understanding of the functional roles of coevolving residue networks, and has implications for the definition of catalytically important residues.

  6. FXR agonist activity of conformationally constrained analogs of GW 4064

    Akwabi-Ameyaw, Adwoa; Bass, Jonathan Y.; Caldwell, Richard D.; Caravella, Justin A.; Chen, Lihong; Creech, Katrina L.; Deaton, David N.; Madauss, Kevin P.; Marr, Harry B.; McFadyen, Robert B.; Miller, Aaron B.; Navas, III, Frank; Parks, Derek J.; Spearing, Paul K.; Todd, Dan; Williams, Shawn P.; Wisely, G. Bruce; (GSKNC)

    2010-09-27

    Two series of conformationally constrained analogs of the FXR agonist GW 4064 1 were prepared. Replacement of the metabolically labile stilbene with either benzothiophene or naphthalene rings led to the identification of potent full agonists 2a and 2g.

  7. The influence of conformational fluctuations on enzymatic activity: modelling the functional motion of {beta}-secretase

    Neri, M [SISSA and INFM, Via Beirut 2-4, I-34014 Trieste (Italy); Cascella, M [SISSA and INFM, Via Beirut 2-4, I-34014 Trieste (Italy); EPFL, Lausanne (Switzerland); Micheletti, C [SISSA and INFM, Via Beirut 2-4, I-34014 Trieste (Italy)

    2005-05-11

    Considerable insight into the functional activity of proteins and enzymes can be obtained by studying the low energy conformational distortions that the biopolymer can sustain. We carry out the characterization of these large scale structural changes for a protein of considerable pharmaceutical interest, the human {beta}-secretase. Starting from the crystallographic structure of the protein, we use the recently introduced {beta}-Gaussian model to identify, with negligible computational expenditure, the most significant distortions occurring in thermal equilibrium and the associated timescales. The application of this strategy helps us to gain considerable insight into the putative functional movements and, furthermore, allows us to identify a handful of key regions in the protein which have an important mechanical influence on the enzymatic activity despite being spatially distant from the active site. The results obtained within the Gaussian model are validated through an extensive comparison against an all-atom molecular dynamics simulation.

  8. The influence of conformational fluctuations on enzymatic activity: modelling the functional motion of β-secretase

    Considerable insight into the functional activity of proteins and enzymes can be obtained by studying the low energy conformational distortions that the biopolymer can sustain. We carry out the characterization of these large scale structural changes for a protein of considerable pharmaceutical interest, the human β-secretase. Starting from the crystallographic structure of the protein, we use the recently introduced β-Gaussian model to identify, with negligible computational expenditure, the most significant distortions occurring in thermal equilibrium and the associated timescales. The application of this strategy helps us to gain considerable insight into the putative functional movements and, furthermore, allows us to identify a handful of key regions in the protein which have an important mechanical influence on the enzymatic activity despite being spatially distant from the active site. The results obtained within the Gaussian model are validated through an extensive comparison against an all-atom molecular dynamics simulation

  9. Search for DNA conformational features for functional sites. Investigation of the TATA box

    Ponomarenko, M.P.; Ponomarenko, J.V.; Kel, A.E.; Kolchanov, N.A. [Institute of Cytology and Genetics, Novosibirsk (Russian Federation)

    1996-12-31

    A method for searching for DNA conformational features significant for functional sites is developed. The method uses helical angles averaged for known X-ray structures. Nucleotide sequences are assigned mean angles in a given region. Choice of the significant angles is based on their capabilities to discriminate functional sites from random sequences. The yeast, invertebrate, and vertebrate TATA boxes are analyzed using this method. Regions neighboring the TATA boxes are found to have smaller helical twist and roll angles. The results agree with the experimental data on Dickerson-Drew dodecamers. There is a significant decrease in the length of a small roll angle region with increasing complexity of taxon organization. 28 refs., 3 figs., 3 tabs.

  10. Stabilizing a flexible interdomain hinge region harboring the SMB binding site drives uPAR into its closed conformation.

    Zhao, Baoyu; Gandhi, Sonu; Yuan, Cai; Luo, Zhipu; Li, Rui; Gårdsvoll, Henrik; de Lorenzi, Valentina; Sidenius, Nicolai; Huang, Mingdong; Ploug, Michael

    2015-03-27

    The urokinase-type plasminogen activator receptor (uPAR) is a multidomain glycolipid-anchored membrane protein, which facilitates extracellular matrix remodeling by focalizing plasminogen activation to cell surfaces via its high-affinity interaction with uPA. The modular assembly of its three LU (Ly6/uPAR-like) domains is inherently flexible and binding of uPA drives uPAR into its closed conformation, which presents the higher-affinity state for vitronectin thus providing an allosteric regulatory mechanism. Using a new class of epitope-mapped anti-uPAR monoclonal antibodies (mAbs), we now demonstrate that the reciprocal stabilization is indeed also possible. By surface plasmon resonance studies, we show that these mAbs and vitronectin have overlapping binding sites on uPAR and that they share Arg91 as hotspot residue in their binding interfaces. The crystal structure solved for one of these uPAR·mAb complexes at 3.0Å clearly shows that this mAb preselects the closed uPAR conformation with an empty but correctly assembled large hydrophobic binding cavity for uPA. Accordingly, these mAbs inhibit the uPAR-dependent lamellipodia formation and migration on vitronectin-coated matrices irrespective of the conformational status of uPAR and its occupancy with uPA. This is the first study to the best of our knowledge, showing that the dynamic assembly of the three LU domains in uPARwt can be driven toward the closed form by an external ligand, which is not engaging the hydrophobic uPA binding cavity. As this binding interface is also exploited by the somatomedin B domain of vitronectin, therefore, this relationship should be taken into consideration when exploring uPAR-dependent cell adhesion and migration in vitronectin-rich environments. PMID:25659907

  11. Does ecosystem sensitivity to precipitation at the site-level conform to regional-scale predictions?.

    Wilcox, Kevin R; Blair, John M; Smith, Melinda D; Knapp, Alan K

    2016-03-01

    Central to understanding global C cycle dynamics is the functional relationship between precipitation and net primary production (NPP). At large spatial (regional) scales, the responsiveness of aboveground NPP (ANPP) to interannual variation in annual precipitation (AP; ANPPsens) is inversely related to site-level ANPP, coinciding with turnover of plant communities along precipitation gradients. Within ecosystems experiencing chronic alterations in water availability, plant community change will also occur with unknown consequences for ANPPsens. To examine the role plant community shifts may play in determining alterations in site-level ANPPPsens, we experimentally increased precipitation by approximately 35% for two decades in a native Central U.S. grassland. Consistent with regional models, ANPPsens decreased initially as water availability and ANPP increased. However, ANPPsens shifted back to ambient levels when mesic species increased in abundance in the plant community. Similarly, in grassland sites with distinct mesic and xeric plant communities and corresponding 50% differences in ANPP, ANPPsens did not differ over almost three decades. We conclude that responses in ANPPsens to chronic alterations in water availability within an ecosystem may not conform to regional AP-ANPP patterns, despite expected changes in ANPP and plant communities. The result is unanticipated functional resistance to climate change at the site scale. PMID:27197383

  12. Active Site Engineering in Electrocatalysis

    Verdaguer Casadevall, Arnau; Stephens, Ifan; Chorkendorff, Ib

    The overall goal of this thesis has been to design better catalysts for electrochemical reactions through a fundamental understanding of the materials at atomic scale. This has been achieved by combining electrochemical measurements with a variety of characterization techniques, often in ultra high...... under reaction conditions, which is ultimately controlled by the crystal structure of the underlying alloy.• Oxygen reduction to hydrogen peroxide has been investigated on single site catalysts, mainly alloys of noble metals with Hg. This resulted in a very special structure with isolated atoms of Pt or......, inexistent in other forms of Cu. The presence of strong CO binding sites correlates well with electrochemical activity, which paves the way for the rational development of even better electrocatalysts....

  13. Conformational activation of visual rhodopsin in native disc membranes

    Malmerberg, E.; Bovee-Geurts, P.H.M.; Katona, G.; Deupi, X.; Arnlund, D.; Wickstrand, C.; Johansson, L.C.; Westenhoff, S.; Nazarenko, E.; GF, X.S.; Menzel, A.; Grip, W.J. de; Neutze, R.

    2015-01-01

    Rhodopsin is the G protein-coupled receptor (GPCR) that serves as a dim-light receptor for vision in vertebrates. We probed light-induced conformational changes in rhodopsin in its native membrane environment at room temperature using time-resolved wide-angle x-ray scattering. We observed a rapid co

  14. Coenzyme A Binding to the Aminoglycoside Acetyltransferase (3)-IIIb Increases Conformational Sampling of Antibiotic Binding Site

    Hu, Xiaohu [ORNL; Norris, Adrianne [University of Tennessee, Knoxville (UTK); Baudry, Jerome Y [ORNL; Serpersu, Engin H [University of Tennessee, Knoxville (UTK)

    2011-01-01

    NMR spectroscopy experiments and molecular dynamics simulations were performed to describe the dynamic properties of the aminoglycoside acetyltransferase (3)-IIIb (AAC) in its apo and coenzyme A (CoASH) bound forms. The {sup 15}N-{sup 1}H HSQC spectra indicate a partial structural change and coupling of the CoASH binding site with another region in the protein upon the CoASH titration into the apo enzyme. Molecular dynamics simulations indicate a significant structural and dynamic variation of the long loop in the antibiotic binding domain in the form of a relatively slow (250 ns), concerted opening motion in the CoASH enzyme complex and that binding of the CoASH increases the structural flexibility of the loop, leading to an interchange between several similar equally populated conformations.

  15. Activation of pheromone-sensitive neurons is mediated by conformational activation of pheromone-binding protein.

    Laughlin, John D; Ha, Tal Soo; Jones, David N M; Smith, Dean P

    2008-06-27

    Detection of volatile odorants by olfactory neurons is thought to result from direct activation of seven-transmembrane odorant receptors by odor molecules. Here, we show that detection of the Drosophila pheromone, 11-cis vaccenyl acetate (cVA), is instead mediated by pheromone-induced conformational shifts in the extracellular pheromone-binding protein, LUSH. We show that LUSH undergoes a pheromone-specific conformational change that triggers the firing of pheromone-sensitive neurons. Amino acid substitutions in LUSH that are predicted to reduce or enhance the conformational shift alter sensitivity to cVA as predicted in vivo. One substitution, LUSH(D118A), produces a dominant-active LUSH protein that stimulates T1 neurons through the neuronal receptor components Or67d and SNMP in the complete absence of pheromone. Structural analysis of LUSH(D118A) reveals that it closely resembles cVA-bound LUSH. Therefore, the pheromone-binding protein is an inactive, extracellular ligand converted by pheromone molecules into an activator of pheromone-sensitive neurons and reveals a distinct paradigm for detection of odorants. PMID:18585358

  16. Effects of perfluorooctane sulfonate on the conformation and activity of bovine serum albumin.

    Wang, Yanqing; Zhang, Hongmei; Kang, Yijun; Cao, Jian

    2016-06-01

    Perfluorooctane sulfonate (PFOS) is among the most prominent contaminates in human serum and has been reported to possess potential toxicity to the human body. In this study, the effects of PFOS on the conformation and activity of bovine serum albumin (BSA) were investigated in vitro. The results indicated that the binding interaction of PFOS with BSA destroyed the tertiary and secondary structures of protein with the loss of α-helix structure and the increasing of hydrophobic microenvironment of the Trp or Tyr residues. During the thermal denaturation protein, PFOS increases the protein stability of BSA. The proportion of α-helix decreased on increasing the PFOS concentration and the microenvironment of the Trp or Tyr residues becomes more hydrophobic. The results from molecular modeling indicated that BSA had not only one possible binding site to bind with PFOS by the polar interaction, hydrogen bonds and hydrophobic forces. In addition, the BSA relative activities were decreased with the increase of PFOS concentration. Such loss of BSA activity in the presence of PFOS indicated that one of the binding sites in BSA is located in subdomain IIIA, which is in good agreement with the fluorescence spectroscopic experiments and molecular modeling results. This study offers a comprehensive picture of the interactions of PFOS with serum albumin and provides insights into the toxicological effect of perfluoroalkylated substances. PMID:27031195

  17. Cofactor bypass variants reveal a conformational control mechanism governing cell wall polymerase activity.

    Markovski, Monica; Bohrhunter, Jessica L; Lupoli, Tania J; Uehara, Tsuyoshi; Walker, Suzanne; Kahne, Daniel E; Bernhardt, Thomas G

    2016-04-26

    To fortify their cytoplasmic membrane and protect it from osmotic rupture, most bacteria surround themselves with a peptidoglycan (PG) exoskeleton synthesized by the penicillin-binding proteins (PBPs). As their name implies, these proteins are the targets of penicillin and related antibiotics. We and others have shown that the PG synthases PBP1b and PBP1a of Escherichia coli require the outer membrane lipoproteins LpoA and LpoB, respectively, for their in vivo function. Although it has been demonstrated that LpoB activates the PG polymerization activity of PBP1b in vitro, the mechanism of activation and its physiological relevance have remained unclear. We therefore selected for variants of PBP1b (PBP1b*) that bypass the LpoB requirement for in vivo function, reasoning that they would shed light on LpoB function and its activation mechanism. Several of these PBP1b variants were isolated and displayed elevated polymerization activity in vitro, indicating that the activation of glycan polymer growth is indeed one of the relevant functions of LpoB in vivo. Moreover, the location of amino acid substitutions causing the bypass phenotype on the PBP1b structure support a model in which polymerization activation proceeds via the induction of a conformational change in PBP1b initiated by LpoB binding to its UB2H domain, followed by its transmission to the glycosyl transferase active site. Finally, phenotypic analysis of strains carrying a PBP1b* variant revealed that the PBP1b-LpoB complex is most likely not providing an important physical link between the inner and outer membranes at the division site, as has been previously proposed. PMID:27071112

  18. Nicotinamide Cofactors Suppress Active-Site Labeling of Aldehyde Dehydrogenases.

    Stiti, Naim; Chandrasekar, Balakumaran; Strubl, Laura; Mohammed, Shabaz; Bartels, Dorothea; van der Hoorn, Renier A L

    2016-06-17

    Active site labeling by (re)activity-based probes is a powerful chemical proteomic tool to globally map active sites in native proteomes without using substrates. Active site labeling is usually taken as a readout for the active state of the enzyme because labeling reflects the availability and reactivity of active sites, which are hallmarks for enzyme activities. Here, we show that this relationship holds tightly, but we also reveal an important exception to this rule. Labeling of Arabidopsis ALDH3H1 with a chloroacetamide probe occurs at the catalytic Cys, and labeling is suppressed upon nitrosylation and oxidation, and upon treatment with other Cys modifiers. These experiments display a consistent and strong correlation between active site labeling and enzymatic activity. Surprisingly, however, labeling is suppressed by the cofactor NAD(+), and this property is shared with other members of the ALDH superfamily and also detected for unrelated GAPDH enzymes with an unrelated hydantoin-based probe in crude extracts of plant cell cultures. Suppression requires cofactor binding to its binding pocket. Labeling is also suppressed by ALDH modulators that bind at the substrate entrance tunnel, confirming that labeling occurs through the substrate-binding cavity. Our data indicate that cofactor binding adjusts the catalytic Cys into a conformation that reduces the reactivity toward chloroacetamide probes. PMID:26990764

  19. The effect of protonation site and conformation on surface-induced dissociation in a small, lysine containing peptide

    Shaikh, Kulsum; Blackwood, Jacob; Barnes, George L.

    2015-09-01

    Simulations of surface induced dissociation (SID) of protonated peptides have provided significant insight into the energy transfer and mechanism of SID; however, they have been limited to glycine and alanine containing peptides. The chemical simplicity of these systems forces N-terminus protonation. Here we present results from simulations involving a lysine containing peptide that allowed for multiple protonation sites and conformations. We found that when the excess proton is located on the basic lysine side chain, fragmentation dynamics are typically slower and occur through a 'charge-remote' pathway. Additionally, conformation alone has a significant effect on the observed proton transfer pathways.

  20. Activation of the A2A adenosine G-protein-coupled receptor by conformational selection.

    Ye, Libin; Van Eps, Ned; Zimmer, Marco; Ernst, Oliver P; Prosser, R Scott

    2016-05-12

    Conformational selection and induced fit are two prevailing mechanisms to explain the molecular basis for ligand-based activation of receptors. G-protein-coupled receptors are the largest class of cell surface receptors and are important drug targets. A molecular understanding of their activation mechanism is critical for drug discovery and design. However, direct evidence that addresses how agonist binding leads to the formation of an active receptor state is scarce. Here we use (19)F nuclear magnetic resonance to quantify the conformational landscape occupied by the adenosine A2A receptor (A2AR), a prototypical class A G-protein-coupled receptor. We find an ensemble of four states in equilibrium: (1) two inactive states in millisecond exchange, consistent with a formed (state S1) and a broken (state S2) salt bridge (known as 'ionic lock') between transmembrane helices 3 and 6; and (2) two active states, S3 and S3', as identified by binding of a G-protein-derived peptide. In contrast to a recent study of the β2-adrenergic receptor, the present approach allowed identification of a second active state for A2AR. Addition of inverse agonist (ZM241385) increases the population of the inactive states, while full agonists (UK432097 or NECA) stabilize the active state, S3', in a manner consistent with conformational selection. In contrast, partial agonist (LUF5834) and an allosteric modulator (HMA) exclusively increase the population of the S3 state. Thus, partial agonism is achieved here by conformational selection of a distinct active state which we predict will have compromised coupling to the G protein. Direct observation of the conformational equilibria of ligand-dependent G-protein-coupled receptor and deduction of the underlying mechanisms of receptor activation will have wide-reaching implications for our understanding of the function of G-protein-coupled receptor in health and disease. PMID:27144352

  1. Puckering Energetics and Optical Activities of [7]Circulene Conformers.

    Hatanaka, Masashi

    2016-02-25

    The structural preference of [7]circulene is analyzed by taking into account vibronic interactions. DFT calculations reveal that pseudo-Jahn-Teller effects cause the D7h-symmetry structure to relax to C2- and Cs-symmetry structures, which are both ca. 9 kcal/mol lower in energy than the D7h structure. In energy terms, the C2-symmetry structure is 0.05 kcal/mol lower than that of the Cs-symmetry. The active vibrations are attributed to low-frequency puckering modes that are coupled with π-σ excitation states. The optical activities of the C2-symmetry structure were simulated by configuration interaction calculations, and the simulated CD/ORD spectra were reasonable and consistent with the experimental data. The optical rotatory strengths obeyed the helix rule; that is, the left-handed helix shows negative Cotton effects through the antisymmetric excited states. The calculated spectra will serve as a foundation for further investigation of optical activities of negatively curved structures. PMID:26829071

  2. Conformational change in full-length mouse prion: A site-directed spin-labeling study

    The structure of the mouse prion (moPrP) was studied using site-directed spin-labeling electron spin resonance (SDSL-ESR). Since a previous NMR study by Hornemanna et al., [Hornemanna, Korthb, Oeschb, Rieka, Widera, Wuethricha, Glockshubera, Recombinant full-length murine prion protein, mPrP (23-231): purification and spectroscopic characterization, FEBS Lett. 413 (1997) 277-281] has indicated that N96, D143, and T189 in moPrP are localized in a Cu2+ binding region, Helix1 and Helix2, respectively, three recombinant moPrP mutations (N96C, D143C, and T189C) were expressed in an Escherichia coli system, and then refolded by dialysis under low pH and purified by reverse-phase HPLC. By using the preparation, we succeeded in preserving a target cystein residue without alteration of the α-helix structure of moPrP and were able to apply SDSL-ESR with a methane thiosulfonate spin label to the full-length prion protein. The rotational correlation times (τ) of 1.1, 3.3, and 4.8 ns were evaluated from the X-band ESR spectra at pH 7.4 and 20 deg C for N96R1, D143R1, and T189R1, respectively. τ reflects the fact that the Cu2+ binding region is more flexible than Helix1 or Helix2. ESR spectra recorded at various temperatures revealed two phases together with a transition point at around 20 deg C in D143R1 and T189R1, but not in N96R1. With the variation of pH from 4.0 to 7.8, ESR spectra of T189R1 at 20 deg C showed a gradual increase of τ from 2.9 to 4.8 ns. On the other hand, the pH-dependent conformational changes in N96R1 and D143R1 were negligible. These results indicated that T189 located in Helix2 possessed a structure sensitive to physiological pH changes; simultaneously, N96 in the Cu2+ binding region and D143 in Helix1 were conserved

  3. Conformational adaptation of Asian macaque TRIMCyp directs lineage specific antiviral activity.

    Laura M J Ylinen

    Full Text Available TRIMCyps are anti-retroviral proteins that have arisen independently in New World and Old World primates. All TRIMCyps comprise a CypA domain fused to the tripartite domains of TRIM5alpha but they have distinct lentiviral specificities, conferring HIV-1 restriction in New World owl monkeys and HIV-2 restriction in Old World rhesus macaques. Here we provide evidence that Asian macaque TRIMCyps have acquired changes that switch restriction specificity between different lentiviral lineages, resulting in species-specific alleles that target different viruses. Structural, thermodynamic and viral restriction analysis suggests that a single mutation in the Cyp domain, R69H, occurred early in macaque TRIMCyp evolution, expanding restriction specificity to the lentiviral lineages found in African green monkeys, sooty mangabeys and chimpanzees. Subsequent mutations have enhanced restriction to particular viruses but at the cost of broad specificity. We reveal how specificity is altered by a scaffold mutation, E143K, that modifies surface electrostatics and propagates conformational changes into the active site. Our results suggest that lentiviruses may have been important pathogens in Asian macaques despite the fact that there are no reported lentiviral infections in current macaque populations.

  4. SCR activity of conformed CuOx/ZrO2-SO4 catalysts

    Rasmussen, Søren Birk; Yates, Malcolm; Due-Hansen, Johannes;

    2010-01-01

    CuOX/ZrO2-SO4 catalysts have been synthesised as conformed materials with the use of sepiolite as agglomerant and the performance in the NH3-SCR reaction with relation to biomass fired boiler units have been studied. The optimal Cu-loading of the catalysts is 3 wt.% CuO, both in terms of activity...

  5. Long range dynamic effects of point-mutations trap a response regulator in an active conformation

    Bobay, Benjamin G.; Thompson, Richele J.; Hoch, James A.; Cavanagh, John

    2010-01-01

    When a point-mutation in a protein elicits a functional change, it is most common to assign this change to local structural perturbations. Here we show that point-mutations, distant from an essential highly dynamic kinase recognition loop in the response regulator Spo0F, lock this loop in an active conformation. This ‘conformational trapping’ results in functionally hyperactive Spo0F. Consequently, point-mutations are seen to affect functionally critical motions both close to and far from the...

  6. Denaturation studies of active-site labeled papain using electron paramagnetic resonance and fluorescence spectroscopy.

    Ping, Z A; Butterfiel, D A

    1991-01-01

    A spin-labeled p-chloromercuribenzoate (SL-PMB) and a fluorescence probe, 6-acryloyl-2-dimethylaminonaphthalene (Acrylodan), both of which bind to the single SH group located in the active site of papain, were used to investigate the interaction of papain (EC 3.4.22.2) with two protein denaturants. It was found that the active site of papain was highly stable in urea solution, but underwent a large conformational change in guanidine hydrochloride solution. Electron paramagnetic resonance and ...

  7. Density functional theory studies of MTSL nitroxide side chain conformations attached to an activation loop

    Concilio, Maria Grazia; Bayliss, Richard; Burgess, Selena G

    2016-01-01

    A quantum-mechanical (QM) method rooted on density functional theory (DFT) has been employed to determine conformations of the methane-thiosulfonate spin label (MTSL) attached to a fragment extracted from the activation loop of Aurora-A kinase. The features of the calculated energy surface revealed low energy barriers between isoenergetic minima and the system could be described in a population of 76 rotamers that can be also considered for other systems since it was found that the X3, X4 and X5 do not depend on the previous two dihedral angles. Conformational states obtained were seen to comparable to those obtained in the {\\alpha}-helix systems studied previously, indicating that the protein backbone does not affect the torsional profiles significantly and suggesting the possibility to use determined conformations for other protein systems for further modelling studies.

  8. Preferential binding of allosteric modulators to active and inactive conformational states of metabotropic glutamate receptors

    Klein-Seetharaman Judith

    2008-02-01

    Full Text Available Abstract Metabotropic glutamate receptors (mGluRs are G protein coupled receptors that play important roles in synaptic plasticity and other neuro-physiological and pathological processes. Allosteric mGluR ligands are particularly promising drug targets because of their modulatory effects – enhancing or suppressing the response of mGluRs to glutamate. The mechanism by which this modulation occurs is not known. Here, we propose the hypothesis that positive and negative modulators will differentially stabilize the active and inactive conformations of the receptors, respectively. To test this hypothesis, we have generated computational models of the transmembrane regions of different mGluR subtypes in two different conformations. The inactive conformation was modeled using the crystal structure of the inactive, dark state of rhodopsin as template and the active conformation was created based on a recent model of the light-activated state of rhodopsin. Ligands for which the nature of their allosteric effects on mGluRs is experimentally known were docked to the modeled mGluR structures using ArgusLab and Autodock softwares. We find that the allosteric ligand binding pockets of mGluRs are overlapping with the retinal binding pocket of rhodopsin, and that ligands have strong preferences for the active and inactive states depending on their modulatory nature. In 8 out of 14 cases (57%, the negative modulators bound the inactive conformations with significant preference using both docking programs, and 6 out of 9 cases (67%, the positive modulators bound the active conformations. Considering results by the individual programs only, even higher correlations were observed: 12/14 (86% and 8/9 (89% for ArgusLab and 10/14 (71% and 7/9 (78% for AutoDock. These findings strongly support the hypothesis that mGluR allosteric modulation occurs via stabilization of different conformations analogous to those identified in rhodopsin where they are induced by

  9. Phenolic Lipids Affect the Activity and Conformation of Acetylcholinesterase from Electrophorus electricus (Electric eel)

    Maria Stasiuk; Alicja Janiszewska; Arkadiusz Kozubek

    2014-01-01

    Phenolic lipids were isolated from rye grains, cashew nutshell liquid (CNSL) from Anacardium occidentale, and fruit bodies of Merrulius tremellosus, and their effects on the electric eel acetylcholinesterase activity and conformation were studied. The observed effect distinctly depended on the chemical structure of the phenolic lipids that were available for interaction with the enzyme. All of the tested compounds reduced the activity of acetylcholinesterase. The degree of inhibition varied, ...

  10. Workers’ Conformism

    Nikolay Ivantchev

    2013-10-01

    Full Text Available Conformism was studied among 46 workers with different kinds of occupations by means of two modified scales measuring conformity by Santor, Messervey, and Kusumakar (2000 – scale for perceived peer pressure and scale for conformism in antisocial situations. The hypothesis of the study that workers’ conformism is expressed in a medium degree was confirmed partly. More than a half of the workers conform in a medium degree for taking risk, and for the use of alcohol and drugs, and for sexual relationships. More than a half of the respondents conform in a small degree for anti-social activities (like a theft. The workers were more inclined to conform for risk taking (10.9%, then – for the use of alcohol, drugs and for sexual relationships (8.7%, and in the lowest degree – for anti-social activities (6.5%. The workers who were inclined for the use of alcohol and drugs tended also to conform for anti-social activities.

  11. Characterization of Reuse Activities at Contaminated Sites

    Angela Vitulli; Charlotte Dougherty; Kimberly Bosworth

    2004-01-01

    Given the increased focus on reuse activity within EPA and state site cleanup programs, policy makers would benefit from looking across programs to better understand the extent and nature of reuse; examine site characteristics that influence reuse; leverage lessons learned; and coordinate reuse activities, data collection, and information management. This research paper begins to examine these issues. It reports the results of a preliminary review and analysis of available EPA and state progr...

  12. Fingerprinting differential active site constraints of ATPases

    Hacker, Stephan M.; Hardt, Norman; Buntru, Alexander; Pagliarini, Dana; Möckel, Martin; Mayer, Thomas U; Scheffner, Martin; Hauck, Christof R.; Marx, Andreas

    2013-01-01

    The free energy provided by adenosine triphosphate (ATP) hydrolysis is central to many cellular processes and, therefore, the number of enzymes utilizing ATP as a substrate is almost innumerable. Modified analogues of ATP are a valuable means to understand the biological function of ATPases. Although these enzymes have evolved towards binding to ATP, large differences in active site architectures were found. In order to systematically access the specific active site constraints of different A...

  13. Small-angle neutron and X-ray scattering reveal conformational changes in rhodopsin activation

    Shrestha, Utsab R.; Bhowmik, Debsindhu; Perera, Suchitrhanga M. C. D.; Chawla, Udeep; Struts, Andrey V.; Graziono, Vito; Pingali, Sai Venkatesh; Heller, William T.; Qian, Shuo; Brown, Michael F.; Chu, Xiang-Qiang

    2015-03-01

    Understanding G-protein-coupled receptor (GPCR) activation plays a crucial role in the development of novel improved molecular drugs. During photo-activation, the retinal chromophore of the visual GPCR rhodopsin isomerizes from 11-cis to all-trans conformation, yielding an equilibrium between inactive Meta-I and active Meta-II states. The principal goals of this work are to address whether the activation of rhodopsin leads to a single state or a conformational ensemble, and how protein organizational structure changes with detergent environment in solution. We use both small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS) techniques to answer the above questions. For the first time we observe the change in protein conformational ensemble upon photo-activation by SANS with contrast variation, which enables the separate study of the protein structure within the detergent assembly. In addition, SAXS study of protein structure within detergent assembly suggests that the detergent molecules form a belt of monolayer (micelle) around protein with different geometrical shapes to keep the protein in folded state.

  14. The transcription initiation sites of eggplant latent viroid strands map within distinct motifs in their in vivo RNA conformations.

    López-Carrasco, Amparo; Gago-Zachert, Selma; Mileti, Giuseppe; Minoia, Sofia; Flores, Ricardo; Delgado, Sonia

    2016-01-01

    Eggplant latent viroid (ELVd), like other members of family Avsunviroidae, replicates in plastids through a symmetric rolling-circle mechanism in which elongation of RNA strands is most likely catalyzed by a nuclear-encoded polymerase (NEP) translocated to plastids. Here we have addressed where NEP initiates transcription of viroid strands. Because this step is presumably directed by sequence/structural motifs, we have previously determined the conformation of the monomeric linear (+) and (-) RNAs of ELVd resulting from hammerhead-mediated self-cleavage. In silico predictions with 3 softwares led to similar bifurcated conformations for both ELVd strands. In vitro examination by non-denaturing PAGE showed that they migrate as prominent single bands, with the ELVd (+) RNA displaying a more compact conformation as revealed by its faster electrophoretic mobility. In vitro SHAPE analysis corroborated the ELVd conformations derived from thermodynamics-based predictions in silico. Moreover, sequence analysis of 94 full-length natural ELVd variants disclosed co-variations, and mutations converting canonical into wobble pairs or vice versa, which confirmed in vivo most of the stems predicted in silico and in vitro, and additionally helped to introduce minor structural refinements. Therefore, results from the 3 experimental approaches were essentially consistent among themselves. Application to RNA preparations from ELVd-infected tissue of RNA ligase-mediated rapid amplification of cDNA ends, combined with pretreatments to modify the 5' ends of viroid strands, mapped the transcription initiation sites of ELVd (+) and (-) strands in vivo at different sequence/structural motifs, in contrast with the situation previously observed in 2 other members of the family Avsunviroidae. PMID:26618399

  15. Proton binding sites and conformational analysis of H+K+-ATPase

    It is proposed that the hydronium ion, H3O+, binds to the E1 conformation of the α-subunit of gastric proton pump. The H3O+ binding cavities are characterized parametrically based on valence, sequence, geometry, and size considerations from comparative modeling. The cavities have scope for accommodating monovalent cations of different ionic radii. The H3O+ transport is proposed to be aided by arenes which are arranged regularly along the pump starting from N-domain through the transmembrane region. Step-by-step structural changes accompanying H3O+ occlusion are studied in detail. The observations corroborate well with earlier experimental studies

  16. Design, synthesis, and biological activities of conformationally restricted analogs of primaquine with a 1,10-phenanthroline framework

    Sall, C.; Yapi, A. D.; Desbois, N.; Chevalley, Séverine; Chezal, J.M.; Tan, K.; Teulade, J. C.; Valentin, A; Y. Blache

    2008-01-01

    A series of primaquine analogs was prepared, according to a conformationally restricted conformation of primaquine. In vitro antiplasmodial activities were evaluated and showed that all compounds were active on different strains of Plasmodium falciparum. In particular compounds 5 and 15 possessing a methoxy group were more active than was primaquine. Furthermore, analog 5 displayed good in vitro gametocytocidal activity. In addition selectivity indexes were calculated in respect with cytotoxi...

  17. Metal active site elasticity linked to activation of homocysteine in methionine synthases

    Koutmos, Markos; Pejchal, Robert; Bomer, Theresa M.; Matthews, Rowena G.; Smith, Janet L.; Ludwig, Martha L. (Michigan)

    2008-04-02

    Enzymes possessing catalytic zinc centers perform a variety of fundamental processes in nature, including methyl transfer to thiols. Cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthases are two such enzyme families. Although they perform the same net reaction, transfer of a methyl group from methyltetrahydrofolate to homocysteine (Hcy) to form methionine, they display markedly different catalytic strategies, modular organization, and active site zinc centers. Here we report crystal structures of zinc-replete MetE and MetH, both in the presence and absence of Hcy. Structural investigation of the catalytic zinc sites of these two methyltransferases reveals an unexpected inversion of zinc geometry upon binding of Hcy and displacement of an endogenous ligand in both enzymes. In both cases a significant movement of the zinc relative to the protein scaffold accompanies inversion. These structures provide new information on the activation of thiols by zinc-containing enzymes and have led us to propose a paradigm for the mechanism of action of the catalytic zinc sites in these and related methyltransferases. Specifically, zinc is mobile in the active sites of MetE and MetH, and its dynamic nature helps facilitate the active site conformational changes necessary for thiol activation and methyl transfer.

  18. Targeting the autolysis loop of urokinase-type plasminogen activator with conformation-specific monoclonal antibodies

    Bøtkjær, Kenneth Alrø; Fogh, Sarah; Bekes, Erin C;

    2011-01-01

    Tight regulation of serine proteases is essential for their physiological function, and unbalanced states of protease activity have been implicated in a variety of human diseases. One key example is the presence of uPA (urokinase-type plasminogen activator) in different human cancer types...... to harbour the epitopes for three conformation-specific monoclonal antibodies, two with a preference for the zymogen form pro-uPA, and one with a preference for active uPA. All three antibodies were shown to have overlapping epitopes, with three common residues being crucial for all three antibodies...

  19. Simultaneous measurement of DNA motor protein conformation and activity with combined optical trap and single-molecule fluorescence

    Chemla, Yann

    2013-03-01

    We present single-molecule measurements of Superfamily 1 UvrD helicase DNA unwinding that reveal directly how helicase stoichiometry and conformation regulate motor activity. Using a new instrument that combines high resolution optical tweezers with single-molecule fluorescence microscopy, we record DNA unwinding activity with base pair-scale resolution (via optical tweezers) simultaneously with helicase stoichiometry and conformation (via fluorescence). Quantifying the fluorescence signal from labeled UvrD, we observe that pairs of UvrD molecules are required for long distance unwinding but that individual molecules exhibit limited, non-processive unwinding activity. UvrD is also known to exhibit two different conformations, `closed' and `open', based on the orientation of its 2B regulatory domain. The function of these conformations has remained elusive. Measuring the fluorescence of FRET labeled proteins, we detect directly the conformation of the 2B domain of individual UvrD molecules during unwinding activity. We observe that UvrD is in the `closed' conformation during DNA unwinding but surprisingly switches to the `open' conformation upon reversal of helicase direction, i.e. when UvrD switches strands and translocates on the opposing strand with the DNA junction rezipping behind it. We hypothesize that the 2B domain acts as a conformational switch that controls DNA unwinding vs. re-annealing. Work supported by NSF (PHY-082261, Center for the Physics of Living Cells) and NIH (R21 RR025341A)

  20. Ligands for pheromone-sensing neurons are not conformationally activated odorant binding proteins.

    Gomez-Diaz, Carolina; Reina, Jaime H; Cambillau, Christian; Benton, Richard

    2013-01-01

    Pheromones form an essential chemical language of intraspecific communication in many animals. How olfactory systems recognize pheromonal signals with both sensitivity and specificity is not well understood. An important in vivo paradigm for this process is the detection mechanism of the sex pheromone (Z)-11-octadecenyl acetate (cis-vaccenyl acetate [cVA]) in Drosophila melanogaster. cVA-evoked neuronal activation requires a secreted odorant binding protein, LUSH, the CD36-related transmembrane protein SNMP, and the odorant receptor OR67d. Crystallographic analysis has revealed that cVA-bound LUSH is conformationally distinct from apo (unliganded) LUSH. Recombinantly expressed mutant versions of LUSH predicted to enhance or diminish these structural changes produce corresponding alterations in spontaneous and/or cVA-evoked activity when infused into olfactory sensilla, leading to a model in which the ligand for pheromone receptors is not free cVA, but LUSH that is "conformationally activated" upon cVA binding. Here we present evidence that contradicts this model. First, we demonstrate that the same LUSH mutants expressed transgenically affect neither basal nor pheromone-evoked activity. Second, we compare the structures of apo LUSH, cVA/LUSH, and complexes of LUSH with non-pheromonal ligands and find no conformational property of cVA/LUSH that can explain its proposed unique activated state. Finally, we show that high concentrations of cVA can induce neuronal activity in the absence of LUSH, but not SNMP or OR67d. Our findings are not consistent with the model that the cVA/LUSH complex acts as the pheromone ligand, and suggest that pheromone molecules alone directly activate neuronal receptors. PMID:23637570

  1. Immobilization of enzymes using non-ionic colloidal liquid aphrons (CLAs): Activity kinetics, conformation, and energetics.

    Ward, Keeran; Xi, Jingshu; Stuckey, David C

    2016-05-01

    This study seeks to examine the ability of non-ionic/non-polar Colloidial Liquid Aphrons (CLAs) to preserve enzyme functionality upon immobilization and release. CLAs consisting of micron-sized oil droplets surrounded by a thin aqueous layer stabilized by a mixture of surfactants, were formulated by direct addition (pre-manufacture addition) using 1% Tween 80/mineral oil and 1% Tween 20 and the enzymes lipase, aprotinin and α-chymotrypsin. The results of activity assays for both lipase and α-chymotrypsin showed that kinetic activity increased upon immobilization by factors of 7 and 5.5, respectively, while aprotinin retained approximately 85% of its native activity. The conformation of the enzymes released through desorption showed no significant alterations compared to their native state. Changes in pH and temperature showed that optimum conditions did not change after immobilization, while analysis of activation energy for the immobilized enzyme showed an increase in activity at higher temperatures. Furthermore, the effect of bound water within the aphron structure allowed for some degree of enzyme hydration, and this hydration was needed for an active conformation with results showing a decrease in ΔH* for the immobilized system compared to its native counterpart. Biotechnol. Bioeng. 2016;113: 970-978. © 2015 Wiley Periodicals, Inc. PMID:26497856

  2. Managing Siting Activities for Nuclear Power Plants

    One of the IAEA's statutory objectives is to ''seek to accelerate and enlarge the contribution of atomic energy to peace, health and prosperity throughout the world''. One way this objective is achieved is through the publication of a range of technical series. Two of these are the IAEA Nuclear Energy Series and the IAEA Safety Standards Series. According to Article III.A.6 of the IAEA Statute, the safety standards establish 'standards of safety for protection of health and minimization of danger to life and property.' The safety standards include the Safety Fundamentals, Safety Requirements and Safety Guides. These standards are written primarily in a regulatory style, and are binding on the IAEA for its own programmes. The principal users are the regulatory bodies in Member States and other national authorities. The IAEA Nuclear Energy Series comprises reports designed to encourage and assist R and D on, and application of, nuclear energy for peaceful uses. This includes practical examples to be used by owners and operators of utilities in Member States, implementing organizations, academia, and government officials, among others. This information is presented in guides, reports on technology status and advances, and best practices for peaceful uses of nuclear energy based on inputs from international experts. The IAEA Nuclear Energy Series complements the IAEA Safety Standards Series. The introduction of nuclear power brings new challenges to States - one of them being the selection of appropriates sites. It is a project that needs to begin early, be well managed, and deploy good communications with all stakeholders; including regulators. This is important, not just for those States introducing nuclear power for the first time, but for any State looking to build a new nuclear power plant. The purpose of the siting activities goes beyond choosing a suitable site and acquiring a licence. A large part of the project is about producing and maintaining a validated

  3. Efficient oxygen electrocatalysis on special active sites

    Halck, Niels Bendtsen

    cobalt incorporated in ruthenium dioxide at high overpotentials during the oxygen reduction reaction (ORR). Density functional theory calculations were used to explain this phenomenon. The special active sites concepts are used to propose a general unified approach to increase the efficiency for oxygen...

  4. A vitellogenin polyserine cleavage site: highly disordered conformation protected from proteolysis by phosphorylation.

    Havukainen, Heli; Underhaug, Jarl; Wolschin, Florian; Amdam, Gro; Halskau, Øyvind

    2012-06-01

    Vitellogenin (Vg) is an egg-yolk precursor protein in most oviparous species. In honeybee (Apis mellifera), the protein (AmVg) also affects social behavior and life-span plasticity. Despite its manifold functions, the AmVg molecule remains poorly understood. The subject of our structure-oriented AmVg study is its polyserine tract - a little-investigated repetitive protein segment mostly found in insects. We previously reported that AmVg is tissue specifically cleaved in the vicinity of this tract. Here, we show that, despite its potential for an open, disordered structure, AmVg is unexpectedly resistant to trypsin/chymotrypsin digestion at the tract. Our findings suggest that multiple phosphorylation plays a role in this resilience. Sequence variation is highly pronounced at the polyserine region in insect Vgs. We demonstrate that sequence differences in this region can lead to structural variation, as NMR and circular dichroism (CD) evidence assign different conformational propensities to polyserine peptides from the honeybee and the jewel wasp Nasonia vitripennis; the former is extended and disordered and the latter more compact and helical. CD analysis of the polyserine region of bumblebee Bombus ignitus and wasp Pimpla nipponica supports a random coil structure in these species. The spectroscopic results strengthen our model of the AmVg polyserine tract as a flexible domain linker shielded by phosphorylation. PMID:22573762

  5. Conformational dynamics of bacterial and human cytoplasmic models of the ribosomal A-site

    Panecka, J.; Šponer, Jiří; Trylska, J.

    112C, MAY 2015 (2015), s. 96-110. ISSN 0300-9084 R&D Projects: GA ČR(CZ) GBP305/12/G034; GA MŠk(CZ) ED1.1.00/02.0068 Institutional support: RVO:68081707 Keywords : MOLECULAR-DYNAMICS * DECODING SITE * CRYSTAL-STRUCTURE Subject RIV: BO - Biophysics Impact factor: 2.963, year: 2014

  6. Perchlorate Reductase Is Distinguished by Active Site Aromatic Gate Residues.

    Youngblut, Matthew D; Tsai, Chi-Lin; Clark, Iain C; Carlson, Hans K; Maglaqui, Adrian P; Gau-Pan, Phonchien S; Redford, Steven A; Wong, Alan; Tainer, John A; Coates, John D

    2016-04-22

    Perchlorate is an important ion on both Earth and Mars. Perchlorate reductase (PcrAB), a specialized member of the dimethylsulfoxide reductase superfamily, catalyzes the first step of microbial perchlorate respiration, but little is known about the biochemistry, specificity, structure, and mechanism of PcrAB. Here we characterize the biophysics and phylogeny of this enzyme and report the 1.86-Å resolution PcrAB complex crystal structure. Biochemical analysis revealed a relatively high perchlorate affinity (Km = 6 μm) and a characteristic substrate inhibition compared with the highly similar respiratory nitrate reductase NarGHI, which has a relatively much lower affinity for perchlorate (Km = 1.1 mm) and no substrate inhibition. Structural analysis of oxidized and reduced PcrAB with and without the substrate analog SeO3 (2-) bound to the active site identified key residues in the positively charged and funnel-shaped substrate access tunnel that gated substrate entrance and product release while trapping transiently produced chlorate. The structures suggest gating was associated with shifts of a Phe residue between open and closed conformations plus an Asp residue carboxylate shift between monodentate and bidentate coordination to the active site molybdenum atom. Taken together, structural and mutational analyses of gate residues suggest key roles of these gate residues for substrate entrance and product release. Our combined results provide the first detailed structural insight into the mechanism of biological perchlorate reduction, a critical component of the chlorine redox cycle on Earth. PMID:26940877

  7. Efficient oxygen electrocatalysis on special active sites

    Halck, Niels Bendtsen

    Oxygen electrocatalysis will be pivotal in future independent of fossil fuels. Renewable energy production will rely heavily on oxygen electrocatalysis as a method for storing energy from intermittent energy sources such as the wind and sun in the form of chemical bonds and to release the energy ...... electrocatalysis (ORR and OER) using organic functional groups on another class of catalysts. These consist of graphene sheets modified to have a local porphyrine site with different transition metals ions as model systems....... stored in these bonds in an eco-friendly fashion in fuel cells. This thesis explores catalysts for oxygen electrocatalysis and how carefully designed local structures on catalysts surfaces termed special active sites can influence the activity. Density functional theory has been used as a method...... throughout this thesis to understand these local structure effects and their influence on surface reactions. The concept of these special active sites is used to explain how oxygen evolution reaction (OER) catalysts can have activities beyond the limits of what was previously thought possible. The concept is...

  8. Site-specific intercalation at the triplex-duplex junction induces a conformational change which is detectable by hypersensitivity to diethylpyrocarbonate.

    Collier, D A; Mergny, J L; Thuong, N T; Helene, C

    1991-01-01

    Using site-specific intercalation directed by intermolecular triplex formation, the conformation of an intercalation site in DNA was examined by footprinting with the purine-specific (A much greater than G) reagent diethylpyrocarbonate. Site specific intercalation was achieved by covalently linking an intercalator to the 5' end of a homopyrimidine oligodeoxynucleotide, which bound to a homopurinehomopyrimidine stretch in a recombinant plasmid via intermolecular triplex formation. This directs...

  9. Distinct conformational changes in activated agonist-bound and agonist-free glycine receptor subunits

    Pless, Stephan Alexander; Lynch, Joseph W

    2009-01-01

    glycine-free or a glycine-bound subunit. Agonist-free subunits were created by incorporating T204A and R65K mutations, which disrupted glycine binding to both (+) and (-) subunit interfaces. In heteromeric receptors comprising wild-type and R65K,T204A,R271C triple-mutant subunits, the fluorescence...... response exhibited a drastically reduced glycine sensitivity relative to the current response. Two conclusions can be drawn from this. First, because the labeled glycine-free subunits were activated by glycine binding to neighboring wild-type subunits, our results provide evidence for a cooperative...... activation mechanism. However, because the fluorescent label on glycine-free subunits does not reflect movements at the channel gate, we conclude that glycine binding also produces a local non-concerted conformational change that is not essential for receptor activation....

  10. Conformational transition in signal transduction: metastable states and transition pathways in the activation of a signaling protein.

    Banerjee, Rahul; Yan, Honggao; Cukier, Robert I

    2015-06-01

    Signal transduction is of vital importance to the growth and adaptation of living organisms. The key to understand mechanisms of biological signal transduction is elucidation of the conformational dynamics of its signaling proteins, as the activation of a signaling protein is fundamentally a process of conformational transition from an inactive to an active state. A predominant form of signal transduction for bacterial sensing of environmental changes in the wild or inside their hosts is a variety of two-component systems, in which the conformational transition of a response regulator (RR) from an inactive to an active state initiates responses to the environmental changes. Here, RR activation has been investigated using RR468 as a model system by extensive unbiased all-atom molecular dynamics (MD) simulations in explicit solvent, starting from snapshots along a targeted MD trajectory that covers the conformational transition. Markov state modeling, transition path theory, and geometric analyses of the wealth of the MD data have provided a comprehensive description of the RR activation. It involves a network of metastable states, with one metastable state essentially the same as the inactive state and another very similar to the active state that are connected via a small set of intermediates. Five major pathways account for >75% of the fluxes of the conformational transition from the inactive to the active-like state. The thermodynamic stability of the states and the activation barriers between states are found, to identify rate-limiting steps. The conformal transition is initiated predominantly by movements of the β3α3 loop, followed by movements of the β4α4-loop and neighboring α4 helix region, and capped by additional movements of the β3α3 loop. A number of transient hydrophobic and hydrogen bond interactions are revealed, and they may be important for the conformational transition. PMID:25945797

  11. Involved-Site Image-Guided Intensity Modulated Versus 3D Conformal Radiation Therapy in Early Stage Supradiaphragmatic Hodgkin Lymphoma

    Purpose: Image-guided intensity modulated radiation therapy (IG-IMRT) allows for margin reduction and highly conformal dose distribution, with consistent advantages in sparing of normal tissues. The purpose of this retrospective study was to compare involved-site IG-IMRT with involved-site 3D conformal RT (3D-CRT) in the treatment of early stage Hodgkin lymphoma (HL) involving the mediastinum, with efficacy and toxicity as primary clinical endpoints. Methods and Materials: We analyzed 90 stage IIA HL patients treated with either involved-site 3D-CRT or IG-IMRT between 2005 and 2012 in 2 different institutions. Inclusion criteria were favorable or unfavorable disease (according to European Organization for Research and Treatment of Cancer criteria), complete response after 3 to 4 cycles of an adriamycin- bleomycin-vinblastine-dacarbazine (ABVD) regimen plus 30 Gy as total radiation dose. Exclusion criteria were chemotherapy other than ABVD, partial response after ABVD, total radiation dose other than 30 Gy. Clinical endpoints were relapse-free survival (RFS) and acute toxicity. Results: Forty-nine patients were treated with 3D-CRT (54.4%) and 41 with IG-IMRT (45.6%). Median follow-up time was 54.2 months for 3D-CRT and 24.1 months for IG-IMRT. No differences in RFS were observed between the 2 groups, with 1 relapse each. Three-year RFS was 98.7% for 3D-CRT and 100% for IG-IMRT. Grade 2 toxicity events, mainly mucositis, were recorded in 32.7% of 3D-CRT patients (16 of 49) and in 9.8% of IG-IMRT patients (4 of 41). IG-IMRT was significantly associated with a lower incidence of grade 2 acute toxicity (P=.043). Conclusions: RFS rates at 3 years were extremely high in both groups, albeit the median follow-up time is different. Acute tolerance profiles were better for IG-IMRT than for 3D-CRT. Our preliminary results support the clinical safety and efficacy of advanced RT planning and delivery techniques in patients affected with early stage HL, achieving complete

  12. Involved-Site Image-Guided Intensity Modulated Versus 3D Conformal Radiation Therapy in Early Stage Supradiaphragmatic Hodgkin Lymphoma

    Filippi, Andrea Riccardo, E-mail: andreariccardo.filippi@unito.it [Department of Oncology, University of Torino, Torino (Italy); Ciammella, Patrizia [Radiation Therapy Unit, Department of Oncology and Advanced Technology, ASMN Hospital IRCCS, Reggio Emilia (Italy); Piva, Cristina; Ragona, Riccardo [Department of Oncology, University of Torino, Torino (Italy); Botto, Barbara [Hematology, Città della Salute e della Scienza, Torino (Italy); Gavarotti, Paolo [Hematology, University of Torino and Città della Salute e della Scienza, Torino (Italy); Merli, Francesco [Hematology Unit, ASMN Hospital IRCCS, Reggio Emilia (Italy); Vitolo, Umberto [Hematology, Città della Salute e della Scienza, Torino (Italy); Iotti, Cinzia [Radiation Therapy Unit, Department of Oncology and Advanced Technology, ASMN Hospital IRCCS, Reggio Emilia (Italy); Ricardi, Umberto [Department of Oncology, University of Torino, Torino (Italy)

    2014-06-01

    Purpose: Image-guided intensity modulated radiation therapy (IG-IMRT) allows for margin reduction and highly conformal dose distribution, with consistent advantages in sparing of normal tissues. The purpose of this retrospective study was to compare involved-site IG-IMRT with involved-site 3D conformal RT (3D-CRT) in the treatment of early stage Hodgkin lymphoma (HL) involving the mediastinum, with efficacy and toxicity as primary clinical endpoints. Methods and Materials: We analyzed 90 stage IIA HL patients treated with either involved-site 3D-CRT or IG-IMRT between 2005 and 2012 in 2 different institutions. Inclusion criteria were favorable or unfavorable disease (according to European Organization for Research and Treatment of Cancer criteria), complete response after 3 to 4 cycles of an adriamycin- bleomycin-vinblastine-dacarbazine (ABVD) regimen plus 30 Gy as total radiation dose. Exclusion criteria were chemotherapy other than ABVD, partial response after ABVD, total radiation dose other than 30 Gy. Clinical endpoints were relapse-free survival (RFS) and acute toxicity. Results: Forty-nine patients were treated with 3D-CRT (54.4%) and 41 with IG-IMRT (45.6%). Median follow-up time was 54.2 months for 3D-CRT and 24.1 months for IG-IMRT. No differences in RFS were observed between the 2 groups, with 1 relapse each. Three-year RFS was 98.7% for 3D-CRT and 100% for IG-IMRT. Grade 2 toxicity events, mainly mucositis, were recorded in 32.7% of 3D-CRT patients (16 of 49) and in 9.8% of IG-IMRT patients (4 of 41). IG-IMRT was significantly associated with a lower incidence of grade 2 acute toxicity (P=.043). Conclusions: RFS rates at 3 years were extremely high in both groups, albeit the median follow-up time is different. Acute tolerance profiles were better for IG-IMRT than for 3D-CRT. Our preliminary results support the clinical safety and efficacy of advanced RT planning and delivery techniques in patients affected with early stage HL, achieving complete

  13. Epitope mapping of conformational V2-specific anti-HIV human monoclonal antibodies reveals an immunodominant site in V2.

    Luzia M Mayr

    Full Text Available In the case-control study of the RV144 vaccine trial, the levels of antibodies to the V1V2 region of the gp120 envelope glycoprotein were found to correlate inversely with risk of HIV infection. This recent demonstration of the potential role of V1V2 as a vaccine target has catapulted this region into the focus of HIV-1 research. We previously described seven human monoclonal antibodies (mAbs derived from HIV-infected individuals that are directed against conformational epitopes in the V1V2 domain. In this study, using lysates of SF162 pseudoviruses carrying V1V2 mutations, we mapped the epitopes of these seven mAbs. All tested mAbs demonstrated a similar binding pattern in which three mutations (F176A, Y177T, and D180L abrogated binding of at least six of the seven mAbs to ≤15% of SF162 wildtype binding. Binding of six or all of the mAbs was reduced to ≤50% of wildtype by single substitutions at seven positions (168, 180, 181, 183, 184, 191, and 193, while one change, V181I, increased the binding of all mAbs. When mapped onto a model of V2, our results suggest that the epitope of the conformational V2 mAbs is located mostly in the disordered region of the available crystal structure of V1V2, overlapping and surrounding the α4β7 binding site on V2.

  14. Flexibility and Stability Trade-Off in Active Site of Cold-Adapted Pseudomonas mandelii Esterase EstK.

    Truongvan, Ngoc; Jang, Sei-Heon; Lee, ChangWoo

    2016-06-28

    Cold-adapted enzymes exhibit enhanced conformational flexibility, especially in their active sites, as compared with their warmer-temperature counterparts. However, the mechanism by which cold-adapted enzymes maintain their active site stability is largely unknown. In this study, we investigated the role of conserved D308-Y309 residues located in the same loop as the catalytic H307 residue in the cold-adapted esterase EstK from Pseudomonas mandelii. Mutation of D308 and/or Y309 to Ala or deletion resulted in increased conformational flexibility. Particularly, the D308A or Y309A mutant showed enhanced substrate affinity and catalytic rate, as compared with wild-type EstK, via enlargement of the active site. However, all mutant EstK enzymes exhibited reduced thermal stability. The effect of mutation was greater for D308 than Y309. These results indicate that D308 is not preferable for substrate selection and catalytic activity, whereas hydrogen bond formation involving D308 is critical for active site stabilization. Taken together, conformation of the EstK active site is constrained via flexibility-stability trade-off for enzyme catalysis and thermal stability. Our study provides further insights into active site stabilization of cold-adapted enzymes. PMID:27259687

  15. BET is active on Sellafield site

    Several companies, all part of BET Plant Services are carrying out work at the British Nuclear Fuels (BNFL) site at Sellafield, Cumbria, on one of the largest construction projects in Europe. The main development scheme is the THORP (Thermal Oxide Reprocessing Plant) buildings. One of the BET companies has the contract to paint the inside of the fuel storage ponds. It will also coat the surfaces of the MASWEP (Medium Active Solid Waste Encapsulation Plant) complex. Other work includes insulation and fire prevention installation. Scaffolding at the EARP (Enhanced Actinide Removal Plant) site is being provided on a common user basis so all the contractors can use the scaffolding and share the cost. Temporary office and living accommodation blocks have been provide by another BET company. (author)

  16. Interactions of a designed peptide with lipopolysaccharide: Bound conformation and anti-endotoxic activity

    Designed peptides that would selectively interact with lipopolysaccharide (LPS) or endotoxin and fold into specific conformations could serve as important scaffolds toward the development of antisepsis compounds. Here, we describe solution structure of a designed amphipathic peptide, H2N-YVKLWRMIKFIR-CONH2 (YW12D) in complex with endotoxin as determined by transferred nuclear Overhauser effect spectroscopy. The conformation of the isolated peptide is highly flexible, but undergoes a dramatic structural stabilization in the presence of LPS. Structure calculations reveal that the peptide presents two amphipathic surfaces in its bound state to LPS whereby each surface is characterized by two positive charges and a number of aromatic and/or aliphatic residues. ITC data suggests that peptide interacts with two molecules of lipid A. In activity assays, YW12D exhibits neutralization of LPS toxicity with very little hemolysis of red blood cells. Structural and functional properties of YW12D would be applicable in designing low molecular weight non-toxic antisepsis molecules

  17. Structure-activity relationship of ibogaine analogs interacting with nicotinic acetylcholine receptors in different conformational states.

    Arias, Hugo R; Feuerbach, Dominik; Targowska-Duda, Katarzyna M; Jozwiak, Krzysztof

    2011-09-01

    The interaction of ibogaine analogs with nicotinic acetylcholine receptors (AChRs) in different conformational states was studied by functional and structural approaches. The results established that ibogaine analogs: (a) inhibit (±)-epibatidine-induced Ca²⁺ influx in human embryonic muscle AChRs with the following potency sequence (IC(50) in μM): (±)-18-methylaminocoronaridine (5.9±0.3)∼(±)-18-methoxycoronaridine (18-MC) (6.8±0.8)>(-)-ibogaine (17±3)∼(+)-catharanthine (20±1)>(±)-albifloranine (46±13), (b) bind to the [³H]TCP binding site with higher affinity when the Torpedo AChR is in the desensitized state compared to that in the resting state. Similar results were obtained using [³H]18-MC. These and docking results suggest a steric interaction between TCP and ibogaine analogs for the same site, (c) enhance [³H]cytisine binding to resting but not to desensitized AChRs, with desensitizing potencies (apparent EC₅₀) that correlate very well with the pK(i) values in the desensitized state, and (d) there are good bilinear correlations between the ligand molecular volumes and their affinities in the desensitized and resting states, with an optimal volume of ∼345 ų for the ibogaine site. These results indicate that the size of the binding sites for ibogaine analogs, located between the serine and nonpolar rings and shared with TCP, is an important structural feature for binding and for inducing desensitization. PMID:21642011

  18. Active site and laminarin binding in glycoside hydrolase family 55.

    Bianchetti, Christopher M; Takasuka, Taichi E; Deutsch, Sam; Udell, Hannah S; Yik, Eric J; Bergeman, Lai F; Fox, Brian G

    2015-05-01

    The Carbohydrate Active Enzyme (CAZy) database indicates that glycoside hydrolase family 55 (GH55) contains both endo- and exo-β-1,3-glucanases. The founding structure in the GH55 is PcLam55A from the white rot fungus Phanerochaete chrysosporium (Ishida, T., Fushinobu, S., Kawai, R., Kitaoka, M., Igarashi, K., and Samejima, M. (2009) Crystal structure of glycoside hydrolase family 55 β-1,3-glucanase from the basidiomycete Phanerochaete chrysosporium. J. Biol. Chem. 284, 10100-10109). Here, we present high resolution crystal structures of bacterial SacteLam55A from the highly cellulolytic Streptomyces sp. SirexAA-E with bound substrates and product. These structures, along with mutagenesis and kinetic studies, implicate Glu-502 as the catalytic acid (as proposed earlier for Glu-663 in PcLam55A) and a proton relay network of four residues in activating water as the nucleophile. Further, a set of conserved aromatic residues that define the active site apparently enforce an exo-glucanase reactivity as demonstrated by exhaustive hydrolysis reactions with purified laminarioligosaccharides. Two additional aromatic residues that line the substrate-binding channel show substrate-dependent conformational flexibility that may promote processive reactivity of the bound oligosaccharide in the bacterial enzymes. Gene synthesis carried out on ∼30% of the GH55 family gave 34 active enzymes (19% functional coverage of the nonredundant members of GH55). These active enzymes reacted with only laminarin from a panel of 10 different soluble and insoluble polysaccharides and displayed a broad range of specific activities and optima for pH and temperature. Application of this experimental method provides a new, systematic way to annotate glycoside hydrolase phylogenetic space for functional properties. PMID:25752603

  19. Reduction of urease activity by interaction with the flap covering the active site.

    Macomber, Lee; Minkara, Mona S; Hausinger, Robert P; Merz, Kenneth M

    2015-02-23

    With the increasing appreciation for the human microbiome coupled with the global rise of antibiotic resistant organisms, it is imperative that new methods be developed to specifically target pathogens. To that end, a novel computational approach was devised to identify compounds that reduce the activity of urease, a medically important enzyme of Helicobacter pylori, Proteus mirabilis, and many other microorganisms. Urease contains a flexible loop that covers its active site; Glide was used to identify small molecules predicted to lock this loop in an open conformation. These compounds were screened against the model urease from Klebsiella aerogenes, and the natural products epigallocatechin and quercetin were shown to inhibit at low and high micromolar concentrations, respectively. These molecules exhibit a strong time-dependent inactivation of urease that was not due to their oxygen sensitivity. Rather, these compounds appear to inactivate urease by reacting with a specific Cys residue located on the flexible loop. Substitution of this cysteine by alanine in the C319A variant increased the urease resistance to both epigallocatechin and quercetin, as predicted by the computational studies. Protein dynamics are integral to the function of many enzymes; thus, identification of compounds that lock an enzyme into a single conformation presents a useful approach to define potential inhibitors. PMID:25594724

  20. Raman optical activity spectra and conformational elucidation of chiral drugs. The case of the antiangiogenic aeroplysinin-1.

    Nieto-Ortega, Belén; Casado, Juan; Blanch, Ewan W; López Navarrete, Juan T; Quesada, Ana R; Ramírez, Francisco J

    2011-04-01

    We present the determination of the conformational properties of aeroplysinin-1 in aqueous solution by means of a combined experimental and theoretical Raman optical activity (ROA) and vibrational circular dichroism (VCD) study. Aeroplysinin-1 is an antiangiogenic drug extracted from the sponge Aplysina cavernicola which has been proved to be a valuable candidate for the treatment of cancer and other antiangiogenic diseases. Our study shows that this molecule possesses the 1S,6R absolute configuration in aqueous solution, where only two conformers are present to a significant level. We discuss in detail the relationships between the chiro-optical ROA and VCD features, and the structural properties of various energy accessible conformers are described. The present work is one of the first studies in which both ROA and VCD have been used as complementary tools for the determination of absolute configuration and dominant solution-state conformations of an unknown therapeutically significant molecule. PMID:21401047

  1. Thermal activation of a group II intron ribozyme reveals multiple conformational states.

    Franzen, J S; Zhang, M; Chay, T R; Peebles, C L

    1994-09-20

    Conformational changes often accompany biological catalysis. Group II introns promote a variety of reactions in vitro that show an unusually sharp temperature dependence. This suggests that the chemical steps are accompanied by the conversion of a folded-but-inactive form to a differently folded active state. We report here the kinetic analysis of 5'-splice-junction hydrolysis (SJH) by E1:12345, a transcript containing the 5'-exon plus the first five of six intron secondary structure domains. The pseudo-first-order SJH reaction shows (1) activation by added KCl to 1.5 M; (2) cooperative activation by added MgCl2, nHill = 4.1-4.3, and [MgCl2]vmax/2 approximately 0.040 M; and (3) a rather high apparent activation energy, Ea approximately 50 kcal mol-l. In contrast, the 5'-terminal phosphodiester bond of a domain 5 transcript (GGD5) was hydrolyzed with Ea approximately 30 kcal mol-1 under SJH conditions; the 5'-GG leader dinucleotide presumably lacks secondary structure constraints. The effect of adding the chaotropic salt tetraethylammonium chloride (TEA) was also investigated. TEA reduced the melting temperatures of GGD5 and E1:12345. TEA also shifted the profile of rate versus temperature for SJH by E1:12345 toward lower temperatures without affecting the maximum rate. TEA had little effect on the rate of hydrolysis of the 5'-phosphodiester bond of GGD5. The high apparent activation enthalpy and entropy for SJH along with the effect of TEA on these parameters imply that conversion of an inactive form of E1:12345 to an active conformation accompanies enhanced occupation of the transition state as the temperature is raised to that for maximum SJH. Analytical modeling indicates that either a two-state model (open and disordered, with open being active) or a three-state model (compact, open, and disordered) could account for the temperature dependence of kSJH. However, the three-state model is clearly preferable, since it does not require that the activation parameters

  2. c-Abl Tyrosine Kinase Adopts Multiple Active Conformational States in Solution

    2016-01-01

    Protein tyrosine kinases of the Abl family have diverse roles in normal cellular regulation and drive several forms of leukemia as oncogenic fusion proteins. In the crystal structure of the inactive c-Abl kinase core, the SH2 and SH3 domains dock onto the back of the kinase domain, resulting in a compact, assembled state. This inactive conformation is stabilized by the interaction of the myristoylated N-cap with a pocket in the C-lobe of the kinase domain. Mutations that perturb these intramolecular interactions result in kinase activation. Here, we present X-ray scattering solution structures of multidomain c-Abl kinase core proteins modeling diverse active states. Surprisingly, the relative positions of the regulatory N-cap, SH3, and SH2 domains in an active myristic acid binding pocket mutant (A356N) were virtually identical to those of the assembled wild-type kinase core, indicating that Abl kinase activation does not require dramatic reorganization of the downregulated core structure. In contrast, the positions of the SH2 and SH3 domains in a clinically relevant imatinib-resistant gatekeeper mutant (T315I) appear to be reconfigured relative to their positions in the wild-type protein. Our results demonstrate that c-Abl kinase activation can occur either with (T315I) or without (A356N) global allosteric changes in the core, revealing the potential for previously unrecognized signaling diversity. PMID:27166638

  3. Conformation-activity studies on the interaction of berberine with acetylcholinesterase:Physical chemistry approach

    Jin Xiang; Changping Yu; Fang Yang; Ling Yang; Hong Ding

    2009-01-01

    Berberine has been reported as an acetylcholinesterase (AChE) inhibitor.With significantly low cytotoxicity,berberine will be developed for the clinical treatment of Alzheimer disease (AD) with higher efficacy and fewer side effects.This work investigated the structure change events of AChE that occur during the interaction with berberine by isothermal titration calorimetry (ITC),fluorescence titration,and circular dichroism (CD).The results show that the binding of berberine to AChE is mainly driven by a favorable entropy increase with a less weak affinity.Berberine causes a loss in enzymatic activity at a concentration much below the concentration which gradually exposed the tryptophan residues to a more hydrophilic environment and unfolded the protein,which indicates that the inhibition of AChE with berberine includes the main contributions of interaction and minor conformation change of the protein induced by the alkaloid.

  4. Conformational study reveals amino acid residues essential for hemagglutinating and anti-proliferative activities of Clematis montana lectin.

    Lu, Bangmin; Zhang, Bin; Qi, Wei; Zhu, Yanan; Zhao, Yan; Zhou, Nan; Sun, Rong; Bao, Jinku; Wu, Chuanfang

    2014-11-01

    Clematis montana lectin (CML), a novel mannose-binding lectin purified from C. montana Buch.-Ham stem (Ranunculaceae), has been proved to have hemagglutinating activity in rabbit erythrocytes and apoptosis-inducing activity in tumor cells. However, the biochemical properties of CML have not revealed and its structural information still needs to be elucidated. In this study, it was found that CML possessed quite good thermostability and alkaline resistance, and its hemagglutinating activity was bivalent metal cation dependent. In addition, hemagglutination test and fluorescence spectroscopy proved that GuHCl, urea, and sodium dodecyl sulfate could change the conformation of CML and further caused the loss of hemagglutination activity. Moreover, the changes of fluorescence spectrum indicated that the tryptophan (Trp) microenvironment conversion might be related to the conformation and bioactivities of CML. In addition, it was also found that Trp residues, arginine (Arg) residues, and sulfhydryl were important for the hemagglutinating activity of CML, but only Trp was proved to be crucial for the CML conformation. Furthermore, the Trp, Arg, and sulfhydryl-modified CML exhibited 97.17%, 76.99%, and 49.64% loss of its anti-proliferative activity, respectively, which was consistent with the alterations of its hemagglutinating activity. Given these findings, Trp residues on the surface of CML are essential for the active center to form substrate-accessible conformation and suitable environment for carbohydrate binding. PMID:25239139

  5. Interconversion of Active and Inactive Conformations of Urokinase-Type Plasminogen Activator

    Liu, Zhuo; Kromann-Hansen, Tobias; Lund, Ida K;

    2012-01-01

    The catalytic activity of serine proteases depends on a salt-bridge between the amino group of residue 16 and the side chain of Asp194. The salt-bridge stabilizes the oxyanion hole and the S1 specificity pocket of the protease. Some serine proteases exist in only partially active forms, in which...... the amino group of residue 16 is exposed to the solvent. Such a partially active state is assumed by a truncated form of the murine urokinase-type plasminogen activator (muPA), consisting of residues 16-243. Here we investigated the allosteric interconversion between partially active states and the...

  6. The balance of flexibility and rigidity in the active site residues of hen egg white lysozyme

    Qi Jian-Xun; Jiang Fan

    2011-01-01

    The crystallographic temperature factors (B factor) of individual atoms contain important information about the thermal motion of the atoms in a macromolecule. Previously the theory of flexibility of active site has been established based on the observation that the enzyme activity is sensitive to low concentration denaturing agents. It has been found that the loss of enzyme activity occurs well before the disruption of the three-dimensional structural scaffold of the enzyme. To test the theory of conformational flexibility of enzyme active site, crystal structures were perturbed by soaking in low concentration guanidine hydrochloride solutions. It was found that many lysozyme crystals tested could still diffract until the concentration of guanidine hydrochloride reached 3 M. It was also found that the B factors averaged over individually collected data sets were more accurate. Thus it suggested that accurate measurement of crystal temperature factors could be achieved for medium-high or even medium resolution crystals by averaging over multiple data sets. Furthermore, we found that the correctly predicted active sites included not only the more flexible residues, but also some more rigid residues. Both the flexible and the rigid residues in the active site played an important role in forming the active site residue network, covering the majority of the substrate binding residues. Therefore, this experimental prediction method may be useful for characterizing the binding site and the function of a protein, such as drug targeting.

  7. Conformational flexibility of aspartame.

    Toniolo, Claudio; Temussi, Pierandrea

    2016-05-01

    L-Aspartyl-L-phenylalanine methyl ester, better known as aspartame, is not only one of the most used artificial sweeteners, but also a very interesting molecule with respect to the correlation between molecular structure and taste. The extreme conformational flexibility of this dipeptide posed a huge difficulty when researchers tried to use it as a lead compound to design new sweeteners. In particular, it was difficult to take advantage of its molecular model as a mold to infer the shape of the, then unknown, active site of the sweet taste receptor. Here, we follow the story of the 3D structural aspects of aspartame from early conformational studies to recent docking into homology models of the receptor. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 376-384, 2016. PMID:27038223

  8. Conformational Isomerism Can Limit Antibody Catalysis

    Debler, E.W.; Muller, R.; Hilvert, D.; Wilson, I.A.

    2009-05-14

    Ligand binding to enzymes and antibodies is often accompanied by protein conformational changes. Although such structural adjustments may be conducive to enzyme catalysis, much less is known about their effect on reactions promoted by engineered catalytic antibodies. Crystallographic and pre-steady state kinetic analyses of antibody 34E4, which efficiently promotes the conversion of benzisoxazoles to salicylonitriles, show that the resting catalyst adopts two interconverting active-site conformations, only one of which is competent to bind substrate. In the predominant isomer, the indole side chain of Trp{sup L91} occupies the binding site and blocks ligand access. Slow conformational isomerization of this residue, on the same time scale as catalytic turnover, creates a deep and narrow binding site that can accommodate substrate and promote proton transfer using Glu{sup H50} as a carboxylate base. Although 34E4 is among the best catalysts for the deprotonation of benzisoxazoles, its efficiency appears to be significantly limited by this conformational plasticity of its active site. Future efforts to improve this antibody might profitably focus on stabilizing the active conformation of the catalyst. Analogous strategies may also be relevant to other engineered proteins that are limited by an unfavorable conformational pre-equilibrium.

  9. SU-C-BRE-01: 3D Conformal Micro Irradiation Results of Four Treatment Sites for Preclinical Small Animal and Clinical Treatment Plans

    Price, S; Yaddanapudi, S [Washington University School of Medicine, Saint Louis, MO (United States); Rangaraj, D; Izaguirre, E [Scott and White Hospital, Temple, TX (United States)

    2014-06-15

    Purpose: Small animal irradiation can provide preclinical insights necessary for clinical advancement. In order to provide clinically relevant data, these small animal irradiations must be designed such that the treatment methods and results are comparable to clinical protocols, regardless of variations in treatment size and modality. Methods: Small animal treatments for four treatment sites (brain, liver, lung and spine) were investigated, accounting for change in treatment energy and target size. Up to five orthovoltage (300kVp) beams were used in the preclinical treatments, using circular, square, and conformal tungsten apertures, based on the treatment site. Treatments were delivered using the image guided micro irradiator (microIGRT). The plans were delivered to a mouse sized phantom and dose measurements in axial and coronal planes were performed using radiochromic film. The results of the clinical and preclinical protocols were characterized in terms of conformality number, CTV coverage, dose nonuniformity ratio, and organ at risk sparing. Results: Preclinical small animal treatment conformality was within 1–16% of clinical results for all treatment sites. The volume of the CTV receiving 100% of the prescription dose was typically within 10% of clinical values. The dose non-uniformity was consistently higher for preclinical treatments compared to clinical treatments, indicating hot spots in the target. The ratios of the mean dose in the target to the mean dose in an organ at risk were comparable if not better for preclinical versus clinical treatments. Finally, QUANTEC dose constraints were applied and the recommended morbidity limits were satisfied in each small animal treatment site. Conclusion: We have shown that for four treatment sites, preclinical 3D conformal small animal treatments can be clinically comparable if clinical protocols are followed. Using clinical protocols as the standard, preclinical irradiation methods can be altered and iteratively

  10. Conformable actively multiplexed high-density surface electrode array for brain interfacing

    Rogers, John; Kim, Dae-Hyeong; Litt, Brian; Viventi, Jonathan

    2015-01-13

    Provided are methods and devices for interfacing with brain tissue, specifically for monitoring and/or actuation of spatio-temporal electrical waveforms. The device is conformable having a high electrode density and high spatial and temporal resolution. A conformable substrate supports a conformable electronic circuit and a barrier layer. Electrodes are positioned to provide electrical contact with a brain tissue. A controller monitors or actuates the electrodes, thereby interfacing with the brain tissue. In an aspect, methods are provided to monitor or actuate spatio-temporal electrical waveform over large brain surface areas by any of the devices disclosed herein.

  11. The relationship between MRP1 activities and its NBD conformational changes

    2004-01-01

    MIANS,a sulfhydryl-reactive fluorescence,was used to label the cysteines of MRP1(multidrug resistance protein),and the results indicated that an increase in fluorescence intensity and a large emission blue shift took place after two Cys residues of MRP1 reacted with MIANS,which demonstrated that labeled Cys residues in MRP1 reside in a relatively hydrophobic environment.The experimental results obtained from fluorescence resonance energy transfer further uncover that two Cys residues of MRP1 modified by MIANS located in the vicinity of its NBDs,of which one lies close to NBD1,and the other near NBD2.ATP,ADP and anticancer drugs can all reduce the rate of reaction of MRP1 with MIANS.The collisional quenchers,acrylamide,I-,and Cs+ were used to assess local environments of MIANS bound to MRP1 and the results showed that the region around the MIANS-labeled cysteine is positively charged.Both MIANS and NEM,which are sulfhydryl-reactive reagents,inhibited MRP1 ATPase activity,whereas anticancer drugs activated it.These results demonstrated that all nucleotides and drugs could induce changes in conformation of the NBDs in MRP1.Nucleotides can bind directly to NBDs,but drugs may react first with TMDs,which in turn alters the accessibility of the two Cys residues bound by MIANS and affects MRP1 ATPase activity,which is coupled with the transport of its substrates.Taken together,the above experimental results provide direct evidence for further study on the coupling of translocation of the transported species to hydrolysis of ATP in MRP1.

  12. A chlorite mineral surface actively drives the deposition of DNA molecules in stretched conformations

    Muscovite mica is commonly used to immobilize DNA molecules onto a flat surface. This method, however, requires either the use of divalent cations in the buffer solution or the chemical modification of the surface. Here we show that DNA molecules have different binding affinities and assume different conformations when adsorbed to different layered minerals. In particular, the effect of biotite, muscovite, talc, brucite and chlorite upon DNA binding is investigated. Using atomic force microscopy it is possible to quantify the amount of DNA deposited onto a flat surface and it is experimentally confirmed that biotite, talc and brucite have a much higher affinity than muscovite (7-, 20- and 25-fold more volume of DNA deposited, respectively). The deposition of DNA onto chlorite presents areas (brucite-like) with high DNA coverage and areas (mica-like) where DNA molecules are absent. We regularly observed isolated DNA molecules that became stretched across these regions of low affinity. The stretching is not induced by the deposition procedure but is driven by the surface potential gradient between brucite-like and mica-like regions in chlorite. The active stretching of DNA on chlorite is a clear indication of the technological potential carried by these materials when used as substrates for biomolecules

  13. Mechanochemical coupling in the myosin motor domain. I. Insights from equilibrium active-site simulations.

    Haibo Yu

    2007-02-01

    Full Text Available Although the major structural transitions in molecular motors are often argued to couple to the binding of Adenosine triphosphate (ATP, the recovery stroke in the conventional myosin has been shown to be dependent on the hydrolysis of ATP. To obtain a clearer mechanistic picture for such "mechanochemical coupling" in myosin, equilibrium active-site simulations with explicit solvent have been carried out to probe the behavior of the motor domain as functions of the nucleotide chemical state and conformation of the converter/relay helix. In conjunction with previous studies of ATP hydrolysis with different active-site conformations and normal mode analysis of structural flexibility, the results help establish an energetics-based framework for understanding the mechanochemical coupling. It is proposed that the activation of hydrolysis does not require the rotation of the lever arm per se, but the two processes are tightly coordinated because both strongly couple to the open/close transition of the active site. The underlying picture involves shifts in the dominant population of different structural motifs as a consequence of changes elsewhere in the motor domain. The contribution of this work and the accompanying paper [] is to propose the actual mechanism behind these "population shifts" and residues that play important roles in the process. It is suggested that structural flexibilities at both the small and large scales inherent to the motor domain make it possible to implement tight couplings between different structural motifs while maintaining small free-energy drops for processes that occur in the detached states, which is likely a feature shared among many molecular motors. The significantly different flexibility of the active site in different X-ray structures with variable level arm orientations supports the notation that external force sensed by the lever arm may transmit into the active site and influence the chemical steps (nucleotide

  14. Promoter proximal polyadenylation sites reduce transcription activity

    Andersen, Pia Kjølhede; Lykke-Andersen, Søren; Jensen, Torben Heick

    2012-01-01

    transcription requires promoter proximity, as demonstrated using artificial constructs and supported by a genome-wide data set. Importantly, transcription down-regulation can be recapitulated in a gene context devoid of splice sites by placing a functional bona fide pA site/transcription terminator within ∼500...

  15. Substrate Shuttling Between Active Sites of Uroporphyrinogen Decarboxylase in Not Required to Generate Coproporphyrinogen

    Phillips, J.; Warby, C; Whitby, F; Kushner, J; Hill, C

    2009-01-01

    Uroporphyrinogen decarboxylase (URO-D; EC 4.1.1.37), the fifth enzyme of the heme biosynthetic pathway, is required for the production of heme, vitamin B12, siroheme, and chlorophyll precursors. URO-D catalyzes the sequential decarboxylation of four acetate side chains in the pyrrole groups of uroporphyrinogen to produce coproporphyrinogen. URO-D is a stable homodimer, with the active-site clefts of the two subunits adjacent to each other. It has been hypothesized that the two catalytic centers interact functionally, perhaps by shuttling of reaction intermediates between subunits. We tested this hypothesis by construction of a single-chain protein (single-chain URO-D) in which the two subunits were connected by a flexible linker. The crystal structure of this protein was shown to be superimposable with wild-type activity and to have comparable catalytic activity. Mutations that impaired one or the other of the two active sites of single-chain URO-D resulted in approximately half of wild-type activity. The distributions of reaction intermediates were the same for mutant and wild-type sequences and were unaltered in a competition experiment using I and III isomer substrates. These observations indicate that communication between active sites is not required for enzyme function and suggest that the dimeric structure of URO-D is required to achieve conformational stability and to create a large active-site cleft.

  16. Superiority of the S,S conformation in diverse pharmacological processes: Intestinal transport and entry inhibition activity of novel anti-HIV drug lead.

    Fanous, Joseph; Swed, Avi; Joubran, Salim; Hurevich, Mattan; Britan-Rosich, Elena; Kotler, Moshe; Gilon, Chaim; Hoffman, Amnon

    2015-11-30

    Chirality is an important aspect in many pharmacological processes including drug transport and metabolism. The current investigation examined the stereospecific transport and entry inhibitory activity of four diastereomers derived from a small (macrocyclic) molecule that has two chiral centers. These molecules were designed to mimic the interaction between CD4 and gp120 site of HIV-1 and thereby to function as entry inhibitor(s). Intestinal permeability was assessed by ex-vivo model using excised rat intestine mounted in side-by-side diffusion chambers. The entry inhibitory activity was monitored using indicator HeLa-CD4-LTR-beta-gal cells (MAGI assay). The (S/S) diastereomer, named CG-1, exhibited superiority in both unrelated tested biological processes: (I) high transport through the intestine and (II) entry inhibition activity (in the low μM range). The permeability screening revealed a unique transporter-mediated absorption pathway of CG-1, suggesting a significant role of the molecule's conformation on the mechanism of intestinal absorption. Here we highlight that only the S,S enantiomer (CG-1) has both (I) promising anti HIV-1 entry inhibitory properties and (II) high transporter mediated intestinal permeability. Hence we suggest preference in pharmacological processes to the S,S conformation. This report augments the knowledge regarding stereoselectivity in receptor mediated and protein-protein interaction processes. PMID:26392249

  17. Stabilizing a Flexible Interdomain Hinge Region Harboring the SMB Binding Site Drives uPAR into Its Closed Conformation

    Zhao, Baoyu; Gandhi, Sonu; Yuan, Cai;

    2015-01-01

    domains is inherently flexible and binding of uPA drives uPAR into its closed conformation, which presents the higher affinity state for vitronectin thus providing an allosteric regulatory mechanism. Using a new class of epitope mapped monoclonal anti-uPAR antibodies, we now demonstrate...

  18. Activities on the site during construction phase

    A survey is given of the work done on the site from site-opening till turn over of the plant to the client. After a short introduction to time schedules, manpower on site, site facilities and civil work and constructions, the commissioning and trial operation phase is discussed in detail. This phase begins with finishing the assembly of individual systems and components and ends with the trial operation and the acceptance measurement. During this period the subsystems are started-up in a useful sequence, first from cold, then from hot conditions and are finally operated as a total with nuclear energy. The single steps are: a) commissioning of indivudal systems; b) hot functional test 1 (without fuels) c) baseline inspection at the reactor pressure vessel; d) hot functional test 2 (with fuels); e) preparation for first criticality; f) postcriticality test program; g) trial operation: h) acceptance measurement. (HP)

  19. Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

    Fyfe, Cameron D.; Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja [University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom); Roszak, Aleksander W. [University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom); University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom); Cogdell, Richard J.; Wall, Daniel M.; Burchmore, Richard J. S.; Byron, Olwyn; Walker, Daniel, E-mail: daniel.walker@glasgow.ac.uk [University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom)

    2015-06-30

    The X-ray structure of protease-cleaved E. coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group.

  20. Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

    The X-ray structure of protease-cleaved E. coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group

  1. Water molecule network and active site flexibility of apo protein tyrosine phosphatase 1B

    Pedersen, A.K.; Peters, Günther H.J.; Møller, K.B.;

    2004-01-01

    Protein tyrosine phosphatase 1B (PTP1B) plays a key role as a negative regulator of insulin and leptin signalling and is therefore considered to be an important molecular target for the treatment of type 2 diabetes and obesity. Detailed structural information about the structure of PTP1B, including...... the conformation and flexibility of active-site residues as well as the water-molecule network, is a key issue in understanding ligand binding and enzyme kinetics and in structure-based drug design. A 1.95 Angstrom apo PTP1B structure has been obtained, showing four highly coordinated water molecules...

  2. A conformational analysis of mouse Nalp3 domain structures by molecular dynamics simulations, and binding site analysis.

    Sahoo, Bikash R; Maharana, Jitendra; Bhoi, Gopal K; Lenka, Santosh K; Patra, Mahesh C; Dikhit, Manas R; Dubey, Praveen K; Pradhan, Sukanta K; Behera, Bijay K

    2014-05-01

    Scrutinizing various nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) genes in higher eukaryotes is very important for understanding the intriguing mechanism of the host defense against pathogens. The nucleotide-binding domain (NACHT), leucine-rich repeat (LRR), and pyrin domains (PYD)-containing protein 3 (Nalp3), is an intracellular innate immune receptor and is associated with several immune system related disorders. Despite Nalp3's protective role during a pathogenic invasion, the molecular features and structural organization of this crucial protein is poorly understood. Using comparative modeling and molecular dynamics simulations, we have studied the structural architecture of Nalp3 domains, and characterized the dynamic and energetic parameters of adenosine triphosphate (ATP) binding in NACHT, and pathogen-derived ligands muramyl dipeptide (MDP) and imidazoquinoline with LRR domains. The results suggested that walker A, B and extended walker B motifs were the key ATP binding regions in NACHT that mediate self-oligomerization. The analysis of the binding sites of MDP and imidazoquinoline revealed LRR 7-9 to be the most energetically favored site for imidazoquinoline interaction. However, the binding free energy calculations using the Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) method indicated that MDP is incompatible for activating the Nalp3 molecule in its monomeric form, and suggest its complex interaction with NOD2 or other NLRs accounts for MDP recognition. The high binding affinity of ATP with NACHT was correlated to the experimental data for human NLRs. Our binding site prediction for imidazoquinoline in LRR warrants further investigation via in vivo models. This is the first study that provides ligand recognition in mouse Nalp3 and its spatial structural arrangements. PMID:24595807

  3. Improving the neutral phytase activity from Bacillus amyloliquefaciens DSM 1061 by site-directed mutagenesis.

    Xu, Wei; Shao, Rong; Wang, Zupeng; Yan, Xiuhua

    2015-03-01

    Neutral phytase is used as a feed additive for degradation of anti-nutritional phytate in aquatic feed industry. Site-directed mutagenesis of Bacillus amyloliquefaciens DSM 1061 phytase was performed with an aim to increase its activity. Mutation residues were chosen based on multiple sequence alignments and structure analysis of neutral phytsaes from different microorganisms. The mutation sites on surface (D148E, S197E and N156E) and around the active site (D52E) of phytase were selected. Analysis of the phytase variants showed that the specific activities of mutants D148E and S197E remarkably increased by about 35 and 13% over a temperature range of 40-75 °C at pH 7.0, respectively. The k cat of mutants D148E and S197E were 1.50 and 1.25 times than that of the wild-type phytase, respectively. Both D148E and S197E showed much higher thermostability than that of the wild-type phytase. However, mutants N156E and D52E led to significant loss of specific activity of the enzyme. Structural analysis revealed that these mutations may affect conformation of the active site of phytase. The present mutant phytases D148E and S197E with increased activities and thermostabilities have application potential as additives in aquaculture feed. PMID:25613522

  4. Intron-encoded endonuclease I-TevI binds as a monomer to effect sequential cleavage via conformational changes in the td homing site.

    Mueller, J E; Smith, D; Bryk, M; Belfort, M

    1995-11-15

    I-TevI, the intron-encoded endonuclease from the thymidylate synthase (td) gene of bacteriophage T4, binds its DNA substrate across the minor groove in a sequence-tolerant fashion. We demonstrate here that the 28 kDa I-TevI binds the extensive 37 bp td homing site as a monomer and significantly distorts its substrate. In situ cleavage assays and phasing analyses indicate that upon nicking the bottom strand of the td homing site, I-TevI induces a directed bend of 38 degrees towards the major groove near the cleavage site. Formation of the bent I-TevI-DNA complex is proposed to promote top-strand cleavage of the homing site. Furthermore, reductions in the degree of distortion and in the efficiency of binding base-substitution variants of the td homing site indicate that sequences flanking the cleavage site contribute to the I-TevI-induced conformational change. These results, combined with genetic, physical and computer-modeling studies, form the basis of a model, wherein I-TevI acts as a hinged monomer to induce a distortion that widens the minor groove, facilitating access to the top-strand cleavage site. The model is compatible with both unmodified DNA and glucosylated hydroxymethylcytosine-containing DNA, as exists in the T-even phages. PMID:8521829

  5. Safety Oversight of Decommissioning Activities at DOE Nuclear Sites

    The Defense Nuclear Facilities Safety Board (Board) is an independent federal agency established by Congress in 1988 to provide nuclear safety oversight of activities at U.S. Department of Energy (DOE) defense nuclear facilities. The activities under the Board's jurisdiction include the design, construction, startup, operation, and decommissioning of defense nuclear facilities at DOE sites. This paper reviews the Board's safety oversight of decommissioning activities at DOE sites, identifies the safety problems observed, and discusses Board initiatives to improve the safety of decommissioning activities at DOE sites. The decommissioning of former defense nuclear facilities has reduced the risk of radioactive material contamination and exposure to the public and site workers. In general, efforts to perform decommissioning work at DOE defense nuclear sites have been successful, and contractors performing decommissioning work have a good safety record. Decommissioning activities have recently been completed at sites identified for closure, including the Rocky Flats Environmental Technology Site, the Fernald Closure Project, and the Miamisburg Closure Project (the Mound site). The Rocky Flats and Fernald sites, which produced plutonium parts and uranium materials for defense needs (respectively), have been turned into wildlife refuges. The Mound site, which performed R and D activities on nuclear materials, has been converted into an industrial and technology park called the Mound Advanced Technology Center. The DOE Office of Legacy Management is responsible for the long term stewardship of these former EM sites. The Board has reviewed many decommissioning activities, and noted that there are valuable lessons learned that can benefit both DOE and the contractor. As part of its ongoing safety oversight responsibilities, the Board and its staff will continue to review the safety of DOE and contractor decommissioning activities at DOE defense nuclear sites

  6. Savannah River Site prioritization of transition activities

    Effective management of SRS conversion from primarily a production facility to other missions (or Decontamination and Decommissioning (D ampersand D)) requires a systematic and consistent method of prioritizing the transition activities. This report discusses the design of a prioritizing method developed to achieve systematic and consistent methods of prioritizing these activities

  7. Savannah River Site prioritization of transition activities

    Finley, R.H.

    1993-11-01

    Effective management of SRS conversion from primarily a production facility to other missions (or Decontamination and Decommissioning (D&D)) requires a systematic and consistent method of prioritizing the transition activities. This report discusses the design of a prioritizing method developed to achieve systematic and consistent methods of prioritizing these activities.

  8. Molecular dynamics study of the effect of active site protonation on Helicobacter pylori 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase.

    Tekpinar, Mustafa; Yildirim, Ahmet; Wassenaar, Tsjerk A

    2015-12-01

    The protein 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is involved in the quorum sensing of several bacterial species, including Helicobacter pylori. In particular, these bacteria depend on MTAN for synthesis of vitamin K2 homologs. The residue D198 in the active site of MTAN seems to be of crucial importance, by acting as a hydrogen-bond acceptor for the ligand. In this study, we investigated the conformation and dynamics of apo and holo H. pylori MTAN (HpMTAN), and assessed the effect of protonation of D198 by use of molecular dynamics simulations. Our results show that protonation of the active site of HpMTAN can cause a conformational transition from a closed state to an open state even in the absence of substrate, via inter-chain mechanical coupling. PMID:26254213

  9. Juvenile Delinquency and Conformism

    Patacchini, Eleonora; Zenou, Yves

    2010-01-01

    This paper studies whether conformism behavior affects individual outcomes in crime. We present a social network model of peer effects with ex-ante heterogeneous agents and show how conformism and deterrence affect criminal activities. We then bring the model to the data by using a very detailed dataset of adolescent friendship networks. A novel social network-based empirical strategy allows us to identify peer effects for different types of crimes. We find that conformity plays an important ...

  10. Conformational characterization of a single-site mutant of murine epidermal growth factor (EGF) by 1H NMR provides evidence that leucine-47 is involved in the interactions with the EGF receptor

    Epidermal growth factor (EGF) is a small protein containing 53 amino acids and three disulfide bonds. There is significant current interest in structure-function relationships in EGF and EGF-like proteins, including the homologous type-α transforming growth factors. The Leu-47 residue of murine EGF (mEGF) is one of several that are strongly conserved among the EGF-like growth factors, suggesting that it may contribute to the active site of mEGF. In several different binding assays, the activity of the mutant analog in which Leu-47 is replaced by Ser ([Ser47]mEGF) ranges from 8 to 18 times weaker than that of wild-type mEGF. The NMR data summarized in this paper demonstrate that the significant differences in the binding activities of wild-type and [Ser47]mEGF cannot be attributed to structural changes remote from the three-dimensional site of mutation. The only minor conformational changes that are indicated by these data involve side chains of residues proximal to Leu-47 in the three-dimensional structure. Therefore, Leu-47 and/or residues spatially adjacent to Leu-47 constitute part of the active site of mEGF

  11. Conformal house

    Ryttov, Thomas Aaby; Sannino, Francesco

    2010-01-01

    fixed point. As a consistency check we recover the previously investigated bounds of the conformal windows when restricting to a single matter representation. The earlier conformal windows can be imagined to be part now of the new conformal house. We predict the nonperturbative anomalous dimensions at...... the infrared fixed points. We further investigate the effects of adding mass terms to the condensates on the conformal house chiral dynamics and construct the simplest instanton induced effective Lagrangian terms...

  12. Active sites of two orthologous cytochromes P450 2E1: Differences revealed by spectroscopic methods

    Cytochromes P450 2E1 of human and minipig origin were examined by absorption spectroscopy under high hydrostatic pressure and by resonance Raman spectroscopy. Human enzyme tends to denature to the P420 form more easily than the minipig form; moreover, the apparent compressibility of the heme active site (as judged from a redshift of the absorption maximum with pressure) is greater than that of the minipig counterpart. Relative compactness of the minipig enzyme is also seen in the Raman spectra, where the presence of planar heme conformation was inferred from band positions characteristic of the low-spin heme with high degree of symmetry. In this respect, the CYP2E1 seems to be another example of P450 conformational heterogeneity as shown, e.g., by Davydov et al. for CYP3A4 [Biochem. Biophys. Res. Commun. 312 (2003) 121-130]. The results indicate that the flexibility of the CYP active site is likely one of its basic structural characteristics

  13. Discovery and Characterization of a Cell-Permeable, Small-Molecule c-Abl Kinase Activator that Binds to the Myristoyl Binding Site

    Yang, Jingsong; Campobasso, Nino; Biju, Mangatt P.; Fisher, Kelly; Pan, Xiao-Qing; Cottom, Josh; Galbraith, Sarah; Ho, Thau; Zhang, Hong; Hong, Xuan; Ward, Paris; Hofmann, Glenn; Siegfried, Brett; Zappacosta, Francesca; Washio, Yoshiaki; Cao, Ping; Qu, Junya; Bertrand, Sophie; Wang, Da-Yuan; Head, Martha S.; Li, Hu; Moores, Sheri; Lai, Zhihong; Johanson, Kyung; Burton, George; Erickson-Miller, Connie; Simpson, Graham; Tummino, Peter; Copeland, Robert A.; Oliff, Allen (GSKPA)

    2014-10-02

    c-Abl kinase activity is regulated by a unique mechanism involving the formation of an autoinhibited conformation in which the N-terminal myristoyl group binds intramolecularly to the myristoyl binding site on the kinase domain and induces the bending of the {alpha}I helix that creates a docking surface for the SH2 domain. Here, we report a small-molecule c-Abl activator, DPH, that displays potent enzymatic and cellular activity in stimulating c-Abl activation. Structural analyses indicate that DPH binds to the myristoyl binding site and prevents the formation of the bent conformation of the {alpha}I helix through steric hindrance, a mode of action distinct from the previously identified allosteric c-Abl inhibitor, GNF-2, that also binds to the myristoyl binding site. DPH represents the first cell-permeable, small-molecule tool compound for c-Abl activation.

  14. Mechanical Control of ATP Synthase Function: Activation Energy Difference between Tight and Loose Binding Sites

    Beke-Somfai, Tamás

    2010-01-26

    Despite exhaustive chemical and crystal structure studies, the mechanistic details of how FoF1-ATP synthase can convert mechanical energy to chemical, producing ATP, are still not fully understood. On the basis of quantum mechanical calculations using a recent highresolution X-ray structure, we conclude that formation of the P-O bond may be achieved through a transition state (TS) with a planar PO3 - ion. Surprisingly, there is a more than 40 kJ/mol difference between barrier heights of the loose and tight binding sites of the enzyme. This indicates that even a relatively small change in active site conformation, induced by the γ-subunit rotation, may effectively block the back reaction in βTP and, thus, promote ATP. © 2009 American Chemical Society.

  15. The Surface Groups and Active Site of Fibrous Mineral Materials

    DONG Fa-qin; WAN Pu; FENG Qi-ming; SONG Gong-bao; PENG Tong-jiang; LI Ping; LI Guo-wu

    2004-01-01

    The exposed and transformed groups of fibrous brucite,wollastonite,chrysotile asbestos,sepiolite,palygorskite,clinoptilolite,crocidolite and diatomaceous earth mineral materials are analyzed by IR spectra after acid and alikali etching,strong mechanical and polarity molecular interaction.The results show the active sites concentrate on the ends in stick mineral materials and on the defect or hole edge in pipe mineral materials.The inside active site of mineral materials plays a main role in small molecular substance.The shape of minerals influence their distribution and density of active site.The strong mechanical impulsion and weak chemical force change the active site feature of minerals,the powder process enables minerals exposed more surface group and more combined types.The surface processing with the small polarity molecular or the brand of middle molecular may produce ionation and new coordinate bond,and change the active properties and level of original mineral materials.

  16. Membrane phospholipid augments cytochrome P4501a enzymatic activity by modulating structural conformation during detoxification of xenobiotics.

    Manik C Ghosh

    Full Text Available Cytochrome P450 is a superfamily of membrane-bound hemoprotein that gets involved with the degradation of xenobiotics and internal metabolites. Accumulated body of evidence indicates that phospholipids play a crucial role in determining the enzymatic activity of cytochrome P450 in the microenvironment by modulating its structure during detoxification; however, the structure-function relationship of cytochrome P4501A, a family of enzymes responsible for degrading lipophilic aromatic hydrocarbons, is still not well defined. Inducibility of cytochrome P4501A in cultured catfish hepatocytes in response to carbofuran, a widely used pesticide around the world, was studied earlier in our laboratory. In this present investigation, we observed that treating catfish with carbofuran augmented total phospholipid in the liver. We examined the role of phospholipid on the of cytochrome P4501A-marker enzyme which is known as ethoxyresorufin-O-deethylase (EROD in the context of structure and function. We purified the carbofuran-induced cytochrome P4501A protein from catfish liver. Subsequently, we examined the enzymatic activity of purified P4501A protein in the presence of phospholipid, and studied how the structure of purified protein was influenced in the phospholipid environment. Membrane phospholipid appeared to accelerate the enzymatic activity of EROD by changing its structural conformation and thus controlling the detoxification of xenobiotics. Our study revealed the missing link of how the cytochrome P450 restores its enzymatic activity by changing its structural conformation in the phospholipid microenvironment.

  17. Relationship between Oversulfation and Conformation of Low and High Molecular Weight Fucoidans and Evaluation of Their in Vitro Anticancer Activity

    Myoung Lae Cho

    2010-12-01

    Full Text Available Low and high molecular weight fucoidans (F5-30K and F>30K were chemically modified through the addition of sulfate groups, and the effect of oversulfation on the in vitro anticancer activity was investigated. After the addition of sulfate groups, a considerable increase of 35.5 to 56.8% was observed in the sulfate content of the F5-30K fraction, while the sulfate content of the F>30K fraction increased to a lesser extent (from 31.7 to 41.2%. Significant differences in anticancer activity were observed between the oversulfated F5–30K and F>30K fractions, with activities of 37.3–68.0% and 20.6–35.8%, respectively. This variation in the anticancer activity of oversulfated fucoidan derivatives was likely due to differences in their sulfate content. The results suggest that the molecular conformation of these molecules is closely related to the extent of sulfation in the fucan backbones and that the sulfates are preferably substituted when the fucoidan polymers are in a loose molecular conformation.

  18. The active site behaviour of electrochemically synthesised gold nanomaterials.

    Plowman, Blake J; O'Mullane, Anthony P; Bhargava, Suresh K

    2011-01-01

    Even though gold is the noblest of metals, a weak chemisorber and is regarded as being quite inert, it demonstrates significant electrocatalytic activity in its nanostructured form. It is demonstrated here that nanostructured and even evaporated thin films of gold are covered with active sites which are responsible for such activity. The identification of these sites is demonstrated with conventional electrochemical techniques such as cyclic voltammetry as well as a large amplitude Fourier transformed alternating current (FT-ac) method under acidic and alkaline conditions. The latter technique is beneficial in determining if an electrode process is either Faradaic or capacitive in nature. The observed behaviour is analogous to that observed for activated gold electrodes whose surfaces have been severely disrupted by cathodic polarisation in the hydrogen evolution region. It is shown that significant electrochemical oxidation responses occur at discrete potential values well below that for the formation of the compact monolayer oxide of bulk gold and are attributed to the facile oxidation of surface active sites. Several electrocatalytic reactions are explored in which the onset potential is determined by the presence of such sites on the surface. Significantly, the facile oxidation of active sites is used to drive the electroless deposition of metals such as platinum, palladium and silver from their aqueous salts on the surface of gold nanostructures. The resultant surface decoration of gold with secondary metal nanoparticles not only indicates regions on the surface which are rich in active sites but also provides a method to form interesting bimetallic surfaces. PMID:22455038

  19. Spacer conformation in biologically active molecules. Part 2. Structure and conformation of 4-[2-(diphenylmethylamino)ethyl]-1-(2-methoxyphenyl) piperazine and its diphenylmethoxy analog—potential 5-HT 1A receptor ligands

    Karolak-Wojciechowska, J.; Fruziński, A.; Czylkowski, R.; Paluchowska, M. H.; Mokrosz, M. J.

    2003-09-01

    As a part of studies on biologically active molecule structures with aliphatic linking chain, the structures of 4-[2-diphenylmethylamino)ethyl]-1-(2-methoxyphenyl)piperazine dihydrochloride ( 1) and 4-[2-diphenylmethoxy)ethyl]-1-(2-methoxyphenyl)piperazine fumarate ( 2) have been reported. In both compounds, four atomic non-all-carbons linking chains (N)C-C-X-C are present. The conformation of that linking spacer depends on the nature of the X-atom. The preferred conformation for chain with XNH has been found to be fully extended while for that with XO—the bend one. It was confirmed by conformational calculations (strain energy distribution and random search) and crystallographic data, including statistics from CCDC.

  20. Structural studies of conformational changes of proteins upon phosphorylation: Structures of activated CheY, CheY-N16-FliM complex, and AAA {sup +} ATPase domain of NtrC1 in both inactive and active states

    Lee, Seok-Yong

    2003-04-10

    Protein phosphorylation is a general mechanism for signal transduction as well as regulation of cellular function. Unlike phosphorylation in eukaryotic systems that uses Ser/Thr for the sites of modification, two-component signal transduction systems, which are prevalent in bacteria, archea, and lower eukaryotes, use an aspartate as the site of phosphorylation. Two-component systems comprise a histidine kinase and a receiver domain. The conformational change of the receiver domain upon phosphorylation leads to signal transfer to the downstream target, a process that had not been understood well at the molecular level. The transient nature of the phospho-Asp bond had made structural studies difficult. The discovery of an excellent analogue for acylphosphate, BeF{sub 3}{sup -}, enabled structural study of activated receiver domains. The structure of activated Chemotaxis protein Y (CheY) was determined both by NMR spectroscopy and X-ray crystallography. These structures revealed the molecular basis of the conformational change that is coupled to phosphorylation. Phosphorylation of the conserved Asp residue in the active site allows hydrogen bonding of the T87 O{gamma} to phospho-aspartate, which in turn leads to the rotation of Y106 into the ''in'' position (termed Y-T coupling). The structure of activated CheY complexed with the 16 N-terminal residues of FliM (N16-FliM), its target, was also determined by X-ray crystallography and confirmed the proposed mechanism of activation (Y-T coupling). First, N16-FliM binds to the region on CheY that undergoes a significant conformational change. Second, the ''in'' position of Y106 presents a better binding surface for FliM because the sidechain of Y106 in the inactive form of CheY (''out'' position) sterically interferes with binding of N16-FliM. In addition to confirmation of Y-T coupling, the structure of the activated CheY-N16-FliM complex suggested that the N16

  1. Fluorescence energy transfer studies on the active site of papain

    Henes, Jill B.; Briggs, Martha S.; Sligar, Stephen G.; Fruton, Joseph S.

    1980-01-01

    Measurements have been performed of the excited-state lifetimes and fluorescence yields of papain tryptophan units when acyl derivatives of Phe-glycinal are bound at the active site of the enzyme. The enhancement of tryptophan fluorescence in complexes of papain with the acetyl or benzyloxycarbonyl derivatives is not stereospecific with respect to the configuration of the phenylalanyl residue, and the L and D isomers are equally effective as active-site-directed inhibitors of papain action. E...

  2. Key messages from active CO2 storage sites

    Wildenborg, T.; Wollenweber, J. [TNO, Princetonlaan 6, 3584 CB Utrecht (Netherlands); Chadwick, A. [BGS, Environmental Science Centre, Keyworth, Nottingham, NG12 5GG (United Kingdom); Deflandre, J.P. [IFP Energies nouvelles, 1-4 avenue de Bois Preau, 92852 Rueil-Malmaison (France); Eiken, O. [Statoil Research Centre, Rotvoll, Arkitekt Ebbells vei 10, 7005 Trondheim (Norway); Mathieson, A. [BP, Alternative Energy, Chertsey Road, Sunbury on Thames (United Kingdom); Metcalfe, R. [QUINTESSA, The Hub, 14 Station Road, Henley-on-Thames, Oxfordshire (United Kingdom); Schmidt Hattenberger, C. [GFZ German Research Centre for Geosciences, Centre for CO2Storage, Potsdam (Germany)

    2013-07-01

    An extensive programme of modelling, monitoring and verification activities was deployed at a set of active storage sites worldwide including Sleipner, In Salah, Ketzin, Weyburn, K12-B and Snoehvit (EU CO2ReMoVe project). All investigated storage sites were well managed and did not have a negative impact on humans or the environment. Time-lapse seismic and pressure monitoring are key in verifying the deep subsurface performance of the storage sites. Evidence gathered during the site characterisation and operational phases is key to handover responsibility of the storage site to governmental authorities after injection has definitely ceased, which is the focus of the follow-up EU project CO2CARE.

  3. Klipperaas study site. Scope of activities and main results

    During the period from 1977 - 1986 SKB (Swedish Nuclear Fuel and Waste Management Co.) performed surface and borehole investigations of 14 study sites for the purpose of assessing their suitability for a repository of spent nuclear fuel. The next phase in the SKB site selection rpogramme will be to perform detailed characterisation, including characterization from shafts and/or tunnels, of two or three sites. The detailed investigations will continue over several years to provide all the data needed for a licensing application to build a repository. Such an application is foreseen to be given to the authorities around the year 2003. It is presently not clear if any of the study sites will be selected as a site for detailed characterization. Other sites with geological and/or socio-economical characteristics judged more favorable may very well be the ones selected. However, as a part of the background documentation needed for the site selection studies to come, summary reports will be prepared for most study sites. These reports will include scope of activities, main results, uncertainties and need of complementary investigations. This report concern the Klipperaas study site. The main topics are the scope of activities, geologic model, geohydrological model, groundwater chemistry, assessment of solute transport, and rock mechanics

  4. Difference and Influence of Inactive and Active States of Cannabinoid Receptor Subtype CB2: From Conformation to Drug Discovery.

    Hu, Jianping; Feng, Zhiwei; Ma, Shifan; Zhang, Yu; Tong, Qin; Alqarni, Mohammed Hamed; Gou, Xiaojun; Xie, Xiang-Qun

    2016-06-27

    Cannabinoid receptor 2 (CB2), a G protein-coupled receptor (GPCR), is a promising target for the treatment of neuropathic pain, osteoporosis, immune system, cancer, and drug abuse. The lack of an experimental three-dimensional CB2 structure has hindered not only the development of studies of conformational differences between the inactive and active CB2 but also the rational discovery of novel functional compounds targeting CB2. In this work, we constructed models of both inactive and active CB2 by homology modeling. Then we conducted two comparative 100 ns molecular dynamics (MD) simulations on the two systems-the active CB2 bound with both the agonist and G protein and the inactive CB2 bound with inverse agonist-to analyze the conformational difference of CB2 proteins and the key residues involved in molecular recognition. Our results showed that the inactive CB2 and the inverse agonist remained stable during the MD simulation. However, during the MD simulations, we observed dynamical details about the breakdown of the "ionic lock" between R131(3.50) and D240(6.30) as well as the outward/inward movements of transmembrane domains of the active CB2 that bind with G proteins and agonist (TM5, TM6, and TM7). All of these results are congruent with the experimental data and recent reports. Moreover, our results indicate that W258(6.48) in TM6 and residues in TM4 (V164(4.56)-L169(4.61)) contribute greatly to the binding of the agonist on the basis of the binding energy decomposition, while residues S180-F183 in extracellular loop 2 (ECL2) may be of importance in recognition of the inverse agonist. Furthermore, pharmacophore modeling and virtual screening were carried out for the inactive and active CB2 models in parallel. Among all 10 hits, two compounds exhibited novel scaffolds and can be used as novel chemical probes for future studies of CB2. Importantly, our studies show that the hits obtained from the inactive CB2 model mainly act as inverse agonist(s) or neutral

  5. Effect of ultrasound combined with malic acid on the activity and conformation of mushroom (Agaricus bisporus) polyphenoloxidase.

    Zhou, Lei; Liu, Wei; Xiong, Zhiqiang; Zou, Liqiang; Liu, Junping; Zhong, Junzhen; Chen, Jun

    2016-08-01

    Polyphenoloxidase (PPO) plays an important role in the browning of vegetables, fruits and edible fungi. The effects of ultrasound, malic acid, and their combination on the activity and conformation of mushroom (Agaricus bisporus) PPO were studied. The activity of PPO decreased gradually with the increasing of malic acid concentrations (5-60mM). Neither medium concentrations (10, 20, 30mM) malic acid nor individual ultrasound (25kHz, 55.48W/cm(2)) treatment could remarkably inactivate PPO. However, the inactivation during their combination was more significant than the sum of ultrasound inactivation and malic acid inactivation. The inactivation kinetics of PPO followed a first-order kinetics under the combination of ultrasound and malic acid. The conformation of combination treated PPO was changed, which was reflected in the decrease of α-helix, increase of β-sheet contents and disruption of the tertiary structure. Results of molecular microstructure showed that ultrasound broke large molecular groups of PPO into small ones. Moreover, combined treatment disrupted the microstructure of PPO and molecules were connected together. PMID:27241293

  6. Self-objectification, feminist activism and conformity to feminine norms among female vegetarians, semi-vegetarians, and non-vegetarians.

    Brinkman, Britney G; Khan, Aliya; Edner, Benjamin; Rosén, Lee A

    2014-01-01

    Recent research has suggested that vegetarians may be at an increased risk for developing disordered eating or body image issues when compared to non-vegetarians. However, the results of such studies are mixed, and no research has explored potential connections between vegetarianism and self-objectification. In the current study, the authors examine factors that predicted body surveillance, body shame, and appearance control beliefs; three aspects of self-objectification. Surveys were completed by 386 women from the United States who were categorized as vegetarian, semi-vegetarian, or non-vegetarian. The three groups differed regarding dietary motivations, levels of feminist activism, and body shame, but did not differ on their conformity to feminine norms. While conformity to feminine norms predicted body surveillance and body shame levels among all three groups of women, feminist activism predicted appearance control beliefs among non-vegetarians only. These findings suggest that it is important for researchers and clinicians to distinguish among these three groups when examining the relationship between vegetarianism and self-objectification. PMID:24411771

  7. Charge transport in columnar stacked triphenylenes: Effects of conformational fluctuations on charge transfer integrals and site energies

    Senthilkumar, K.; Grozema, F.C.; Bickelhaupt, F.M.; Siebbeles, L.D.A.

    2003-01-01

    Values of charge transfer integrals, spatial overlap integrals and site energies involved in transport of positive charges along columnar stacked triphenylene derivatives are provided. These parameters were calculated directly as the matrix elements of the Kohn–Sham Hamiltonian, defined in terms of

  8. Computational investigation of locked nucleic acid (LNA) nucleotides in the active sites of DNA polymerases by molecular docking simulations.

    Poongavanam, Vasanthanathan; Madala, Praveen K; Højland, Torben; Veedu, Rakesh N

    2014-01-01

    Aptamers constitute a potential class of therapeutic molecules typically selected from a large pool of oligonucleotides against a specific target. With a scope of developing unique shorter aptamers with very high biostability and affinity, locked nucleic acid (LNA) nucleotides have been investigated as a substrate for various polymerases. Various reports showed that some thermophilic B-family DNA polymerases, particularly KOD and Phusion DNA polymerases, accepted LNA-nucleoside 5'-triphosphates as substrates. In this study, we investigated the docking of LNA nucleotides in the active sites of RB69 and KOD DNA polymerases by molecular docking simulations. The study revealed that the incoming LNA-TTP is bound in the active site of the RB69 and KOD DNA polymerases in a manner similar to that seen in the case of dTTP, and with LNA structure, there is no other option than the locked C3'-endo conformation which in fact helps better orienting within the active site. PMID:25036012

  9. Site-specific Interaction Mapping of Phosphorylated Ubiquitin to Uncover Parkin Activation.

    Yamano, Koji; Queliconi, Bruno B; Koyano, Fumika; Saeki, Yasushi; Hirokawa, Takatsugu; Tanaka, Keiji; Matsuda, Noriyuki

    2015-10-16

    Damaged mitochondria are eliminated through autophagy machinery. A cytosolic E3 ubiquitin ligase Parkin, a gene product mutated in familial Parkinsonism, is essential for this pathway. Recent progress has revealed that phosphorylation of both Parkin and ubiquitin at Ser(65) by PINK1 are crucial for activation and recruitment of Parkin to the damaged mitochondria. However, the mechanism by which phosphorylated ubiquitin associates with and activates phosphorylated Parkin E3 ligase activity remains largely unknown. Here, we analyze interactions between phosphorylated forms of both Parkin and ubiquitin at a spatial resolution of the amino acid residue by site-specific photo-crosslinking. We reveal that the in-between-RING (IBR) domain along with RING1 domain of Parkin preferentially binds to ubiquitin in a phosphorylation-dependent manner. Furthermore, another approach, the Fluoppi (fluorescent-based technology detecting protein-protein interaction) assay, also showed that pathogenic mutations in these domains blocked interactions with phosphomimetic ubiquitin in mammalian cells. Molecular modeling based on the site-specific photo-crosslinking interaction map combined with mass spectrometry strongly suggests that a novel binding mechanism between Parkin and ubiquitin leads to a Parkin conformational change with subsequent activation of Parkin E3 ligase activity. PMID:26260794

  10. Structure analysis reveals the flexibility of the ADAMTS-5 active site

    Shieh, Huey-Sheng; Tomasselli, Alfredo G.; Mathis, Karl J.; Schnute, Mark E.; Woodard, Scott S.; Caspers, Nicole; Williams, Jennifer M.; Kiefer, James R.; Munie, Grace; Wittwer, Arthur; Malfait, Anne-Marie; Tortorella, Micky D. (Pfizer)

    2012-03-02

    A ((1S,2R)-2-hydroxy-2,3-dihydro-1H-inden-1-yl) succinamide derivative (here referred to as Compound 12) shows significant activity toward many matrix metalloproteinases (MMPs), including MMP-2, MMP-8, MMP-9, and MMP-13. Modeling studies had predicted that this compound would not bind to ADAMTS-5 (a disintegrin and metalloproteinase with thrombospondin motifs-5) due to its shallow S1' pocket. However, inhibition analysis revealed it to be a nanomolar inhibitor of both ADAMTS-4 and -5. The observed inconsistency was explained by analysis of crystallographic structures, which showed that Compound 12 in complex with the catalytic domain of ADAMTS-5 (cataTS5) exhibits an unusual conformation in the S1' pocket of the protein. This first demonstration that cataTS5 can undergo an induced conformational change in its active site pocket by a molecule like Compound 12 should enable the design of new aggrecanase inhibitors with better potency and selectivity profiles.

  11. Dashboard applications to monitor experiment activities at sites

    Andreeva, Julia; Belforte, Stefano; Boehm, Max; Casajus, Adrian; Flix, Josep; Gaidioz, Benjamin; Grigoras, Costin; Kokoszkiewicz, Lukasz; Lanciotti, Elisa; Rocha, Ricardo; Saiz, Pablo; Santinelli, Roberto; Sidorova, Irina; Sciabà, Andrea; Tsaregorodtsev, Andrei

    2010-04-01

    In the framework of a distributed computing environment, such as WLCG, monitoring has a key role in order to keep under control activities going on in sites located in different countries and involving people based in many different sites. To be able to cope with such a large scale heterogeneous infrastructure, it is necessary to have monitoring tools providing a complete and reliable view of the overall performance of the sites. Moreover, the structure of a monitoring system critically depends on the object to monitor and on the users it is addressed to. In this article we will describe two different monitoring systems both aimed to monitor activities and services provided in the WLCG framework, but designed in order to meet the requirements of different users: Site Status Board has an overall view of the services available in all the sites supporting an experiment, whereas Siteview provides a complete view of all the activities going on at a site, for all the experiments supported by the site.

  12. Dashboard applications to monitor experiment activities at sites

    Andreeva, J; Boehm, M; Casajus, A; Flix, J; Gaidioz, B; Grigoras, C; Kokoszkiewicz, L; Lanciotti, E; Rocha, R; Saiz, P; Santinelli, R; Sidorova, I; Sciabà, A; Tsaregorodtsev, A

    2010-01-01

    In the framework of a distributed computing environment, such as WLCG, monitoring has a key role in order to keep under control activities going on in sites located in different countries and involving people based in many different sites. To be able to cope with such a large scale heterogeneous infrastructure, it is necessary to have monitoring tools providing a complete and reliable view of the overall performance of the sites. Moreover, the structure of a monitoring system critically depends on the object to monitor and on the users it is addressed to. In this article we will describe two different monitoring systems both aimed to monitor activities and services provided in the WLCG framework, but designed in order to meet the requirements of different users: Site Status Board has an overall view of the services available in all the sites supporting an experiment, whereas Siteview provides a complete view of all the activities going on at a site, for all the experiments supported by the site.

  13. Dashboard applications to monitor experiment activities at sites

    In the framework of a distributed computing environment, such as WLCG, monitoring has a key role in order to keep under control activities going on in sites located in different countries and involving people based in many different sites. To be able to cope with such a large scale heterogeneous infrastructure, it is necessary to have monitoring tools providing a complete and reliable view of the overall performance of the sites. Moreover, the structure of a monitoring system critically depends on the object to monitor and on the users it is addressed to. In this article we will describe two different monitoring systems both aimed to monitor activities and services provided in the WLCG framework, but designed in order to meet the requirements of different users: Site Status Board has an overall view of the services available in all the sites supporting an experiment, whereas Siteview provides a complete view of all the activities going on at a site, for all the experiments supported by the site.

  14. Charge transport in columnar stacked triphenylenes: Effects of conformational fluctuations on charge transfer integrals and site energies

    K. Senthilkumar; Grozema, F.C.; Bickelhaupt, F.M.; Siebbeles, L.D.A.

    2003-01-01

    Values of charge transfer integrals, spatial overlap integrals and site energies involved in transport of positive charges along columnar stacked triphenylene derivatives are provided. These parameters were calculated directly as the matrix elements of the Kohn–Sham Hamiltonian, defined in terms of the molecular orbitals on individual triphenylene molecules. This was realized by exploiting the unique feature of the Amsterdam density functional theory program that allows one to use molecular o...

  15. Site-specific conformational determination in thermal unfolding studies of helical peptides using vibrational circular dichroism with isotopic substitution

    Silva, R. A. G. D.; Kubelka, Jan; Bour, Petr; Decatur, Sean M.; Keiderling, Timothy A.

    2000-01-01

    Understanding the detailed mechanism of protein folding requires dynamic, site-specific stereochemical information. The short time response of vibrational spectroscopies allows evaluation of the distribution of populations in rapid equilibrium as the peptide unfolds. Spectral shifts associated with isotopic labels along with local stereochemical sensitivity of vibrational circular dichroism (VCD) allow determination of the segment sequence of unfolding. For a series of alanine-rich peptides t...

  16. Gideaa study site. Scope of activities and main results

    During the period from 1977-1986 SKB (Swedish Nuclear Fuel and Waste Management Co) performed surface and borehole investigations of 14 study sites for the purpose of assessing their suitability for a repository of spent nuclear fuel. The next phase in the SKB site selection programme will be to perform detailed characterization, including characterization from shafts and/or tunnels, of two or three sites. The detailed investigations will continue over several years to provide all the data needed for a licensing application to build a repository. Such an application is foreseen to be given to the authorities around the year 2003. It is presently not clear if anyone of the study sites will be selected as a site for detailed characterization. Other site with geological and/or socio-economical characteristics judged more favourable may very well be the ones selected. However, as a part of the background documentation needed for the site selection studies to come, summary reports will be prepared for most study sites. These reports will include scope of activities, main results, uncertainties and need of complementary investigations. This report concerns the Gideaa study site. (au)

  17. Fjaellveden study site. Scope of activities and main results

    During the period from 1977-1986 SKB (Swedish Nuclear Fuel and Waste Management CO) performed surface and borehole investigations of 14 study sites for the purpose of assessing their suitability for a repository of spent nuclear fuel. The next phase in the SKB site selection programme will be to perform detailed characterization, including characterization from shafts and/or tunnels, of two or three sites. The detailed investigations will continue over several years to provide all the data needed for a licensing application to build repository. Such an application is foreseen to be given to the authorities around the year 2003. It is presently not clear if anyone of the study sites will be selected as a site for detailed characterization. Other sites with geological and/or socio-economical characteristics judged more favourable may very well be the ones selected. However, as a part of the background documentation needed for the site selection studies to come, summary reports will be prepared for most study sites. These reports will include scope of activities, main results, uncertainties and need for complementary investigations. This report concerns the Fjaellveden study site. (au)

  18. Kamlunge study site. Scope of activities and main results

    During the period from 1977-1986 SKB (Swedish nuclear Fuel and Waste Management Co.) performed surface and borehole investigations of 14 study sites for the purpose of assessing their suitability for a repository of spent nuclear fuel. The next phase in the SKB site selection programme will be to perform detailed characterization, including characterization from shafts and/or tunnels, of two or three sites. The detailed investigations will continue over several years to provide all the data needed for a licensing application to build a repository. Such an application is foreseen to be given to the authorities around the year 2003. It is presently not clear if anyone of the study sites will be selected as a site for detailed characterization. Other sites with geological and/or socio-economical characteristics judged more favourable may very well be selected. However, as a part of the background documentation needed for the site selection studies to come, summary reports will be prepared for most study sites. These reports will include scope of activities, main results, uncertainties and need of complementary investigations. This report concerns the Kamlunge study site. (79 refs.) (au)

  19. Influence of sodium alginate pretreated by ultrasound on papain properties: Activity, structure, conformation and molecular weight and distribution.

    Feng, Liping; Cao, Yanping; Xu, Duoxia; You, Sasa; Han, Fu

    2016-09-01

    The aim of the study was to investigate the impact of sodium alginate (ALG) pretreated by ultrasound on the enzyme activity, structure, conformation and molecular weight and distribution of papain. ALG solutions were pretreated with ultrasound at varying power (0.05, 0.15, 0.25, 0.35, 0.45W/cm(2)), 135kHz, 50°C for 20min. The maximum relative activity of papain increased by 10.53% when mixed with ALG pretreated by ultrasound at 0.25W/cm(2), compared with the untreated ALG. The influence of ultrasound pretreated ALG on the conformation and secondary structure of papain were assessed by fluorescence spectroscopy and circular dichroism spectroscopy. The fluorescence spectra revealed that ultrasound pretreated ALG increased the number of tryptophan on papain surface, especially at 0.25W/cm(2). It indicated that ultrasound pretreatment induced molecular unfolding, causing the exposure of more hydrophobic groups and regions from inside to the outside of the papain molecules. Furthermore, ultrasound pretreated ALG resulted in minor changes in the secondary structure of the papain. The content of α-helix was slightly increased after ultrasound pretreatment and no significant change was observed at different ultrasound powers. ALG pretreated by ultrasound enhanced the stability of the secondary structure of papain, especially at 0.25W/cm(2). The free sulfhydryl (SH) content of papain was slightly increased and then decreased with the increase of ultrasonic power. The maximum content of free SH was observed at 0.25W/cm(2), under which the content of the free SH increased by 6.36% compared with the untreated ALG. Dynamic light scattering showed that the effect of ultrasound treatment was mainly the homogenization of the ALG particles in the mixed dispersion. The gel permeation chromatography coupled with the multi-angle laser light scattering photometer analysis showed that the molecular weight (Mw) of papain/ALG was decreased and then increased with the ultrasonic

  20. Altered binding of thioflavin t to the peripheral anionic site of acetylcholinesterase after phosphorylation of the active site by chlorpyrifos oxon or dichlorvos

    The peripheral anionic site of acetylcholinesterase, when occupied by a ligand, is known to modulate reaction rates at the active site of this important enzyme. The current report utilized the peripheral anionic site specific fluorogenic probe thioflavin t to determine if the organophosphates chlorpyrifos oxon and dichlorvos bind to the peripheral anionic site of human recombinant acetylcholinesterase, since certain organophosphates display concentration-dependent kinetics when inhibiting this enzyme. Incubation of 3 nM acetylcholinesterase active sites with 50 nM or 2000 nM inhibitor altered both the Bmax and Kd for thioflavin t binding to the peripheral anionic site. However, these changes resulted from phosphorylation of Ser203 since increasing either inhibitor from 50 nM to 2000 nM did not alter further thioflavin t binding kinetics. Moreover, the organophosphate-induced decrease in Bmax did not represent an actual reduction in binding sites, but instead likely resulted from conformational interactions between the acylation and peripheral anionic sites that led to a decrease in the rigidity of bound thioflavin t. A drop in fluorescence quantum yield, leading to an apparent decrease in Bmax, would accompany the decreased rigidity of bound thioflavin t molecules. The organophosphate-induced alterations in Kd represented changes in binding affinity of thioflavin t, with diethylphosphorylation of Ser203 increasing Kd, and dimethylphosphorylation of Ser203 decreasing Kd. These results indicate that chlorpyrifos oxon and dichlorvos do not bind directly to the peripheral anionic site of acetylcholinesterase, but can affect binding to that site through phosphorylation of Ser203

  1. Comparative study of convolution, superposition, and fast superposition algorithms in conventional radiotherapy, three-dimensional conformal radiotherapy, and intensity modulated radiotherapy techniques for various sites, done on CMS XIO planning system

    Muralidhar K; Murthy Narayana; Raju Alluri; Sresty NVNM

    2009-01-01

    The aim of this study is to compare the dosimetry results that are obtained by using Convolution, Superposition and Fast Superposition algorithms in Conventional Radiotherapy, Three-Dimensional Conformal Radiotherapy (3D-CRT), and Intensity Modulated Radiotherapy (IMRT) for different sites, and to study the suitability of algorithms with respect to site and technique. For each of the Conventional, 3D-CRT, and IMRT techniques, four different sites, namely, Lung, Esophagus, Prostate, and Hypoph...

  2. Role of a cysteine residue in the active site of ERK and the MAPKK family

    Kinases of mitogen-activated protein kinase (MAPK) cascades, including extracellular signal-regulated protein kinase (ERK), represent likely targets for pharmacological intervention in proliferative diseases. Here, we report that FR148083 inhibits ERK2 enzyme activity and TGFβ-induced AP-1-dependent luciferase expression with respective IC50 values of 0.08 and 0.05 μM. FR265083 (1'-2' dihydro form) and FR263574 (1'-2' and 7'-8' tetrahydro form) exhibited 5.5-fold less and no activity, respectively, indicating that both the α,β-unsaturated ketone and the conformation of the lactone ring contribute to this inhibitory activity. The X-ray crystal structure of the ERK2/FR148083 complex revealed that the compound binds to the ATP binding site of ERK2, involving a covalent bond to Sγ of ERK2 Cys166, hydrogen bonds with the backbone NH of Met108, Nζ of Lys114, backbone C=O of Ser153, Nδ2 of Asn154, and hydrophobic interactions with the side chains of Ile31, Val39, Ala52, and Leu156. The covalent bond motif in the ERK2/FR148083 complex assures that the inhibitor has high activity for ERK2 and no activity for other MAPKs such as JNK1 and p38MAPKα/β/γ/δ which have leucine residues at the site corresponding to Cys166 in ERK2. On the other hand, MEK1 and MKK7, kinases of the MAPKK family which also can be inhibited by FR148083, contain a cysteine residue corresponding to Cys166 of ERK2. The covalent binding to the common cysteine residue in the ATP-binding site is therefore likely to play a crucial role in the inhibitory activity for these MAP kinases. These findings on the molecular recognition mechanisms of FR148083 for kinases with Cys166 should provide a novel strategy for the pharmacological intervention of MAPK cascades

  3. Active Sites Environmental Monitoring Program: Mid-FY 1991 report

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1991-10-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from October 1990 through March 1991. The ASEMP was established in 1989 by Solid Waste Operations and the Environmental Sciences Division to provide early detection and performance monitoring at active low-level radioactive waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 as required by chapters II and III of US Department of Energy Order 5820.2A. Monitoring results continue to demonstrate the no LLW is being leached from the storage vaults on the tumulus pads. Loading of vaults on Tumulus II began during this reporting period and 115 vaults had been loaded by the end of March 1991.

  4. Conformational Dynamics of Insulin

    Qing-xin eHua

    2011-10-01

    Full Text Available We have exploited a prandial insulin analogue (insulin lispro, the active component of Humalog®; Eli Lilly and Co. to elucidate the underlying structure and dynamics of insulin as a monomer in solution. Whereas NMR-based modeling recapitulates structural relationships of insulin crystals (T-state protomers, dynamic anomalies are revealed by amide-proton exchange kinetics in D2O. Surprisingly, the majority of hydrogen bonds observed in crystal structures are only transiently maintained in solution, including key T-state-specific inter-chain contacts. Long-lived hydrogen bonds (as defined by global exchange kinetics exist only at a subset of four -helical sites (two per chain flanking an internal disulfide bridge (cystine A20-B19; these sites map within the proposed folding nucleus of proinsulin. The anomalous flexibility of insulin otherwise spans its active surface and may facilitate receptor binding. Because conformational fluctuations promote the degradation of pharmaceutical formulations, we envisage that dynamic re-engineering of insulin may enable design of ultra-stable formulations for humanitarian use in the developing world.

  5. An active site-tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase.

    Murphy, Jesse R; Donini, Stefano; Kappock, T Joseph

    2015-10-01

    Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that `close' the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an `open' structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. As a polar but almost neutral ligand, the active site-tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS. PMID:26457521

  6. Active chemisorption sites in functionalized ionic liquids for carbon capture.

    Cui, Guokai; Wang, Jianji; Zhang, Suojiang

    2016-07-25

    Development of novel technologies for the efficient and reversible capture of CO2 is highly desired. In the last decade, CO2 capture using ionic liquids has attracted intensive attention from both academia and industry, and has been recognized as a very promising technology. Recently, a new approach has been developed for highly efficient capture of CO2 by site-containing ionic liquids through chemical interaction. This perspective review focuses on the recent advances in the chemical absorption of CO2 using site-containing ionic liquids, such as amino-based ionic liquids, azolate ionic liquids, phenolate ionic liquids, dual-functionalized ionic liquids, pyridine-containing ionic liquids and so on. Other site-containing liquid absorbents such as amine-based solutions, switchable solvents, and functionalized ionic liquid-amine blends are also investigated. Strategies have been discussed for how to activate the existent reactive sites and develop novel reactive sites by physical and chemical methods to enhance CO2 absorption capacity and reduce absorption enthalpy. The carbon capture mechanisms of these site-containing liquid absorbents are also presented. Particular attention has been paid to the latest progress in CO2 capture in multiple-site interactions by amino-free anion-functionalized ionic liquids. In the last section, future directions and prospects for carbon capture by site-containing ionic liquids are outlined. PMID:27243042

  7. Conformational Dynamics and Allostery in Pyruvate Kinase.

    Donovan, Katherine A; Zhu, Shaolong; Liuni, Peter; Peng, Fen; Kessans, Sarah A; Wilson, Derek J; Dobson, Renwick C J

    2016-04-22

    Pyruvate kinase catalyzes the final step in glycolysis and is allosterically regulated to control flux through the pathway. Two models are proposed to explain how Escherichia coli pyruvate kinase type 1 is allosterically regulated: the "domain rotation model" suggests that both the domains within the monomer and the monomers within the tetramer reorient with respect to one another; the "rigid body reorientation model" proposes only a reorientation of the monomers within the tetramer causing rigidification of the active site. To test these hypotheses and elucidate the conformational and dynamic changes that drive allostery, we performed time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange studies followed by mutagenic analysis to test the activation mechanism. Global exchange experiments, supported by thermostability studies, demonstrate that fructose 1,6-bisphosphate binding to the allosteric domain causes a shift toward a globally more dynamic ensemble of conformations. Mapping deuterium exchange to peptides within the enzyme highlight site-specific regions with altered conformational dynamics, many of which increase in conformational flexibility. Based upon these and mutagenic studies, we propose an allosteric mechanism whereby the binding of fructose 1,6-bisphosphate destabilizes an α-helix that bridges the allosteric and active site domains within the monomeric unit. This destabilizes the β-strands within the (β/α)8-barrel domain and the linked active site loops that are responsible for substrate binding. Our data are consistent with the domain rotation model but inconsistent with the rigid body reorientation model given the increased flexibility at the interdomain interface, and we can for the first time explain how fructose 1,6-bisphosphate affects the active site. PMID:26879751

  8. ATP and MO25alpha regulate the conformational state of the STRADalpha pseudokinase and activation of the LKB1 tumour suppressor.

    Elton Zeqiraj

    2009-06-01

    Full Text Available Pseudokinases lack essential residues for kinase activity, yet are emerging as important regulators of signal transduction networks. The pseudokinase STRAD activates the LKB1 tumour suppressor by forming a heterotrimeric complex with LKB1 and the scaffolding protein MO25. Here, we describe the structure of STRADalpha in complex with MO25alpha. The structure reveals an intricate web of interactions between STRADalpha and MO25alpha involving the alphaC-helix of STRADalpha, reminiscent of the mechanism by which CDK2 interacts with cyclin A. Surprisingly, STRADalpha binds ATP and displays a closed conformation and an ordered activation loop, typical of active protein kinases. Inactivity is accounted for by nonconservative substitution of almost all essential catalytic residues. We demonstrate that binding of ATP enhances the affinity of STRADalpha for MO25alpha, and conversely, binding of MO25alpha promotes interaction of STRADalpha with ATP. Mutagenesis studies reveal that association of STRADalpha with either ATP or MO25alpha is essential for LKB1 activation. We conclude that ATP and MO25alpha cooperate to maintain STRADalpha in an "active" closed conformation required for LKB1 activation. It has recently been demonstrated that a mutation in human STRADalpha that truncates a C-terminal region of the pseudokinase domain leads to the polyhydramnios, megalencephaly, symptomatic epilepsy (PMSE syndrome. We demonstrate this mutation destabilizes STRADalpha and prevents association with LKB1. In summary, our findings describe one of the first structures of a genuinely inactive pseudokinase. The ability of STRADalpha to activate LKB1 is dependent on a closed "active" conformation, aided by ATP and MO25alpha binding. Thus, the function of STRADalpha is mediated through an active kinase conformation rather than kinase activity. It is possible that other pseudokinases exert their function through nucleotide binding and active conformations.

  9. Conformal Infinity

    Frauendiener Jörg

    2004-01-01

    Full Text Available The notion of conformal infinity has a long history within the research in Einstein's theory of gravity. Today, 'conformal infinity' is related to almost all other branches of research in general relativity, from quantisation procedures to abstract mathematical issues to numerical applications. This review article attempts to show how this concept gradually and inevitably evolved from physical issues, namely the need to understand gravitational radiation and isolated systems within the theory of gravitation, and how it lends itself very naturally to the solution of radiation problems in numerical relativity. The fundamental concept of null-infinity is introduced. Friedrich's regular conformal field equations are presented and various initial value problems for them are discussed. Finally, it is shown that the conformal field equations provide a very powerful method within numerical relativity to study global problems such as gravitational wave propagation and detection.

  10. Conformal Infinity

    Frauendiener Jörg

    2000-08-01

    Full Text Available The notion of conformal infinity has a long history within the research in Einstein's theory of gravity. Today, ``conformal infinity'' is related with almost all other branches of research in general relativity, from quantisation procedures to abstract mathematical issues to numerical applications. This review article attempts to show how this concept gradually and inevitably evolved out of physical issues, namely the need to understand gravitational radiation and isolated systems within the theory of gravitation and how it lends itself very naturally to solve radiation problems in numerical relativity. The fundamental concept of null-infinity is introduced. Friedrich's regular conformal field equations are presented and various initial value problems for them are discussed. Finally, it is shown that the conformal field equations provide a very powerful method within numerical relativity to study global problems such as gravitational wave propagation and detection.

  11. The conformational state of the nucleosome entry–exit site modulates TATA box-specific TBP binding

    Aaron R Hieb; Gansen, Alexander; Böhm, Vera; Langowski, Jörg

    2014-01-01

    The TATA binding protein (TBP) is a critical transcription factor used for nucleating assembly of the RNA polymerase II machinery. TBP binds TATA box elements with high affinity and kinetic stability and in vivo is correlated with high levels of transcription activation. However, since most promoters use less stable TATA-less or TATA-like elements, while also competing with nucleosome occupancy, further mechanistic insight into TBP's DNA binding properties and ability to access chromatin is n...

  12. Resonant active sites in catalytic ammonia synthesis: A structural model

    Cholach, Alexander R.; Bryliakova, Anna A.; Matveev, Andrey V.; Bulgakov, Nikolai N.

    2016-03-01

    Adsorption sites Mn consisted of n adjacent atoms M, each bound to the adsorbed species, are considered within a realistic model. The sum of bonds Σ lost by atoms in a site in comparison with the bulk atoms was used for evaluation of the local surface imperfection, while the reaction enthalpy at that site was used as a measure of activity. The comparative study of Mn sites (n = 1-5) at basal planes of Pt, Rh, Ir, Fe, Re and Ru with respect to heat of N2 dissociative adsorption QN and heat of Nad + Had → NHad reaction QNH was performed using semi-empirical calculations. Linear QN(Σ) increase and QNH(Σ) decrease allowed to specify the resonant Σ for each surface in catalytic ammonia synthesis at equilibrium Nad coverage. Optimal Σ are realizable for Ru2, Re2 and Ir4 only, whereas other centers meet steric inhibition or unreal crystal structure. Relative activity of the most active sites in proportion 5.0 × 10- 5: 4.5 × 10- 3: 1: 2.5: 3.0: 1080: 2270 for a sequence of Pt4, Rh4, Fe4(fcc), Ir4, Fe2-5(bcc), Ru2, Re2, respectively, is in agreement with relevant experimental data. Similar approach can be applied to other adsorption or catalytic processes exhibiting structure sensitivity.

  13. An in silico study of the molecular basis of B-RAF activation and conformational stability

    Fratev, Filip Filipov; Jonsdottir, Svava Osk

    2009-01-01

    B-RAF kinase plays an important role both in tumour induction and maintenance in several cancers and it is an attractive new drug target. However, the structural basis of the B-RAF activation is still not well understood. RESULTS: In this study we suggest a novel molecular basis of B-RAF activation...... found that several mutations, which directly or indirectly destabilized the interactions between these residues within this network, contributed to the changes in B-RAF activity. CONCLUSION: Our results showed that the above mechanisms lead to the disruption of the electrostatic interactions between the...

  14. Vibrational and Electronic optical Activity of the Chiral Disulphide Group: Implications for Disulphide Bridge Conformation

    Bednárová, Lucie; Bouř, Petr; Maloň, Petr

    2010-01-01

    Roč. 22, č. 5 (2010), s. 514-526. ISSN 0899-0042 R&D Projects: GA ČR(CZ) GA203/07/1335; GA ČR GA202/07/0732; GA AV ČR IAA400550702 Institutional research plan: CEZ:AV0Z40550506 Keywords : disulphide bridge * circular dichroism * vibrational optical activity * Raman optical activity Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.892, year: 2010

  15. Enhancement of Chaperone Activity of Plant-Specific Thioredoxin through γ-Ray Mediated Conformational Change

    Seung Sik Lee

    2015-11-01

    Full Text Available AtTDX, a thioredoxin-like plant-specific protein present in Arabidospis is a thermo-stable and multi-functional enzyme. This enzyme is known to act as a thioredoxin and as a molecular chaperone depending upon its oligomeric status. The present study examines the effects of γ-irradiation on the structural and functional changes of AtTDX. Holdase chaperone activity of AtTDX was increased and reached a maximum at 10 kGy of γ-irradiation and declined subsequently in a dose-dependent manner, together with no effect on foldase chaperone activity. However, thioredoxin activity decreased gradually with increasing irradiation. Electrophoresis and size exclusion chromatography analysis showed that AtTDX had a tendency to form high molecular weight (HMW complexes after γ-irradiation and γ-ray-induced HMW complexes were tightly associated with a holdase chaperone activity. The hydrophobicity of AtTDX increased with an increase in irradiation dose till 20 kGy and thereafter decreased further. Analysis of the secondary structures of AtTDX using far UV-circular dichroism spectra revealed that the irradiation remarkably increased the exposure of β-sheets and random coils with a dramatic decrease in α-helices and turn elements in a dose-dependent manner. The data of the present study suggest that γ-irradiation may be a useful tool for increasing holdase chaperone activity without adversely affecting foldase chaperone activity of thioredoxin-like proteins.

  16. Probing the active sites for CO dissociation on ruthenium nanoparticles

    Strebel, Christian Ejersbo; Murphy, Shane; Nielsen, Rasmus Munksgård;

    2012-01-01

    The active sites for CO dissociation were probed on mass-selected Ru nanoparticles on a HOPG support by temperature programmed desorption spectroscopy using isotopically labelled CO. Combined with transmission electron microscopy we gain insight on how the size and morphology of the nanoparticles...

  17. Chemical Modification of Papain and Subtilisin: An Active Site Comparison

    St-Vincent, Mireille; Dickman, Michael

    2004-01-01

    An experiment using methyle methanethiosulfonate (MMTS) and phenylmethylsulfonyl flouride (PMSF) to specifically modify the cysteine and serine residues in the active sites of papain and subtilism respectively is demonstrated. The covalent modification of these enzymes and subsequent rescue of papain shows the beginning biochemist that proteins…

  18. Active site modeling in copper azurin molecular dynamics simulations

    Rizzuti, B; Swart, M; Sportelli, L; Guzzi, R

    2004-01-01

    Active site modeling in molecular dynamics simulations is investigated for the reduced state of copper azurin. Five simulation runs (5 ns each) were performed at room temperature to study the consequences of a mixed electrostatic/constrained modeling for the coordination between the metal and the po

  19. Active Sites Environmental Monitoring Program: FY 1990 annual report

    Wickliff, D.S.; Morrissey, C.M.; Ashwood, T.L.

    1991-10-01

    Chapter 3 of US Department of Energy (DOE) Order 5820.2A (DOE 1988) sets forth requirements for environmental monitoring of active low-level waste (LLW) disposal sites. Active sites are defined as those LLW facilities that were in use on or after the date of the order (September 1988). The transuranic (TRU) waste storage areas in Solid Waste Storage Area (SWSA) 5 North are covered by Chap. 2 of the order. In both chapters, monitoring is required to provide for early warning of leaks before those leaks pose a threat to human health or the environment. Chapter 3 also requires that monitoring be conducted to evaluate the short- and long-term performance of LLW disposal facilities. In accordance with this order, the Solid Waste Operations Department at Oak Ridge National Laboratory (ORNL) has established an Active Sites Environmental Monitoring Program (ASEMP) that is implemented by staff of the Environmental Sciences Division (ESD) at ORNL. This report summarizes data from ASEMP monitoring activities for the final 6 months of FY 1990. A brief summary of the monitoring methodology for each site is presented also.

  20. The nature of the active site in heterogeneous metal catalysis

    Nørskov, Jens Kehlet; Bligaard, Thomas; Larsen, Britt Hvolbæk;

    2008-01-01

    This tutorial review, of relevance for the surface science and heterogeneous catalysis communities, provides a molecular-level discussion of the nature of the active sites in metal catalysis. Fundamental concepts such as "Bronsted-Evans-Polanyi relations'' and "volcano curves'' are introduced, and...

  1. Active Sites Environmental Monitoring Program: FY 1990 annual report

    Chapter 3 of US Department of Energy (DOE) Order 5820.2A (DOE 1988) sets forth requirements for environmental monitoring of active low-level waste (LLW) disposal sites. Active sites are defined as those LLW facilities that were in use on or after the date of the order (September 1988). The transuranic (TRU) waste storage areas in Solid Waste Storage Area (SWSA) 5 North are covered by Chap. 2 of the order. In both chapters, monitoring is required to provide for early warning of leaks before those leaks pose a threat to human health or the environment. Chapter 3 also requires that monitoring be conducted to evaluate the short- and long-term performance of LLW disposal facilities. In accordance with this order, the Solid Waste Operations Department at Oak Ridge National Laboratory (ORNL) has established an Active Sites Environmental Monitoring Program (ASEMP) that is implemented by staff of the Environmental Sciences Division (ESD) at ORNL. This report summarizes data from ASEMP monitoring activities for the final 6 months of FY 1990. A brief summary of the monitoring methodology for each site is presented also

  2. α-Lytic protease can exist in two separately stable conformations with different His57 mobilities and catalytic activities

    Haddad, Kristin Coffman; Sudmeier, James L.; Bachovchin, Daniel A.; Bachovchin, William W.

    2005-01-01

    α-Lytic protease is a bacterial serine protease widely studied as a model system of enzyme catalysis. Here we report that lyophilization induces a structural change in the enzyme that is not reversed by redissolution in water. The structural change reduces the mobility of the active-site histidine residue and the catalytic activity of the enzyme. The application of mild pressure to solutions of the altered enzyme reverses the lyophilization-induced structural change and restores the mobility ...

  3. Detailed conformation dynamics and activation process of wild type c-Abl and T315I mutant

    Yang, Li-Jun; Zhao, Wen-Hua; Liu, Qian

    2014-10-01

    Bcr-Abl is an important target for therapy against chronic myelogenous leukemia (CML) and acute lymphocytic leukemia (ALL). The synergistic effect between myristyl pocket and the ATP pocket has been found. But its detailed information based on molecular level still has not been achieved. In this study, conventional molecular dynamics (CMD) and target molecular dynamics (TMD) simulations were performed to explore the effect of T315I mutation on dynamics and activation process of Abl containing the N-terminal cap (Ncap). The CMD simulation results reveal the increasing flexibility of ATP pocket in kinase domain (KD) after T315I mutation which confirms the disability of ATP-pocket inhibitors to the Abl-T315I mutant. On the contrary, the T315I mutation decreased the flexibility of remote helix αI which suggests the synergistic effect between them. The mobility of farther regions containing Ncap, SH3, SH2 and SH2-KD linker were not affected by T315I mutation. The TMD simulation results show that the activation process of wild type Abl and Abl-T315I mutant experienced global conformation change. Their differences were elucidated by the activation motion of subsegments including A-loop, P-loop and Ncap. Besides, the T315I mutation caused decreasing energy barrier and increasing intermediate number in activation process, which results easier activation process. The TMD and CMD results indicate that a drug targeting only the ATP pocket is not enough to inhibit the Abl-T315I mutant. An effective way to inhibit the abnormal activity of Abl-T315I mutant is to combine the ATP-pocket inhibitors with inhibitors binding at non-ATP pockets mainly related to Ncap, SH2-KD linker and myristyl pocket.

  4. Electronic and Vibrational Optical Activity of Several Peptides Related to Neurohypophyseal Hormones: Disulfide Group Conformation

    Pazderková, Markéta; Bednárová, Lucie; Dlouhá, Helena; Flegel, Martin; Lebl, M.; Hlaváček, Jan; Setnička, V.; Urbanová, M.; Hynie, S.; Klenerová, V.; Baumruk, V.; Maloň, Petr

    2012-01-01

    Roč. 97, č. 11 (2012), s. 923-932. ISSN 0006-3525 R&D Projects: GA ČR GAP205/10/1276 Grant ostatní: GA UK(CZ) 578212 Institutional research plan: CEZ:AV0Z40550506 Keywords : neurohypophyseal hormones * disulfide bridge * Raman optical activity * vibrational circular dichroism Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.879, year: 2012

  5. Protein Conformational Populations and Functionally Relevant Sub-states

    Agarwal, Pratul K [ORNL; Burger, Virginia [University of Pittsburgh School of Medicine, Pittsburgh PA; Savol, Andrej [University of Pittsburgh School of Medicine, Pittsburgh PA; Ramanathan, Arvind [ORNL; Chennubhotla, Chakra [University of Pittsburgh School of Medicine, Pittsburgh PA

    2013-01-01

    it to attain the transition state, therefore promoting the reaction mechanism. In the long term, this emerging view of proteins with conformational substates has broad implications for improving our understanding of enzymes, enzyme engineering, and better drug design. Researchers have already used photoactivation to modulate protein conformations as a strategy to develop a hypercatalytic enzyme. In addition, the alteration of the conformational substates through binding of ligands at locations other than the active site provides the basis for the design of new medicines through allosteric modulation.

  6. Mapping the active site of vaccinia virus RNA triphosphatase

    The RNA triphosphatase component of vaccinia virus mRNA capping enzyme (the product of the viral D1 gene) belongs to a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi, protozoa, Chlorella virus, and baculoviruses. The family is defined by two glutamate-containing motifs (A and C) that form the metal-binding site. Most of the family members resemble the fungal and Chlorella virus enzymes, which have a complex active site located within the hydrophilic interior of a topologically closed eight-stranded β barrel (the so-called ''triphosphate tunnel''). Here we queried whether vaccinia virus capping enzyme is a member of the tunnel subfamily, via mutational mapping of amino acids required for vaccinia triphosphatase activity. We identified four new essential side chains in vaccinia D1 via alanine scanning and illuminated structure-activity relationships by conservative substitutions. Our results, together with previous mutational data, highlight a constellation of six acidic and three basic amino acids that likely compose the vaccinia triphosphatase active site (Glu37, Glu39, Arg77, Lys107, Glu126, Asp159, Lys161, Glu192, and Glu194). These nine essential residues are conserved in all vertebrate and invertebrate poxvirus RNA capping enzymes. We discerned no pattern of clustering of the catalytic residues of the poxvirus triphosphatase that would suggest structural similarity to the tunnel proteins (exclusive of motifs A and C). We infer that the poxvirus triphosphatases are a distinct lineage within the metal-dependent RNA triphosphatase family. Their unique active site, which is completely different from that of the host cell's capping enzyme, recommends the poxvirus RNA triphosphatase as a molecular target for antipoxviral drug discovery

  7. Hydrophobic Side-Chain Length Determines Activity and Conformational Heterogeneity of a Vancomycin Derivative Bound to the Cell Wall of Staphylococcus aureus§

    Kim, Sung Joon; Schaefer, Jacob

    2008-01-01

    Disaccharide modified glycopeptides with hydrophobic sidechains are active against vancomycin-resistant enterococci and vancomycin-resistant S. aureus. The activity depends on the length of the sidechain. The benzyl sidechain of N-(4-fluorobenzyl)vancomycin (FBV) has the minimal length sufficient for enhancement in activity against vancomycin-resistant pathogens. The conformation of FBV bound to the peptidoglycan in whole cells of S. aureus has been determined using rotational-echo double res...

  8. Decommissioning and decontamination activity, Gnome Site, Eddy County, New Mexico

    The purpose of this assessment is to present a brief description of the proposed activity and its potential impacts on the environment. This assessment will constitute an evaluation as to whether or not a formal Environmental Statement need be prepared. As background to the proposed activity, Project Gnome was an underground nuclear test conducted in December 1961 as part of the PLOWSHARE Program. The project site is located about 25 miles southeast of Carlsbad, New Mexico. By means of an excavated shaft and tunnel, a 3-kiloton nuclear explosive was emplaced and detonated in a salt bed about 1200 feet below the surface. The uncontaminated rock and salt muck from the original excavation and subsequent contaminated muck and minor construction debris from reentry activities into the nuclear cavity is commingled and stored in a pile near the Gnome/Coach Shaft. Other areas on the site are known to have been contaminated. In 1969, a program was conducted to cleanup and dispose of all surface contamination to whatever depth it occurred in excess of 0.1 mR/hr. Contaminated materials and soil were collected and disposed into the Gnome shaft, which was filled and sealed. Since then, NV has proposed to DOE/HQ much lower criteria for residual radioactive contamination for the Gnome Site. These proposed criteria were to collect and dispose of surficial materials which contain more than 2 x 10-5 microcuries per gram of soil for beta/gamma emitters and 3 x 10-2 microcuries per milliliter of tritium in soil moisture. According to the latest reconnaissance in 1972, low concentrations of Cs-137, Sr-90 and tritium were present at various locations on the site in excess of these proposed guidelines. Other operational areas within the site are suspected of containing radioactive contamination in much lesser volume, which are to be determined by careful probing and monitoring, as described in the next section

  9. Structural Study of a Flexible Active Site Loop in Human Indoleamine 2,3-Dioxygenase and Its Functional Implications.

    Álvarez, Lucía; Lewis-Ballester, Ariel; Roitberg, Adrián; Estrin, Darío A; Yeh, Syun-Ru; Marti, Marcelo A; Capece, Luciana

    2016-05-17

    Human indoleamine 2,3-dioxygenase catalyzes the oxidative cleavage of tryptophan to N-formyl kynurenine, the initial and rate-limiting step in the kynurenine pathway. Additionally, this enzyme has been identified as a possible target for cancer therapy. A 20-amino acid protein segment (the JK loop), which connects the J and K helices, was not resolved in the reported hIDO crystal structure. Previous studies have shown that this loop undergoes structural rearrangement upon substrate binding. In this work, we apply a combination of replica exchange molecular dynamics simulations and site-directed mutagenesis experiments to characterize the structure and dynamics of this protein region. Our simulations show that the JK loop can be divided into two regions: the first region (JK loop(C)) displays specific and well-defined conformations and is within hydrogen bonding distance of the substrate, while the second region (JK loop(N)) is highly disordered and exposed to the solvent. The peculiar flexible nature of JK loop(N) suggests that it may function as a target for post-translational modifications and/or a mediator for protein-protein interactions. In contrast, hydrogen bonding interactions are observed between the substrate and Thr379 in the highly conserved "GTGG" motif of JK loop(C), thereby anchoring JK loop(C) in a closed conformation, which secures the appropriate substrate binding mode for catalysis. Site-directed mutagenesis experiments confirm the key role of this residue, highlighting the importance of the JK loop(C) conformation in regulating the enzymatic activity. Furthermore, the existence of the partially and totally open conformations in the substrate-free form suggests a role of JK loop(C) in controlling substrate and product dynamics. PMID:27112409

  10. Methodology for contaminated sites of military activity territories restoration

    Major part of Eastern Europe countries meet environmental problems related to sites of military activity. Major part of these sites is characterised with degradation of natural landscapes and contamination of geological environment with toxic and hazardous waste representing actual and potential danger for population and environment. Actual danger is caused with localisation of toxic waste, hazardous materials and waste which are preventing normal land use. Potential danger is related to successive dispersion of contamination in biosphere as well as origin of new derivatives and products having toxic and hazardous properties. The list of such sites and objects comprises bases of land, air and naval forces. These objects include a network of infrastructures: storages of fuels and lubricants (surface, underground), filling stations, pipe lines, reparation stations, garages, decontamination stations, underground storages of different purposes, depots (for ammunition, chemical products), hospitals, constructions, firing grounds (tank, artillery, aircraft bombing etc.) and waste disposal sites. Special programs aimed at military industries and bases contaminated sites remediation have been carrying out in developed countries (USA, United Kingdom, Germany etc.). This experience was used in the frames of joint programs having been founded in several countries of Central and Eastern Europe (Chesh Republic, Slovakia, Lithuania etc.). (author)

  11. Exploiting Innocuous Activity for Correlating Users Across Sites

    Goga, Oana; Lei, Howard; Parthasarathi, Sree Hari Krishnan; Friedland, Gerald; Sommer, Robin; Teixeira, Renata

    2013-01-01

    International audience We study how potential attackers can identify accounts on different social network sites that all belong to the same user, exploiting only innocuous activity that inherently comes with posted content. We examine three specific features on Yelp, Flickr, and Twitter: the geo-location attached to a user's posts, the timestamp of posts, and the user's writing style as captured by language models. We show that among these three features the location of posts is the most po...

  12. Active sites in char gasification: Final technical report

    Wojtowicz, M.; Lilly, W.D.; Perkins, M.T.; Hradil, G.; Calo, J.M.; Suuberg, E.M.

    1987-09-01

    Among the key variables in the design of gasifiers and combustors is the reactivity of the chars which must be gasified or combusted. Significant loss of unburned char is unacceptable in virtually any process; the provision of sufficient residence time for complete conversion is essential. A very wide range of reactivities are observed, depending upon the nature of the char in a process. The current work focuses on furthering the understanding of gasification reactivities of chars. It has been well established that the reactivity of char to gasification generally depends upon three principal factors: (1) the concentration of ''active sites'' in the char; (2) mass transfer within the char; and (3) the type and concentration of catalytic impurities in the char. The present study primarily addresses the first factor. The subject of this research is the origin, nature, and fate of active sites in chars derived from parent hydrocarbons with coal-like structure. The nature and number of the active sites and their reactivity towards oxygen are examined in ''model'' chars derived from phenol-formaldehyde type resins. How the active sites are lost by the process of thermal annealing during heat treatment of chars are studied, and actual rate for the annealing process is derived. Since intrinsic char reactivities are of primary interest in the present study, a fair amount of attention was given to the model char synthesis and handling so that the effect of catalytic impurities and oxygen-containing functional groups in the chemical structure of the material were minimized, if not completely eliminated. The project would not be considered complete without comparing characteristic features of synthetic chars with kinetic behavior exhibited by natural chars, including coal chars.

  13. Resolving the Structure of Active Sites on Platinum Catalytic Nanoparticles

    Chang, Lan Yun; Barnard, Amanda S.; Gontard, Lionel Cervera; Dunin-Borkowski, Rafal E.

    2010-01-01

    Accurate understanding of the structure of active sites is fundamentally important in predicting catalytic properties of heterogeneous nanocatalysts. We present an accurate determination of both experimental and theoretical atomic structures of surface monatomic steps on industrial platinum...... nanoparticles. This comparison reveals that the edges of nanoparticles can significantly alter the atomic positions of monatomic steps in their proximity, which can lead to substantial deviations in the catalytic properties compared with the extended surfaces....

  14. The purification of affinity-labelled active-site peptides

    The isolation of the labelled peptide from the protein digest, following the affinity labelling of the active sites of enzymes or antibodies, is described. Single-step affinity chromatography utilises the affinity of the native enzymes or antibody for the ligand used to label the same protein. The labelled peptide is the only one in the digest that displays affinity for the immobilised protein and can be released with eluants that dissociate the protein-ligand complex. (Auth.)

  15. Structure, chain conformation, and immunomodulatory activity of the polysaccharide purified from Bacillus Calmette Guerin formulation.

    Liu, Wei; Wang, Hong; Yu, Juping; Liu, Yameng; Lu, Weisheng; Chai, Yin; Liu, Chao; Pan, Chun; Yao, Wenbing; Gao, Xiangdong

    2016-10-01

    A polysaccharide, coded as BDP, purified from the injection powder of Bacillus Calmette Guerin (BCG) polysaccharide and nucleic acid, was composed mainly of α-D-(1→4)-linked glucan with (1→6)-linked branches and trace amounts of fucose and mannose from the results of FT-IR, HPAEC-PAD and NMR spectrum. The Mw, Mn, Mz, and [Formula: see text] were determined to be 1.320×10(5)g/mol, 1.012×10(5)g/mol, 2.139×10(5)g/mol, and 21.8±3.2%nm by using HPSEC-MALLS, respectively. The ν value from [Formula: see text] was calculated to be 0.52±0.01, which firstly clarified that BDP existed as random coils in 0.9% NaCl aqueous solution. AFM and SEM combined with Congo-red test also revealed that the polysaccharide was irregular globular like or curly structure. Furthermore, in vitro tests on RAW264.7 murine macrophages cells revealed that BDP exhibited significant immunomodulatory activity. PMID:27312624

  16. Brownian aggregation rate of colloid particles with several active sites

    We theoretically analyze the aggregation kinetics of colloid particles with several active sites. Such particles (so-called “patchy particles”) are well known as chemically anisotropic reactants, but the corresponding rate constant of their aggregation has not yet been established in a convenient analytical form. Using kinematic approximation for the diffusion problem, we derived an analytical formula for the diffusion-controlled reaction rate constant between two colloid particles (or clusters) with several small active sites under the following assumptions: the relative translational motion is Brownian diffusion, and the isotropic stochastic reorientation of each particle is Markovian and arbitrarily correlated. This formula was shown to produce accurate results in comparison with more sophisticated approaches. Also, to account for the case of a low number of active sites per particle we used Monte Carlo stochastic algorithm based on Gillespie method. Simulations showed that such discrete model is required when this number is less than 10. Finally, we applied the developed approach to the simulation of immunoagglutination, assuming that the formed clusters have fractal structure

  17. Site characterization techniques used in environmental remediation activities

    As a result of decades of nuclear energy research, weapons production, as well as ongoing operations, a significant amount of radioactive contamination has occurred throughout the United States Department of Energy (DOE) complex. DOE facility are in the process of assessing and potentially remediating various sites according to the regulations imposed by a Federal Facility Agreement and Consent order (FFA/CO) between DOE, the state in which the facility is located, and the U.S. Environmental Protection Agency (EPA). In support of these active site remediation efforts, the DOE has devoted considerable resources towards the development of innovative site characterization techniques that support environmental restoration activities. These resources and efforts have focused on various aspects of this complex problem. Research and technology development conducted at the Idaho National Engineering and Environmental Laboratory (INEEL) has resulted in the ability and state-of-the-art equipment required to obtain real-time, densely spaced, in situ characterization data (i.e. detection, speciation, and location) of various radionuclides and contaminants. The Remedial Action Monitoring System (RAMS), developed by the INEEL, consists of enhanced sensor technology, measurement modeling and interpretation techniques, and a suite of deployment platforms which can be interchanged to directly support remedial cleanup and site verification operations. In situ characterization techniques have advanced to the point where they are being actively deployed in support of remedial operations. The INEEL has deployed its system at various DOE and international sites. The deployment of in situ characterization systems during environmental restoration operations has shown that this approach results in several significant benefits versus conventional sampling techniques. A flexible characterization system permits rapid modification to satisfy physical site conditions, available site resources

  18. Seismic activity parameters of the Finnish potential repository sites

    Posiva Oy has started a project for estimating the possible earthquake induced rock movements on the deposition holes containing canisters of spent nuclear fuel. These estimates will be made for the four investigation sites, Romuvaara, Kivetty, Olkiluoto and Haestholmen. This study deals with the current and future seismicity associated with the above mentioned sites. Seismic belts that participate the seismic behaviour of the studied sites have been identified and the magnitude-frequency distributions of these belts have been estimated. The seismic activity parameters of the sites have been deduced from the characteristics of the seismic belts in order to forecast the seismicity during the next 100,000 years. The report discusses the possible earthquakes induced by future glaciation. The seismic interpretation seems to indicate that the previous postglacial faults in Finnish Lapland have been generated in compressional environment. The orientation of the rather uniform compression has been NW-SE, which coincide with the current stress field. It seems that, although the impact of postglacial crustal rebound must have been significant, the impact of plate tectonics has been dominant. A major assumption of this study has been that future seismicity will generally resemble the current seismicity. However, when the postglacial seismicity is concerned, the magnitude-frequency distribution is likely different and the expected maximum magnitude will be higher. Maximum magnitudes of future postglacial earthquakes have been approximated by strain release examinations. Seismicity has been examined within the framework of the lineament maps, in order to associate the future significant earthquakes with active fault zones in the vicinity of the potential repository sites. (orig.)

  19. Seismic activity parameters of the Finnish potential repository sites

    Saari, J. [Fortum Engineering Oy, Vantaa (Finland)

    2000-10-01

    Posiva Oy has started a project for estimating the possible earthquake induced rock movements on the deposition holes containing canisters of spent nuclear fuel. These estimates will be made for the four investigation sites, Romuvaara, Kivetty, Olkiluoto and Haestholmen. This study deals with the current and future seismicity associated with the above mentioned sites. Seismic belts that participate the seismic behaviour of the studied sites have been identified and the magnitude-frequency distributions of these belts have been estimated. The seismic activity parameters of the sites have been deduced from the characteristics of the seismic belts in order to forecast the seismicity during the next 100,000 years. The report discusses the possible earthquakes induced by future glaciation. The seismic interpretation seems to indicate that the previous postglacial faults in Finnish Lapland have been generated in compressional environment. The orientation of the rather uniform compression has been NW-SE, which coincide with the current stress field. It seems that, although the impact of postglacial crustal rebound must have been significant, the impact of plate tectonics has been dominant. A major assumption of this study has been that future seismicity will generally resemble the current seismicity. However, when the postglacial seismicity is concerned, the magnitude-frequency distribution is likely different and the expected maximum magnitude will be higher. Maximum magnitudes of future postglacial earthquakes have been approximated by strain release examinations. Seismicity has been examined within the framework of the lineament maps, in order to associate the future significant earthquakes with active fault zones in the vicinity of the potential repository sites. (orig.)

  20. Oxygen reduction and evolution at single-metal active sites

    Calle-Vallejo, F.; Martínez, J.I.; García Lastra, Juan Maria;

    2013-01-01

    A worldwide spread of clean technologies such as low-temperature fuel cells and electrolyzers depends strictly on their technical reliability and economic affordability. Currently, both conditions are hardly fulfilled mainly due to the same reason: the oxygen electrode, which has large overpotent......A worldwide spread of clean technologies such as low-temperature fuel cells and electrolyzers depends strictly on their technical reliability and economic affordability. Currently, both conditions are hardly fulfilled mainly due to the same reason: the oxygen electrode, which has large...... overpotentials and is made of precious materials. A possible solution is the use of non-noble electrocatalysts with single-metal active sites. Here, on the basis of DFT calculations of adsorbed intermediates and a thermodynamic analysis, we compare the oxygen reduction (ORR) and evolution (OER) activities of...... functionalized graphitic materials and gas-phase porphyrins with late transition metals. We find that both kinds of materials follow approximately the same activity trends, and active sites with transition metals from groups 7 to 9 may be good ORR and OER electrocatalysts. However, spin analyses show more...

  1. Stimulatory effects of opioid neuropeptides on locomotory activity and conformational changes in invertebrate and human immunocytes: evidence for a subtype of delta receptor.

    Stefano, G B; Cadet, P; Scharrer, B

    1989-01-01

    The presence of opioid neuropeptides was shown to stimulate conformational changes and locomotory activity in immunocytes of two representatives of invertebrates as well as in human leukocytes. Cells were examined by use of phase-contrast and Nomarski optics coupled with a Zeiss Axiophot microscope, and of the Zeiss Videoplan/Vidas Image Analysis system. Immunocompetent blood cells, activated by exogenous opioids or stressful stimuli presumed to engage endogenous opioids, showed flattening, e...

  2. HDAC Inhibitors without an Active Site Zn2+-Binding Group

    Vickers, Chris J.; Olsen, Christian Adam; Leman, Luke J.;

    2012-01-01

    Natural and synthetic histone deacetylase (HDAC) inhibitors generally derive their strong binding affinity and high potency from a key functional group that binds to the Zn2+ ion within the enzyme active site. However, this feature is also thought to carry the potential liability of undesirable off......-target interactions with other metalloenzymes. As a step toward mitigating this issue, here, we describe the design, synthesis, and structure−activity characterizations of cyclic α3β-tetrapeptide HDAC inhibitors that lack the presumed indispensable Zn2+-binding group. The lead compounds (e.g., 15 and 26) display good...... potency against class 1 HDACs and are active in tissue culture against various human cancer cell lines. Importantly, enzymological analysis of 26 indicates that the cyclic α3β-tetrapeptide is a fast-on/ off competitive inhibitor of HDACs 1−3 with Ki values of 49, 33, and 37 nM, respectively. Our proof...

  3. Current activities handbook: formerly utilized sites remedial action program

    None

    1981-02-27

    This volume is one of a series produced under contract with the DOE, by Politech Corporation to develop a legislative and regulatory data base to assist the FUSRAP management in addressing the institutional and socioeconomic issues involved in carrying out the Formerly Utilized Sites Remedial Action Program. This Information Handbook series contains information about all relevant government agencies at the Federal and state levels, the pertinent programs they administer, each affected state legislature, and current Federal and state legislative and regulatory initiatives. This volume is a compilation of information about the activities each of the thirteen state legislatures potentially affected by the Formerly Utilized Sites Remedial Action Program. It contains a description of the state legislative procedural rules and a schedule of each legislative session; a summary of pending relevant legislation; the name and telephone number of legislative and state agency contacts; and the full text of all bills identified.

  4. Overview of the activities carried out at the FEBEX site

    Missana, T.; Buil, B.; Garralon, A.; Gomez, P. [CIEMAT, Dept. de Medioambien te, 28040 Madrid (Spain); Perez-Estaun, A.; Carbonell, R. [Inst. Jaume Almera, CSIC (Spain); Suso, J.; Carretero, G.; Bueno, J.; Martinez, L. [AITEMIN (Spain) ; Hernan, P. [ENRESA (Spain)

    2007-06-15

    One of the main aim of WP 4.1 and 4.2 is to study solute migration mechanisms in crystalline host-rock in realistic conditions. Many organisations are participating in a joint study that is being performed in the FEBEX gallery (NAGRA's Grimsel Test Site, GTS, Switzerland). The FEBEX experiment reproduces at a real scale a high-level waste repository in granite and was installed more than 9 years ago. At moment, it represents the most realistic environment where the processes affecting radionuclide migration from the bentonite to granite can be studied. This paper summarises the main activities carried out at the FEBEX site during the second year of the project.

  5. Current activities handbook: formerly utilized sites remedial action program

    This volume is one of a series produced under contract with the DOE, by Politech Corporation to develop a legislative and regulatory data base to assist the FUSRAP management in addressing the institutional and socioeconomic issues involved in carrying out the Formerly Utilized Sites Remedial Action Program. This Information Handbook series contains information about all relevant government agencies at the Federal and state levels, the pertinent programs they administer, each affected state legislature, and current Federal and state legislative and regulatory initiatives. This volume is a compilation of information about the activities each of the thirteen state legislatures potentially affected by the Formerly Utilized Sites Remedial Action Program. It contains a description of the state legislative procedural rules and a schedule of each legislative session; a summary of pending relevant legislation; the name and telephone number of legislative and state agency contacts; and the full text of all bills identified

  6. Electron capture dissociation and drift tube ion mobility-mass spectrometry coupled with site directed mutations provide insights into the conformational diversity of a metamorphic protein

    Harvey, Sophie R.; Porrini, Massimiliano; Tyler, Robert C.; MacPhee, Cait E.; Volkman, Brian F.; Barran, Perdita E.

    2015-01-01

    Ion mobility mass spectrometry can be combined with data from top-down sequencing to discern adopted conformations of proteins in the absence of solvent. This multi-technique approach has particular applicability for conformationally dynamic systems. Previously, we demonstrated the use of drift tube ion mobility-mass spectrometry (DT IM-MS) and electron capture dissociation (ECD) to study the metamorphic protein lymphotactin (Ltn). Ltn exists in equilibrium between distinct monomeric (Ltn10) ...

  7. 10 CFR 63.16 - Review of site characterization activities. 2

    2010-01-01

    ... 10 Energy 2 2010-01-01 2010-01-01 false Review of site characterization activities. 2 63.16... site characterization activities. 2 2 In addition to the review of site characterization activities... investigation and site characterization, to allow early identification of potential licensing issues for...

  8. EFFECTS OF CONFORMATION OF POLYMER LIGANDS IN COPPER(Ⅱ) COMPLEXES ON CATALYLIC ACTIVITIES AND MECHANISM OF OXIDATIVE COUPLING OF β-NAPHTHOL

    BIAN Kejian; LUO Chunqiao; CAO Mengjun

    1987-01-01

    Copper(Ⅱ) complexes of sericin, chitosan, 6-and 2-aminodeoxystarch were used as catalysts in oxidative coupling of β-naphthol, the effects of conformation of the polymer ligands in these complexes on activities of the catalysts and mechanisms of the reaction were studied. It was found that if the catalysts react with the substrate by mechanism similar to the enzymic catalysis they must be composed of polymer ligands with highly coiled, especially with densely helicoidal,conformations. While catalysts composed of loosely coiled or helicoidal ligands react with the substrate through molecular collision and have relatively lower activities only. Under nitrogen,catalysts from sericin and chitosam reacting with β-naphthol give optically active β-binaphthol,rotating polarized light to right, but the stereoselectivities are rather low.

  9. Identification of covalent active site inhibitors of dengue virus protease

    Koh-Stenta, Xiaoying; Joy, Joma; Wang, Si Fang; Kwek, Perlyn Zekui; Wee, John Liang Kuan; Wan, Kah Fei; Gayen, Shovanlal; Chen, Angela Shuyi; Kang, CongBao; Lee, May Ann; Poulsen, Anders; Vasudevan, Subhash G; Hill, Jeffrey; Nacro, Kassoum

    2015-01-01

    Dengue virus (DENV) protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described. PMID:26677315

  10. Identification of covalent active site inhibitors of dengue virus protease.

    Koh-Stenta, Xiaoying; Joy, Joma; Wang, Si Fang; Kwek, Perlyn Zekui; Wee, John Liang Kuan; Wan, Kah Fei; Gayen, Shovanlal; Chen, Angela Shuyi; Kang, CongBao; Lee, May Ann; Poulsen, Anders; Vasudevan, Subhash G; Hill, Jeffrey; Nacro, Kassoum

    2015-01-01

    Dengue virus (DENV) protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described. PMID:26677315

  11. Binding of 3,4,5,6-Tetrahydroxyazepanes to the Acid-[beta]-glucosidase Active Site: Implications for Pharmacological Chaperone Design for Gaucher Disease

    Orwig, Susan D.; Tan, Yun Lei; Grimster, Neil P.; Yu, Zhanqian; Powers, Evan T.; Kelly, Jeffery W.; Lieberman, Raquel L. (Scripps); (GIT)

    2013-03-07

    Pharmacologic chaperoning is a therapeutic strategy being developed to improve the cellular folding and trafficking defects associated with Gaucher disease, a lysosomal storage disorder caused by point mutations in the gene encoding acid-{beta}-glucosidase (GCase). In this approach, small molecules bind to and stabilize mutant folded or nearly folded GCase in the endoplasmic reticulum (ER), increasing the concentration of folded, functional GCase trafficked to the lysosome where the mutant enzyme can hydrolyze the accumulated substrate. To date, the pharmacologic chaperone (PC) candidates that have been investigated largely have been active site-directed inhibitors of GCase, usually containing five- or six-membered rings, such as modified azasugars. Here we show that a seven-membered, nitrogen-containing heterocycle (3,4,5,6-tetrahydroxyazepane) scaffold is also promising for generating PCs for GCase. Crystal structures reveal that the core azepane stabilizes GCase in a variation of its proposed active conformation, whereas binding of an analogue with an N-linked hydroxyethyl tail stabilizes GCase in a conformation in which the active site is covered, also utilizing a loop conformation not seen previously. Although both compounds preferentially stabilize GCase to thermal denaturation at pH 7.4, reflective of the pH in the ER, only the core azepane, which is a mid-micromolar competitive inhibitor, elicits a modest increase in enzyme activity for the neuronopathic G202R and the non-neuronopathic N370S mutant GCase in an intact cell assay. Our results emphasize the importance of the conformational variability of the GCase active site in the design of competitive inhibitors as PCs for Gaucher disease.

  12. PCR-based site-specific mutagenesis of peptide antibiotics FALL-39 and its biologic activities

    Yun-xia YANG; Yun FENG; Bo-yao WANG; Qi WU

    2004-01-01

    AIM: To construct PGEX-1λT-FALL-39 expression vector and its mutant vector, and study the relationship of function and structure. METHODS: A cDNA encoding mature FALL-39 was cloned from SPCA- 1 cell mRNA and the prokaryotic expression vector PGEX- 1λT-FALL-39 was constructed. Two kinds of polymerase chain reaction (PCR) for the site-direction mutagenesis were used to construct FALL-39 mutant expression vector, FALL-39-Lys-32 and FALL-39-Lys-24. Minimal effective concentration, minimal inhibitory concentration, and minimal bactericidal concentration were used to assay the antibacterial activities of these peptides. Effects of different solution on the antibacterial activity of FALL-39 and FALL-39-Lys-32 were observed by CFU determination. The hemolytic effects of these peptides were also examined on human red blood cells. RESULTS: Two site-specific mutants FALL-39-Lys-32 and FALL-39-Lys24 were obtained by PCR-induced mutagenesis. In comparison with two-step PCR which required two pairs of primers, one step PCR which required one pair of primers is a simple and efficient method for the PCR based site-specific mutagenesis. Using the prokaryotic expression system, the E coli-based products of recombinant FALL39 and its mutant peptides were also obtained. The antibacterial assay showed that FALL-39-Lys-32 and FALL-39-Lys24 were more potential in the antibacterial activity against E coli ML35p and Pseltdomonas aeruginosa ATCC27853 than that of FALL-39, and no increase in hemolysis was observed at the antibacterial concentrations. The antibacterial activity of FALL-39-Lys-32 against E coli was more potent than that of FALL-39 in NaCl-containing LB medium, while its activity was almost the same as FALL-39 in SO2-4 containing Medium E. CONCLUSION: PCR-based mutagensis is a useful model system for studying the structure and function relationship of antimicrobial peptides. Keeping α-helical conformation of FALL-39 and increasing net positive charge can increase the

  13. Polarizability of the active site of cytochrome c reduces the activation barrier for electron transfer

    Dinpajooh, Mohammadhasan; Martin, Daniel R.; Matyushov, Dmitry V.

    2016-06-01

    Enzymes in biology’s energy chains operate with low energy input distributed through multiple electron transfer steps between protein active sites. The general challenge of biological design is how to lower the activation barrier without sacrificing a large negative reaction free energy. We show that this goal is achieved through a large polarizability of the active site. It is polarized by allowing a large number of excited states, which are populated quantum mechanically by electrostatic fluctuations of the protein and hydration water shells. This perspective is achieved by extensive mixed quantum mechanical/molecular dynamics simulations of the half reaction of reduction of cytochrome c. The barrier for electron transfer is consistently lowered by increasing the number of excited states included in the Hamiltonian of the active site diagonalized along the classical trajectory. We suggest that molecular polarizability, in addition to much studied electrostatics of permanent charges, is a key parameter to consider in order to understand how enzymes work.

  14. Polarizability of the active site of cytochrome c reduces the activation barrier for electron transfer

    Dinpajooh, Mohammadhasan; Martin, Daniel R.; Matyushov, Dmitry V.

    2016-01-01

    Enzymes in biology’s energy chains operate with low energy input distributed through multiple electron transfer steps between protein active sites. The general challenge of biological design is how to lower the activation barrier without sacrificing a large negative reaction free energy. We show that this goal is achieved through a large polarizability of the active site. It is polarized by allowing a large number of excited states, which are populated quantum mechanically by electrostatic fluctuations of the protein and hydration water shells. This perspective is achieved by extensive mixed quantum mechanical/molecular dynamics simulations of the half reaction of reduction of cytochrome c. The barrier for electron transfer is consistently lowered by increasing the number of excited states included in the Hamiltonian of the active site diagonalized along the classical trajectory. We suggest that molecular polarizability, in addition to much studied electrostatics of permanent charges, is a key parameter to consider in order to understand how enzymes work. PMID:27306204

  15. Study the active site of flavonoid applying radiation chemistry

    Flavonoid are a large and important class of naturally occurring, low molecular weight benzo-γ-pyrone derivatives which are reported to have a myriad of biological activities, but the study on the active sites of flavonoids is still ambiguous. In this paper, rutin, quercetin and baicalin have been selected as model compounds. It is well known that rutin is used in inhibiting arteriosclerosis and baicalin is antibacterial and antiviral. They have similar basic structure, but their medicinal properties are so different, why? As most flavonoids contain carbonyl group, which can capture electron effectively, we predict that flavonoids can capture electron to form radical anion. The formation of anion radical may have influence on the mitochondrial electron transport chain. The difference in the ability of forming anion radical may cause the difference in their medicinal effects. (author)

  16. SITE-DIRECTED MUTAGENESIS OF PROPOSED ACTIVE-SITE RESIDUES OF PENICILLIN-BINDING PROTEIN-5 FROM ESCHERICHIA-COLI

    VANDERLINDEN, MPG; DEHAAN, L; DIDEBERG, O; KECK, W

    1994-01-01

    Alignment of the amino acid sequence of penicillin-binding protein 5 (PBP5) with the sequences of other members of the family of active-site-serine penicillin-interacting enzymes predicted the residues playing a role in the catalytic mechanism of PBP5. Apart from the active-site (Ser(44)), Lys(47),

  17. Identification of covalent active site inhibitors of dengue virus protease

    Koh-Stenta X

    2015-12-01

    Full Text Available Xiaoying Koh-Stenta,1 Joma Joy,1 Si Fang Wang,1 Perlyn Zekui Kwek,1 John Liang Kuan Wee,1 Kah Fei Wan,2 Shovanlal Gayen,1 Angela Shuyi Chen,1 CongBao Kang,1 May Ann Lee,1 Anders Poulsen,1 Subhash G Vasudevan,3 Jeffrey Hill,1 Kassoum Nacro11Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR, Singapore; 2Novartis Institute for Tropical Diseases, Singapore; 3Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, SingaporeAbstract: Dengue virus (DENV protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described.Keywords: flavivirus protease, small molecule optimization, covalent inhibitor, active site binding, pyrazole ester derivatives

  18. Maxey Flats low-level waste disposal site closure activities

    The Maxey Flats Radioactive Waste Disposal Facility in Fleming County, Kentucky is in the process of being closed. The facility opened for commercial business in the spring of 1963 and received approximately 4.75 million cubic feet of radioactive waste by the time it was closed in December of 1977. During fourteen years of operation approximately 2.5 million curies of by-product material, 240,000 kilograms of source material, and 430 kilograms of special nuclear material were disposed. The Commonwealth purchased the lease hold estate and rights in May 1978 from the operating company. This action was taken to stabilize the facility and prepare it for closure consisting of passive care and monitoring. To prepare the site for closure, a number of remedial activities had to be performed. The remediation activities implemented have included erosion control, surface drainage modifications, installation of a temporary plastic surface cover, leachate removal, analysis, treatment and evaporation, US DOE funded evaporator concentrates solidification project and their on-site disposal in an improved disposal trench with enhanced cover for use in a humid environment situated in a fractured geology, performance evaluation of a grout injection demonstration, USGS subsurface geologic investigation, development of conceptual closure designs, and finally being added to the US EPA National Priority List for remediation and closure under Superfund. 13 references, 3 figures

  19. Effects of the substitution of amino acid residues, through chemical synthesis, on the conformation and activity of antimicrobial peptides

    Regina C. Adão

    2012-06-01

    Full Text Available Antimicrobial peptides make up an assorted group of molecules which contain from 12 to 50 amino acid residues and which may be produced by microorganisms, plants and animals. From the discovery that these biomolecules are lethal to bacteria, inhibiting the pathogenic organism’s growth, and are also related to innate and adapted defense mechanisms, the investigation of such molecules came to be an emergent research field, in which more than 1800 antimicrobial peptides have so far been discovered throughout the last three decades. These molecules are potential representatives of a new generation of antibiotic agents and the main motivation for such use is their activity against a wide variety of pathogens, including Gram-positive and Gram-negative bacteria as well as fungi and viruses. An important class of comprising some of these peptides may be found in anurans, from which it has been isolated, a considerable number of antimicrobial peptides with diverse sequences and structures, including linear and dimeric ones. In this work monomeric chains (CH1 e CH2 of the heterodimeric antimicrobial peptide distinctin (isolated in 1999 from Phyllomedusa distincta anurans, as well as its mutated monomers (CH1-S and CH2-S and the heterodimer itself were synthesized. The distinctin is the peptide with two chains of different sequences (Table 1 bound each other by disulfide bond from the cystein residues constituting the heterodimer. To investigate the effects on the biological activity by amino acids substitution at normal distinctin CH1 and CH2 chains, both were synthesized as well as their similar chains (CH1-S and CH2-S in which the cystein (Fig.1 a residues of each chain were changed by serin residues (Fig. 1 b. The new chains were named mutants. The synthesis was carried out in solid phase, using Fmoc strategy. The heterodimer distinctin was obtained from CH1 and CH2 chains coupling through cystein residues air oxidation. The results from HPLC

  20. Active serine involved in the stabilization of the active site loop in the Humicola lanuginosa lipase

    Peters, Günther H.j.; Svendsen, A.; Langberg, H.; Vind, J.; Patkar, S.A.; Toxvaerd, S.; Kinnunen, P.K.J.

    1998-01-01

    reveal that the hinges of the active site lid are more flexible in the wild-type Hll than in S146A. In contrast, larger fluctuations are observed in the middle region of the active site loop in S 146A than in Hll. These findings reveal that the single mutation (S146A) of the active site serine leads to...

  1. The tertiary origin of the allosteric activation of E. coli glucosamine-6-phosphate deaminase studied by sol-gel nanoencapsulation of its T conformer.

    Sergio Zonszein

    Full Text Available The role of tertiary conformational changes associated to ligand binding was explored using the allosteric enzyme glucosamine-6-phosphate (GlcN6P deaminase from Escherichia coli (EcGNPDA as an experimental model. This is an enzyme of amino sugar catabolism that deaminates GlcN6P, giving fructose 6-phosphate and ammonia, and is allosterically activated by N-acetylglucosamine 6-phosphate (GlcNAc6P. We resorted to the nanoencapsulation of this enzyme in wet silica sol-gels for studying the role of intrasubunit local mobility in its allosteric activation under the suppression of quaternary transition. The gel-trapped enzyme lost its characteristic homotropic cooperativity while keeping its catalytic properties and the allosteric activation by GlcNAc6P. The nanoencapsulation keeps the enzyme in the T quaternary conformation, making possible the study of its allosteric activation under a condition that is not possible to attain in a soluble phase. The involved local transition was slowed down by nanoencapsulation, thus easing the fluorometric analysis of its relaxation kinetics, which revealed an induced-fit mechanism. The absence of cooperativity produced allosterically activated transitory states displaying velocity against substrate concentration curves with apparent negative cooperativity, due to the simultaneous presence of subunits with different substrate affinities. Reaction kinetics experiments performed at different tertiary conformational relaxation times also reveal the sequential nature of the allosteric activation. We assumed as a minimal model the existence of two tertiary states, t and r, of low and high affinity, respectively, for the substrate and the activator. By fitting the velocity-substrate curves as a linear combination of two hyperbolic functions with Kt and Kr as KM values, we obtained comparable values to those reported for the quaternary conformers in solution fitted to MWC model. These results are discussed in the

  2. A single active catalytic site is sufficient to promote transport in P-glycoprotein.

    Bársony, Orsolya; Szalóki, Gábor; Türk, Dóra; Tarapcsák, Szabolcs; Gutay-Tóth, Zsuzsanna; Bacsó, Zsolt; Holb, Imre J; Székvölgyi, Lóránt; Szabó, Gábor; Csanády, László; Szakács, Gergely; Goda, Katalin

    2016-01-01

    P-glycoprotein (Pgp) is an ABC transporter responsible for the ATP-dependent efflux of chemotherapeutic compounds from multidrug resistant cancer cells. Better understanding of the molecular mechanism of Pgp-mediated transport could promote rational drug design to circumvent multidrug resistance. By measuring drug binding affinity and reactivity to a conformation-sensitive antibody we show here that nucleotide binding drives Pgp from a high to a low substrate-affinity state and this switch coincides with the flip from the inward- to the outward-facing conformation. Furthermore, the outward-facing conformation survives ATP hydrolysis: the post-hydrolytic complex is stabilized by vanadate, and the slow recovery from this state requires two functional catalytic sites. The catalytically inactive double Walker A mutant is stabilized in a high substrate affinity inward-open conformation, but mutants with one intact catalytic center preserve their ability to hydrolyze ATP and to promote drug transport, suggesting that the two catalytic sites are randomly recruited for ATP hydrolysis. PMID:27117502

  3. Interactions of plasminogen activator inhibitor-1 with vitronectin involve an extensive binding surface and induce mutual conformational rearrangements

    Blouse, Grant E; Dupont, Daniel Miotto; Schar, Christine R; Jensen, Jan K; Minor, Kenneth H; Anagli, John Y; Gårdsvoll, Henrik; Ploug, Michael; Peterson, Cynthia B; Andreasen, Peter A

    2009-01-01

    full-length vitronectin is present. Nonetheless, in all cases, the initial fast interaction is followed by slower fluorescence changes attributed to a conformational change in PAI-1. Complementary experiments using an engineered, fluorescently silent PAI-1 with non-natural amino acids showed that...

  4. The ClpP N-Terminus Coordinates Substrate Access with Protease Active Site Reactivity

    Energy-dependent protein degradation machines, such as the Escherichia coli protease ClpAP, require regulated interactions between the ATPase component (ClpA) and the protease component (ClpP) for function. Recent studies indicate that the ClpP N-terminus is essential in these interactions, yet the dynamics of this region remain unclear. Here, we use synchrotron hydroxyl radical footprinting and kinetic studies to characterize functionally important conformational changes of the ClpP N-terminus. Footprinting experiments show that the ClpP N-terminus becomes more solvent-exposed upon interaction with ClpA. In the absence of ClpA, deletion of the ClpP N-terminus increases the initial degradation rate of large peptide substrates 5-15-fold. Unlike ClpAP, ClpP?N exhibits a distinct slow phase of product formation that is eliminated by the addition of hydroxylamine, suggesting that truncation of the N-terminus leads to stabilization of the acyl-enzyme intermediate. These results indicate that (1) the ClpP N-terminus acts as a 'gate' controlling substrate access to the active sites, (2) binding of ClpA opens this 'gate', allowing substrate entry and formation of the acyl-enzyme intermediate, and (3) closing of the N-terminal 'gate' stimulates acyl-enzyme hydrolysis.

  5. Metavanadate at the active site of the phosphatase VHZ.

    Kuznetsov, Vyacheslav I; Alexandrova, Anastassia N; Hengge, Alvan C

    2012-09-01

    Vanadate is a potent modulator of a number of biological processes and has been shown by crystal structures and NMR spectroscopy to interact with numerous enzymes. Although these effects often occur under conditions where oligomeric forms dominate, the crystal structures and NMR data suggest that the inhibitory form is usually monomeric orthovanadate, a particularly good inhibitor of phosphatases because of its ability to form stable trigonal-bipyramidal complexes. We performed a computational analysis of a 1.14 Å structure of the phosphatase VHZ in complex with an unusual metavanadate species and compared it with two classical trigonal-bipyramidal vanadate-phosphatase complexes. The results support extensive delocalized bonding to the apical ligands in the classical structures. In contrast, in the VHZ metavanadate complex, the central, planar VO(3)(-) moiety has only one apical ligand, the nucleophilic Cys95, and a gap in electron density between V and S. A computational analysis showed that the V-S interaction is primarily ionic. A mechanism is proposed to explain the formation of metavanadate in the active site from a dimeric vanadate species that previous crystallographic evidence has shown to be able to bind to the active sites of phosphatases related to VHZ. Together, the results show that the interaction of vanadate with biological systems is not solely reliant upon the prior formation of a particular inhibitory form in solution. The catalytic properties of an enzyme may act upon the oligomeric forms primarily present in solution to generate species such as the metavanadate ion observed in the VHZ structure. PMID:22876963

  6. An Allosteric Cross-Talk Between the Activation Loop and the ATP Binding Site Regulates the Activation of Src Kinase

    Pucheta-Martínez, Encarna; Saladino, Giorgio; Morando, Maria Agnese; Martinez-Torrecuadrada, Jorge; Lelli, Moreno; Sutto, Ludovico; D’Amelio, Nicola; Gervasio, Francesco Luigi

    2016-04-01

    Phosphorylation of the activation loop is a fundamental step in the activation of most protein kinases. In the case of the Src tyrosine kinase, a prototypical kinase due to its role in cancer and its historic importance, phosphorylation of tyrosine 416 in the activation loop is known to rigidify the structure and contribute to the switch from the inactive to a fully active form. However, whether or not phosphorylation is able per-se to induce a fully active conformation, that efficiently binds ATP and phosphorylates the substrate, is less clear. Here we employ a combination of solution NMR and enhanced-sampling molecular dynamics simulations to fully map the effects of phosphorylation and ATP/ADP cofactor loading on the conformational landscape of Src tyrosine kinase. We find that both phosphorylation and cofactor binding are needed to induce a fully active conformation. What is more, we find a complex interplay between the A-loop and the hinge motion where the phosphorylation of the activation-loop has a significant allosteric effect on the dynamics of the C-lobe.

  7. 10 CFR 60.18 - Review of site characterization activities. 2

    2010-01-01

    ... 10 Energy 2 2010-01-01 2010-01-01 false Review of site characterization activities. 2 60.18... IN GEOLOGIC REPOSITORIES Licenses Preapplication Review § 60.18 Review of site characterization activities. 2 2 In addition to the review of site characterization activities specified in this section,...

  8. Relationship between SU Subdomains That Regulate the Receptor-Mediated Transition from the Native (Fusion-Inhibited) to the Fusion-Active Conformation of the Murine Leukemia Virus Glycoprotein

    Lavillette, Dimitri; Ruggieri, Alessia; Boson, Bertrand; Maurice, Marielle; Cosset, François-Loïc

    2002-01-01

    Envelope glycoproteins (Env) of retroviruses are trimers of SU (surface) and TM (transmembrane) heterodimers and are expressed on virions in fusion-competent forms that are likely to be metastable. Activation of the viral receptor-binding domain (RBD) via its interaction with a cell surface receptor is thought to initiate a cascade of events that lead to refolding of the Env glycoprotein into its stable fusion-active conformation. While the fusion-active conformation of the TM subunit has bee...

  9. Hazardous Material Storage Facilities and Sites - WASTE_SOLID_ACTIVE_PERMITTED_IDEM_IN: Active Permitted Solid Waste Sites in Indiana (Indiana Department of Environmental Management, Point Shapefile)

    NSGIC GIS Inventory (aka Ramona) — WASTE_SOLID_ACTIVE_PERMITTED_IDEM_IN is a point shapefile that contains active permitted solid waste site locations in Indiana, provided by personnel of Indiana...

  10. Composite active site of chondroitin lyase ABC accepting both epimers of uronic acid

    Shaya, D.; Hahn, Bum-Soo; Bjerkan, Tonje Marita; Kim, Wan Seok; Park, Nam Young; Sim, Joon-Soo; Kim, Yeong-Shik; Cygler, M. (Catholic Univ of Korea); (NUST); (McGill); (Nat); (Natural Products Res Inst, Korea)

    2008-03-19

    Enzymes have evolved as catalysts with high degrees of stereospecificity. When both enantiomers are biologically important, enzymes with two different folds usually catalyze reactions with the individual enantiomers. In rare cases a single enzyme can process both enantiomers efficiently, but no molecular basis for such catalysis has been established. The family of bacterial chondroitin lyases ABC comprises such enzymes. They can degrade both chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans at the nonreducing end of either glucuronic acid (CS) or its epimer iduronic acid (DS) by a {beta}-elimination mechanism, which commences with the removal of the C-5 proton from the uronic acid. Two other structural folds evolved to perform these reactions in an epimer-specific fashion: ({alpha}/{alpha}){sub 5} for CS (chondroitin lyases AC) and {beta}-helix for DS (chondroitin lyases B); their catalytic mechanisms have been established at the molecular level. The structure of chondroitinase ABC from Proteus vulgaris showed surprising similarity to chondroitinase AC, including the presence of a Tyr-His-Glu-Arg catalytic tetrad, which provided a possible mechanism for CS degradation but not for DS degradation. We determined the structure of a distantly related Bacteroides thetaiotaomicron chondroitinase ABC to identify additional structurally conserved residues potentially involved in catalysis. We found a conserved cluster located {approx}12 {angstrom} from the catalytic tetrad. We demonstrate that a histidine in this cluster is essential for catalysis of DS but not CS. The enzyme utilizes a single substrate-binding site while having two partially overlapping active sites catalyzing the respective reactions. The spatial separation of the two sets of residues suggests a substrate-induced conformational change that brings all catalytically essential residues close together.

  11. Thermal-induced conformational changes in the product release area drive the enzymatic activity of xylanases 10B: Crystal structure, conformational stability and functional characterization of the xylanase 10B from Thermotoga petrophila RKU-1

    Santos, Camila Ramos; Meza, Andreia Navarro [Laboratorio Nacional de Biociencias (LNBio), Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP (Brazil); Hoffmam, Zaira Bruna; Silva, Junio Cota; Alvarez, Thabata Maria; Ruller, Roberto [Laboratorio Nacional de Ciencia e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP (Brazil); Giesel, Guilherme Menegon; Verli, Hugo [Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil); Squina, Fabio Marcio [Laboratorio Nacional de Ciencia e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP (Brazil); Prade, Rolf Alexander [Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK (United States); Murakami, Mario Tyago, E-mail: mario.murakami@lnbio.org.br [Laboratorio Nacional de Biociencias (LNBio), Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP (Brazil)

    2010-12-10

    Research highlights: {yields} The hyperthermostable xylanase 10B from Thermotoga petrophila RKU-1 produces exclusively xylobiose at the optimum temperature. {yields} Circular dichroism spectroscopy suggests a coupling effect of temperature-induced structural changes with its enzymatic behavior. {yields} Crystallographic and molecular dynamics studies indicate that conformational changes in the product release area modulate the enzyme action mode. -- Abstract: Endo-xylanases play a key role in the depolymerization of xylan and recently, they have attracted much attention owing to their potential applications on biofuels and paper industries. In this work, we have investigated the molecular basis for the action mode of xylanases 10B at high temperatures using biochemical, biophysical and crystallographic methods. The crystal structure of xylanase 10B from hyperthermophilic bacterium Thermotoga petrophila RKU-1 (TpXyl10B) has been solved in the native state and in complex with xylobiose. The complex crystal structure showed a classical binding mode shared among other xylanases, which encompasses the -1 and -2 subsites. Interestingly, TpXyl10B displayed a temperature-dependent action mode producing xylobiose and xylotriose at 20 {sup o}C, and exclusively xylobiose at 90 {sup o}C as assessed by capillary zone electrophoresis. Moreover, circular dichroism spectroscopy suggested a coupling effect of temperature-induced structural changes with this particular enzymatic behavior. Molecular dynamics simulations supported the CD analysis suggesting that an open conformational state adopted by the catalytic loop (Trp297-Lys326) provokes significant modifications in the product release area (+1,+2 and +3 subsites), which drives the enzymatic activity to the specific release of xylobiose at high temperatures.

  12. Thermal-induced conformational changes in the product release area drive the enzymatic activity of xylanases 10B: Crystal structure, conformational stability and functional characterization of the xylanase 10B from Thermotoga petrophila RKU-1

    Research highlights: → The hyperthermostable xylanase 10B from Thermotoga petrophila RKU-1 produces exclusively xylobiose at the optimum temperature. → Circular dichroism spectroscopy suggests a coupling effect of temperature-induced structural changes with its enzymatic behavior. → Crystallographic and molecular dynamics studies indicate that conformational changes in the product release area modulate the enzyme action mode. -- Abstract: Endo-xylanases play a key role in the depolymerization of xylan and recently, they have attracted much attention owing to their potential applications on biofuels and paper industries. In this work, we have investigated the molecular basis for the action mode of xylanases 10B at high temperatures using biochemical, biophysical and crystallographic methods. The crystal structure of xylanase 10B from hyperthermophilic bacterium Thermotoga petrophila RKU-1 (TpXyl10B) has been solved in the native state and in complex with xylobiose. The complex crystal structure showed a classical binding mode shared among other xylanases, which encompasses the -1 and -2 subsites. Interestingly, TpXyl10B displayed a temperature-dependent action mode producing xylobiose and xylotriose at 20 oC, and exclusively xylobiose at 90 oC as assessed by capillary zone electrophoresis. Moreover, circular dichroism spectroscopy suggested a coupling effect of temperature-induced structural changes with this particular enzymatic behavior. Molecular dynamics simulations supported the CD analysis suggesting that an open conformational state adopted by the catalytic loop (Trp297-Lys326) provokes significant modifications in the product release area (+1,+2 and +3 subsites), which drives the enzymatic activity to the specific release of xylobiose at high temperatures.

  13. Characterization of Metal Binding in the Active Sites of acireductone dioxygenase Isoforms from Klebsiella ATCC 8724

    S Chai; T Ju; M Dang; R Goldsmith; M Maroney; T Pochapsky

    2011-12-31

    The two acireductone dioxygenase (ARD) isozymes from the methionine salvage pathway of Klebsiella ATCC 8724 present an unusual case in which two enzymes with different structures and distinct activities toward their common substrates (1,2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene and dioxygen) are derived from the same polypeptide chain. Structural and functional differences between the two isozymes are determined by the type of M{sup 2+} metal ion bound in the active site. The Ni{sup 2+}-bound NiARD catalyzes an off-pathway shunt from the methionine salvage pathway leading to the production of formate, methylthiopropionate, and carbon monoxide, while the Fe{sup 2+}-bound FeARD catalyzes the on-pathway formation of methionine precursor 2-keto-4-methylthiobutyrate and formate. Four potential protein-based metal ligands were identified by sequence homology and structural considerations. Based on the results of site-directed mutagenesis experiments, X-ray absorption spectroscopy (XAS), and isothermal calorimetry measurements, it is concluded that the same four residues, His96, His98, Glu102 and His140, provide the protein-based ligands for the metal in both the Ni- and Fe-containing forms of the enzyme, and subtle differences in the local backbone conformations trigger the observed structural and functional differences between the FeARD and NiARD isozymes. Furthermore, both forms of the enzyme bind their respective metals with pseudo-octahedral geometry, and both may lose a histidine ligand upon binding of substrate under anaerobic conditions. However, mutations at two conserved nonligand acidic residues, Glu95 and Glu100, result in low metal contents for the mutant proteins as isolated, suggesting that some of the conserved charged residues may aid in transfer of metal from in vivo sources or prevent the loss of metal to stronger chelators. The Glu100 mutant reconstitutes readily but has low activity. Mutation of Asp101 results in an active enzyme that incorporates

  14. A comparative study of drug resistance mechanism associated with active site and non-active site mutations: I388N and D425G mutants of acetyl-coenzyme-A carboxylase.

    Zhu, Xiao-Lei; Yang, Guang-Fu

    2012-03-01

    A major concern in the development of acetyl-CoA carboxylase-inhibiting (ACCase; EC 6.4.1.2) herbicides is the emergence of resistance as a result of the selection of distinct mutations within the CT domain. Mutations associated with resistance have been demonstrated to include both active sites and non-active sites, including Ile-1781-Leu, Trp- 2027-Cys, Ile-2041-Asn, Asp-2078-Gly, and Gly-2096-Ala (numbered according to the Alopecurus myosuroides plastid ACCase). In the present study, extensive computational simulations, including molecular dynamics (MD) simulations and molecular mechanics-Poisson-Boltzmann surface area (MM/PBSA) calculations, were carried out to compare the molecular mechanisms of active site mutation (I388N) and non-active site mutation (D425G) in Alopecurus myosuroides resistance to some commercial herbicides targeting ACCase, including haloxyfop (HF), diclofop (DF) and fenoxaprop (FR). All of the computational model and energetic results indicated that both I388N and D425G mutations have effects on the conformational change of the binding pocket. The π-π interaction between ligand and Phe377 and Tyr161' residues, which make an important contribution to the binding affinity, was decreased after mutation. As a result, the mutant-type ACCase has a lower affinity for the inhibitor than the wild-type enzyme, which accounts for the molecular basis of herbicidal resistance. The structural and mechanistic insights obtained from the present study will deepen our understanding of the interactions between ACCase and herbicides, which provides a molecular basis for the future design of a promising inhibitor with low resistance risk. PMID:22242795

  15. Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography

    Keedy, Daniel A.; Kenner, Lillian R.; Warkentin, Matthew; Woldeyes, Rahel A.; Hopkins, Jesse B.; Thompson, Michael C.; Brewster, Aaron S.; Van Benschoten, Andrew H.; Baxter, Elizabeth L.; Uervirojnangkoorn, Monarin; McPhillips, Scott E.; Song, Jinhu; Alonso-Mori, Roberto; Holton, James M.; Weis, William I.; Brunger, Axel T.; Soltis, S. Michael; Lemke, Henrik; Gonzalez, Ana; Sauter, Nicholas K.; Cohen, Aina E.; van den Bedem, Henry; Thorne, Robert E.; Fraser, James S.

    2015-09-30

    Determining the interconverting conformations of dynamic proteins in atomic detail is a major challenge for structural biology. Conformational heterogeneity in the active site of the dynamic enzyme cyclophilin A (CypA) has been previously linked to its catalytic function, but the extent to which the different conformations of these residues are correlated is unclear. Here we compare the conformational ensembles of CypA by multitemperature synchrotron crystallography and fixed-target X-ray free-electron laser (XFEL) crystallography. The diffraction-before-destruction nature of XFEL experiments provides a radiation-damage-free view of the functionally important alternative conformations of CypA, confirming earlier synchrotron-based results. We monitored the temperature dependences of these alternative conformations with eight synchrotron datasets spanning 100-310 K. Multiconformer models show that many alternative conformations in CypA are populated only at 240 K and above, yet others remain populated or become populated at 180 K and below. These results point to a complex evolution of conformational heterogeneity between 180-–240 K that involves both thermal deactivation and solvent-driven arrest of protein motions in the crystal. The lack of a single shared conformational response to temperature within the dynamic active-site network provides evidence for a conformation shuffling model, in which exchange between rotamer states of a large aromatic ring in the middle of the network shifts the conformational ensemble for the other residues in the network. Together, our multitemperature analyses and XFEL data motivate a new generation of temperature- and time-resolved experiments to structurally characterize the dynamic underpinnings of protein function.

  16. Conformational preferences of synthetic peptides derived from the immunodominant site of the circumsporozoite protein of Plasmodium falciparum by sup 1 H NMR

    Dyson, H.J.; Satterthwait, A.C.; Lerner, R.A.; Wright, P.E. (Research Institute of Scripps Clinic, La Jolla, CA (USA))

    1990-08-28

    Proton nuclear magnetic resonance and ultraviolet circular dichroism spectroscopy have been used to probe the conformational ensemble of the tandemly repeated tetrapeptide unit of the circumsporozoite coat protein of the malaria parasite Plasmodium falciparum. Peptides based on the Asn-Ala-Asn-Pro and Asn-Pro-Asn-Ala cadences and composed of one to three tetrapeptide units were synthesized and examined using one- and two-dimensional NMR spectroscopy. The chemical shift of the amide protons, the temperature dependence of the amide proton chemical shift, and the patterns of NOE connectivities in the various peptides give evidence for the presence of a substantial population of folded conformers in several of the peptides in water solution at pH 5.0. Correlations between the behavior of the tandemly repeated units in different peptides have been used to infer the structure(s) of the folded conformers. The data are consistent with the presence of turnlike structures stabilized by hydrogen bonding of the backbone amid protons of the alanines and the asparagine residues preceding them. Specific differences in the strengths of NOEs between peptides of different lengths indicate that the folded structure is considerably stabilized by the presence of the asparagine residue following the alanine. Differences between peptides with different cadences of the tandemly repeating unit indicate that a repeating structural motif is formed by the Asn-Pro-Asn-Ala-(Asn) cadence.

  17. The mechanisms of substrates interaction with the active site of Mycobacterium tuberculosis tyrosyl-tRNA synthetase studied by molecular dynamics simulations

    Mykuliak V. V.

    2014-03-01

    Full Text Available Aim. To study the mechanisms of substrates interaction with the active site of Mycobacterium tuberculosis tyrosyl-tRNA synthetase (MtTyrRS. Methods. Complexes of MtTyrRS with tyrosine, ATP and tyrosyl adenylate were constructed by superposition of the MtTyrRS structure and crystallographic structures of bacterial TyrRS. All complexes of MtTyrRS with substrates were investigated by molecular dynamics (MD simulations in solution. Results. It was shown the formation of network of hydrogen bonds between substrates and the MtTyrRS active center, which were stable in the course of MD simulations. ATP binds in the active site both by hydrogen bonds and via electrostatic interactions with Lys231 and Lys234 of catalytic KFGKS motif. Conclusions. The L-tyrosine binding site in the enzyme active site is negatively charged, whereas the ATP binding site contains positive Lys231 and Lys234 residues of catalytic KFGKS motif. The occupancy of H-bonds between substrates and the enzyme evidences a significant conformational mobility of the active site.

  18. Detection limit for activation measurements in ultralow background sites

    Trache, Livius; Chesneanu, D.; Margineanu, R.; Pantelica, A.; Ghita, D. G.; Burducea, I.; Straticiuc, M.; Tang, X. D.

    2014-09-01

    We used 12C +13C fusion at the beam energies E = 6, 7 and 8 MeV to determine the sensitivity and the limits of activation method measurements in ultralow background sites. A 13C beam of 0.5 μA from the 3 MV Tandem accelerator of the Horia Hulubei National Institute of Physics and Nuclear Engineering - IFIN HH impinged on thick graphite targets. After about 24 hrs of irradiation targets were measured in two different laboratories: one with a heavy shielded Ge detector in the institute (at the surface) and one located underground in the microBequerel laboratory, in the salt mine of Slanic-Prahova, Romania. The 1369- and 2754 keV peaks from 24Na deactivation were clearly observed in the γ-ray spectra obtained for acquisitions lasting a few hours, or a few days. Determination of the detection limit in evaluating the cross sections for the target irradiated at Ec . m = 3 MeV indicates the fact that it is possible to measure gamma spectrum in underground laboratory down to Ec . m = 2 . 6 MeV. Cleaning the spectra with beta-gamma coincidences and increasing beam intensity 20 times will take as further down. The measurements are motivated by the study of the 12 C +12 C reaction at astrophysical energies.

  19. Comparative study of convolution, superposition, and fast superposition algorithms in conventional radiotherapy, three-dimensional conformal radiotherapy, and intensity modulated radiotherapy techniques for various sites, done on CMS XIO planning system

    Muralidhar K

    2009-01-01

    Full Text Available The aim of this study is to compare the dosimetry results that are obtained by using Convolution, Superposition and Fast Superposition algorithms in Conventional Radiotherapy, Three-Dimensional Conformal Radiotherapy (3D-CRT, and Intensity Modulated Radiotherapy (IMRT for different sites, and to study the suitability of algorithms with respect to site and technique. For each of the Conventional, 3D-CRT, and IMRT techniques, four different sites, namely, Lung, Esophagus, Prostate, and Hypopharynx were analyzed. Treatment plans were created using 6MV Photon beam quality using the CMS XiO (Computerized Medical System, St.Louis, MO treatment planning system. The maximum percentage of variation recorded between algorithms was 3.7% in case of Ca.Lung, for the IMRT Technique. Statistical analysis was performed by comparing the mean relative difference, Conformity Index, and Homogeneity Index for target structures. The fast superposition algorithm showed excellent results for lung and esophagus cases for all techniques. For the prostate, the superposition algorithm showed better results in all techniques. In the conventional case of the hypopharynx, the convolution algorithm was good. In case of Ca. Lung, Ca Prostate, Ca Esophagus, and Ca Hypopharynx, OARs got more doses with the superposition algorithm; this progressively decreased for fast superposition and convolution algorithms, respectively. According to this study the dosimetric results using different algorithms led to significant variation and therefore care had to be taken while evaluating treatment plans. The choice of a dose calculation algorithm may in certain cases even influence clinical results.

  20. Small-Angle Neutron Scattering Reveals pH-Dependent Conformational Changes in Trichoderma reesei Cellobiohydrolase I: Implications for Enzymatic Activity

    Pingali, Sai Venkatesh [ORNL; O' Neill, Hugh Michael [ORNL; McGaughey, Joseph [ORNL; Urban, Volker S [ORNL; Rempe, Caroline S [ORNL; Petridis, Loukas [ORNL; Smith, Jeremy C [ORNL; Evans, Barbara R [ORNL; Heller, William T [ORNL

    2011-01-01

    Cellobiohydrolase I (Cel7A) of the fungus Trichoderma reesei (now classified as an anamorph of Hypocrea jecorina) hydrolyzes crystalline cellulose to soluble sugars, making it of key interest for producing fermentable sugars from biomass for biofuel production. The activity of the enzyme is pH-dependent, with its highest activity occurring at pH 4 5. To probe the response of the solution structure of Cel7A to changes in pH, we measured small angle neutron scattering of it in a series of solutions having pH values of 7.0, 6.0, 5.3, and 4.2. As the pH decreases from 7.0 to 5.3, the enzyme structure remains well defined, possessing a spatial differentiation between the cellulose binding domain and the catalytic core that only changes subtly. At pH 4.2, the solution conformation of the enzyme changes to a structure that is intermediate between a properly folded enzyme and a denatured, unfolded state, yet the secondary structure of the enzyme is essentially unaltered. The results indicate that at the pH of optimal activity, the catalytic core of the enzyme adopts a structure in which the compact packing typical of a fully folded polypeptide chain is disrupted and suggest that the increased range of structures afforded by this disordered state plays an important role in the increased activity of Cel7A through conformational selection.

  1. Finnsjoen study site. Scope of activities and main results

    The Finnsjoen study site was selected in 1977 to provide input to the KBS-1 and KBS-2 performance assessments. The site was later used as a test site for testing new instruments and new site characterization methods, as well as a research site for studying mainly groundwater flow and groundwater transport. All together, the Finnsjoen studies have involved 11 cored boreholes, down to max 700 m depth, and extensive borehole geophysical, geochemical and geohydraulic measurements, as well as rock stress measurements and tracer tests. This report presents the scope of the Finnsjoen studies together with main results. Conceptual uncertainties in assumptions and models are discussed with emphasis on the models used for the performance assessment SKB91. Of special interest for the Finnsjoen study site is the strong influence caused by a subhorizontal fracture zone on groundwater flow, transport and chemistry

  2. Impact of an extruded nucleotide on cleavage activity and dynamic catalytic core conformation of the hepatitis delta virus ribozyme

    Šefčíková, J.; Krasovská, Maryna V.; Špačková, Naďa; Šponer, Jiří; Walter, N.G.

    2007-01-01

    Roč. 85, 5-6 (2007), s. 392-406. ISSN 0006-3525 R&D Projects: GA ČR(CZ) GA203/05/0388; GA ČR(CZ) GA203/05/0009; GA AV ČR(CZ) 1QS500040581; GA MŠk(CZ) LC512 Institutional research plan: CEZ:AV0Z50040702 Keywords : conformational dynamics * hepatitis delta virus * molecular dynamics Subject RIV: BO - Biophysics Impact factor: 2.389, year: 2007

  3. Attitudinal Conformity and Anonymity

    Tyson, Herbert; Kaplowitz, Stan

    1977-01-01

    Tested college students for conformity when conditions contributing to conformity were absent. Found that social pressures (responding in public, being surveyed by fellow group members) are necessary to produce conformity. (RL)

  4. Regulation of conformation and activity of nuclear NF-kappaBeta p65 by phosphorylation, chaperones and p65 DNA-binding

    Milanovic, Maja

    2014-01-01

    The TF NF-kB is an important regulator of immunity, stress responses as well as apoptosis, cell cycle progression and oncogenesis. NF-kB is activated by various stimuli and regulates expression many different target genes. The first part of this work shows that stimulation of cells with the cytokines TNF or IL-1 results in a profound conformational switch of the NF-kB subunit p65, as revealed by limited proteolysis assays. The cytokine-triggered reconfiguration of p65 mainly occurs for p65 co...

  5. Interconversion of active and inactive 30 S ribosomal subunits is accompanied by a conformational change in the decoding region of 16 S rRNA

    Moazed, D; Van Stolk, B J; Douthwaite, S;

    1986-01-01

    Zamir, Elson and their co-workers have shown that 30 S ribosomal subunits are reversibly inactivated by depletion of monovalent or divalent cations. We have re-investigated the conformation of 16 S rRNA in the active and inactive forms of the 30 S subunit, using a strategy that is designed......' regions of 16 S rRNA. The inactive form also shows significantly decreased reactivity at positions 1533 to 1538 (the Shine-Dalgarno region), in agreement with earlier findings. The principal changes in reactivity involve the universally conserved nucleotides G926, C1395, A1398 and G1401. The three purines...

  6. Retraction: Open and closed conformations reveal induced fit movements in butyrate kinase 2 activation. J. Diao, Y. D. Ma, and M. S. Hasson.

    2012-06-01

    The following article from Proteins: Structure, Function, and Bioinformatics, "Open and closed conformations reveal induced fit movements in butyrate kinase 2 activation," by Jiasheng Diao, Yunglin D. Ma, and Miriam S. Hasson, published online on 21 October 2010 in Wiley Online Library (onlinelibrary.wiley.com), has been retracted by agreement between the journal Editor in Chief, Bertrand Garcia-Moreno, and Wiley Periodicals. The retraction has been agreed because it was established by internal investigation performed by Purdue University that the authors of this article are not the owners of the data and have no right to publication. PMID:19847916

  7. Accelerator production of tritium activities at the Savannah River Site

    The Savannah River site (SRS) has been chosen as the site to host the accelerator production of tritium (APT). This facility, which will produce tritium for national defense purposes, is a natural extension of the site's original mission. All of the tritium for U.S. weapons needs has been produced at SRS in the heavy water reactors (now shut down) that operated until 1988. Much of the tritium-handling infrastructure still exists at SRS, and the tritium recycling and purification facilities are new and fully operational. This paper summarizes the reasons for the choice of the site and describes some of the early SRS efforts to learn the new technology, to weave into it a strong operations/production ethic, and to prepare the site to host the APT

  8. Health physics and public health activities at hazardous wastes sites

    The Agency for Toxic Substances and Disease Registry (ATSDR) has worked with the U.S. Environmental Protection Agency (EPA) at several sites contaminated with radioactive materials. The Navajo Brown Vandever (B-V) uranium mine site near Bluewater, New Mexico, and the Austin Avenue Radiation Site (AAR) in Lansdowne, Pennsylvania were the subject of ATSDR health advisories. The sites were contamined with uranium or uranium byproducts but the identification of potential health effects and actions taken to prevent or reduce exposures were approached from different perspectives. At B-V contaminants included uranium and mine tailings, radium, and radon. Contaminants at the site and physical hazards were removed. At AAR, radium and radon were located in residential settings. Residents who might have had annual exposures greater than accepted standards or recommendations were relocated and contaminated building demolished

  9. Robotics and Automation Activities at the Savannah River Site: A Site Report for SUBWOG 39F

    The Savannah River Site has successfully used robots, teleoperators, and remote video to reduce exposure to ionizing radiation, improve worker safety, and improve the quality of operations. Previous reports have described the use of mobile teleoperators in coping with a high level liquid waste spill, the removal of highly contaminated equipment, and the inspection of nuclear reactor vessels. This report will cover recent applications at the Savannah River, as well as systems which SRS has delivered to other DOE site customers

  10. Conformational Fluctuations in G-Protein-Coupled Receptors

    Brown, Michael F.

    2014-03-01

    G-protein-coupled receptors (GPCRs) comprise almost 50% of pharmaceutical drug targets, where rhodopsin is an important prototype and occurs naturally in a lipid membrane. Rhodopsin photoactivation entails 11-cis to all-trans isomerization of the retinal cofactor, yielding an equilibrium between inactive Meta-I and active Meta-II states. Two important questions are: (1) Is rhodopsin is a simple two-state switch? Or (2) does isomerization of retinal unlock an activated conformational ensemble? For an ensemble-based activation mechanism (EAM) a role for conformational fluctuations is clearly indicated. Solid-state NMR data together with theoretical molecular dynamics (MD) simulations detect increased local mobility of retinal after light activation. Resultant changes in local dynamics of the cofactor initiate large-scale fluctuations of transmembrane helices that expose recognition sites for the signal-transducing G-protein. Time-resolved FTIR studies and electronic spectroscopy further show the conformational ensemble is strongly biased by the membrane lipid composition, as well as pH and osmotic pressure. A new flexible surface model (FSM) describes how the curvature stress field of the membrane governs the energetics of active rhodopsin, due to the spontaneous monolayer curvature of the lipids. Furthermore, influences of osmotic pressure dictate that a large number of bulk water molecules are implicated in rhodopsin activation. Around 60 bulk water molecules activate rhodopsin, which is much larger than the number of structural waters seen in X-ray crystallography, or inferred from studies of bulk hydrostatic pressure. Conformational selection and promoting vibrational motions of rhodopsin lead to activation of the G-protein (transducin). Our biophysical data give a paradigm shift in understanding GPCR activation. The new view is: dynamics and conformational fluctuations involve an ensemble of substates that activate the cognate G-protein in the amplified visual

  11. The active sites of supported silver particle catalysts in formaldehyde oxidation.

    Chen, Yaxin; Huang, Zhiwei; Zhou, Meijuan; Hu, Pingping; Du, Chengtian; Kong, Lingdong; Chen, Jianmin; Tang, Xingfu

    2016-08-01

    Surface silver atoms with upshifted d-orbitals are identified as the catalytically active sites in formaldehyde oxidation by correlating their activity with the number of surface silver atoms, and the degree of the d-orbital upshift governs the catalytic performance of the active sites. PMID:27406403

  12. On Terminal Alkynes That Can React with Active-Site Cysteine Nucleophiles in Proteases

    Reggy Ekkebus; van Kasteren, Sander I.; Yogesh Kulathu; Arjen Scholten; Ilana Berlin; Geurink, Paul P; Annemieke de Jong; Soenita Goerdayal; Jacques Neefjes; Heck, Albert J.R.; David Komander; Huib Ovaa

    2013-01-01

    Active-site directed probes are powerful in studies of enzymatic function. We report an active-site directed probe based on a warhead so far considered unreactive. By replacing the C-terminal carboxylate of ubiquitin (Ub) with an alkyne functionality, a selective reaction with the active-site cysteine residue of de-ubiquitinating enzymes was observed. The resulting product was shown to be a quaternary vinyl thioether, as determined by X-ray crystallography. Proteomic analysis of proteins boun...

  13. Conformational changes in DNA gyrase revealed by limited proteolysis

    Kampranis, S C; Maxwell, A

    1998-01-01

    We have used limited proteolysis to identify conformational changes in DNA gyrase. Gyrase exhibits a proteolytic fingerprint dominated by two fragments, one of approximately 62 kDa, deriving from the A protein, and another of approximately 25 kDa from the B protein. Quinolone binding to the enzyme......-DNA complex induces a conformational change which is reflected in the protection of the C-terminal 47-kDa domain of the B protein. An active site mutant (Tyr122 to Ser in the A protein) that binds quinolones but cannot cleave DNA still gives the quinolone proteolytic pattern, while stabilization of a cleaved......-DNA intermediate by calcium ions does not reveal any protection, suggesting that the quinolone-induced conformational change is different from an "open-gate" state of the enzyme. A quinolone-resistant mutant of gyrase fails to give the characteristic quinolone-associated proteolytic signature. The ATP...

  14. Human population and activities in Forsmark. Site description

    The Swedish Nuclear Fuel and Waste Management Co (SKB) is in the process of selecting a safe and environmentally acceptable location for a deep repository of radioactive waste. Two alternative locations are under investigation. These are Forsmark, Oesthammars kommun (kommun = municipality) and Simpevarp/Laxemar, Oskarshamns kommun. SKB has expressed the importance of describing the humans and their activities in these areas and therefore has this synthesis concerning the human population in Forsmark been produced.The description is a statistical synthesis, mainly based upon statistical data from SCB (Statistics Sweden) that has been collected, processed and analysed. The statistical data has not been verified through site inspections and interviews. When using statistical data, it is advisable to note that the data becomes more unreliable if the areas are small, with small populations.The data in this description is essential for future evaluations of the impact on the environment and its human population (Environmental Impact Assessments). The data is also important when modelling the potential flows of radio nuclides and calculating the risk of exposure in future safety assessments.The actual area for the study is in this report called 'the Forsmark area', an area of 19.5 km2 near Forsmark nuclear power plant. The land use in the Forsmark area differs notably from the land use in Uppsala laen (laen = county). Only 0.04% of the total area is developed (built-up) compared to 4.9% in Uppsala laen and only 4% is agricultural land compared to 25% in the county. Furthermore, there are far more forest, wetlands and water areas in the Forsmark area. The forest area represents as much as 72.5% of the total area.The Forsmark area is uninhabited, and its surroundings are very sparsely populated. In 2002, the population density in Forsmark was 1.8 inhabitants per square kilometre, which was 24 times lower than in Uppsala laen. The population density in the parish has been

  15. Human population and activities in Forsmark. Site description

    Miliander, Sofia; Punakivi, Mari; Kylaekorpi, Lasse; Rydgren, Bernt [SwedPower AB, Stockholm (Sweden)

    2004-12-01

    The Swedish Nuclear Fuel and Waste Management Co (SKB) is in the process of selecting a safe and environmentally acceptable location for a deep repository of radioactive waste. Two alternative locations are under investigation. These are Forsmark, Oesthammars kommun (kommun = municipality) and Simpevarp/Laxemar, Oskarshamns kommun. SKB has expressed the importance of describing the humans and their activities in these areas and therefore has this synthesis concerning the human population in Forsmark been produced.The description is a statistical synthesis, mainly based upon statistical data from SCB (Statistics Sweden) that has been collected, processed and analysed. The statistical data has not been verified through site inspections and interviews. When using statistical data, it is advisable to note that the data becomes more unreliable if the areas are small, with small populations.The data in this description is essential for future evaluations of the impact on the environment and its human population (Environmental Impact Assessments). The data is also important when modelling the potential flows of radio nuclides and calculating the risk of exposure in future safety assessments.The actual area for the study is in this report called 'the Forsmark area', an area of 19.5 km{sup 2} near Forsmark nuclear power plant. The land use in the Forsmark area differs notably from the land use in Uppsala laen (laen = county). Only 0.04% of the total area is developed (built-up) compared to 4.9% in Uppsala laen and only 4% is agricultural land compared to 25% in the county. Furthermore, there are far more forest, wetlands and water areas in the Forsmark area. The forest area represents as much as 72.5% of the total area.The Forsmark area is uninhabited, and its surroundings are very sparsely populated. In 2002, the population density in Forsmark was 1.8 inhabitants per square kilometre, which was 24 times lower than in Uppsala laen. The population density in the

  16. Substitution of Tyr254 with Phe at the active site of flavocytochrome b2: consequences on catalysis of lactate dehydrogenation

    Dubois, J.; Chapman, S.K.; Mathews, F.S.; Reid, G.A.; Lederer, F. (INSERM U 25, CNRS UA 122, Hopital Necker, Paris (France))

    1990-07-10

    A role for Tyr254 in L-lactate dehydrogenation catalyzed by flavocytochrome b2 has recently been proposed on the basis of the known active-site structure and of studies that had suggested a mechanism involving the initial formation of a lactate carbanion. This role is now examined after replacement of Tyr254 with phenylalanine. The kcat is decreased about 40-fold, Km for lactate appears unchanged, and the mainly rate-limiting step is still alpha-hydrogen abstraction, as judged from the steady-state deuterium isotope effect. Modeling studies with lactate introduced into the active site indicate two possible substrate conformations with different hydrogen-bonding partners for the substrate hydroxyl. If the hydrogen bond is formed with Tyr254, as was initially postulated, the mechanism must involve removal by His373 of the C2 hydrogen, with carbanion formation. If, in the absence of the Tyr254 phenol group, the hydrogen bond is formed with His373 N3, the substrate is positioned in such a way that the reaction must proceed by hydride transfer. Therefore the mechanism of the Y254F enzyme was investigated so as to distinguish between the two mechanistic possibilities. 2-Hydroxy-3-butynoate behaves with the mutant as a suicide reagent, as with the wild-type enzyme. Similarly, the mutant protein also catalyzes the reduction and the dehydrohalogenation of bromopyruvate under transhydrogenation conditions.

  17. 76 FR 7226 - Commercial Wind Lease Issuance and Site Characterization Activities; Atlantic Outer Continental...

    2011-02-09

    ... site characterization and assessment data the lessee may submit a construction and operations plan (COP... 30 CFR 285.620-.629. Although BOEMRE does not authorize site characterization activities (i.e... on the environmental effects of reasonably foreseeable site characterization surveys that may...

  18. Conformal transformations and conformal invariance in gravitation

    Dabrowski, Mariusz P.; Garecki, Janusz; Blaschke, David B.

    2008-01-01

    Conformal transformations are frequently used tools in order to study relations between various theories of gravity and the Einstein relativity. In this paper we discuss the rules of these transformations for geometric quantities as well as for the matter energy-momentum tensor. We show the subtlety of the matter energy-momentum conservation law which refers to the fact that the conformal transformation "creates" an extra matter term composed of the conformal factor which enters the conservat...

  19. The status of Yucca Mountain site characterization activities

    The U.S. Department of Energy (DOE) Office of Civilian Radioactive Waste Management is continuing its studies to determine if Yucca Mountain in Nevada can safely isolate high-level nuclear waste for the next ten thousand years. As mandated by Congress in 1987, DOE is studying the rocks, the climate, and the water table at Yucca Mountain to ensure that the site is suitable before building a repository about 305 meters (1,000 feet) below the surface. Yucca Mountain, located 161 kilometers (100 miles) northwest of Las Vegas, lies on the western edge of the Nevada Test Site. Nevada and DOE have been in litigation for almost two years over three environmental permits needed to conduct studies, but recent court decisions have allowed limited work to take place. This paper will examine progress made on the Yucca Mountain Site Characterization Project (YMP) during the past year and continuing into 1992, discuss the complex legal issues that are delaying progress, and describe new site drilling work. Title I and II design work on the underground exploratory studies facility (ESF) also will be discussed

  20. Preliminary siting activities for new waste handling facilities at the Idaho National Engineering Laboratory

    Taylor, D.D.; Hoskinson, R.L.; Kingsford, C.O.; Ball, L.W.

    1994-09-01

    The Idaho Waste Processing Facility, the Mixed and Low-Level Waste Treatment Facility, and the Mixed and Low-Level Waste Disposal Facility are new waste treatment, storage, and disposal facilities that have been proposed at the Idaho National Engineering Laboratory (INEL). A prime consideration in planning for such facilities is the selection of a site. Since spring of 1992, waste management personnel at the INEL have been involved in activities directed to this end. These activities have resulted in the (a) identification of generic siting criteria, considered applicable to either treatment or disposal facilities for the purpose of preliminary site evaluations and comparisons, (b) selection of six candidate locations for siting,and (c) site-specific characterization of candidate sites relative to selected siting criteria. This report describes the information gathered in the above three categories for the six candidate sites. However, a single, preferred site has not yet been identified. Such a determination requires an overall, composite ranking of the candidate sites, which accounts for the fact that the sites under consideration have different advantages and disadvantages, that no single site is superior to all the others in all the siting criteria, and that the criteria should be assigned different weighing factors depending on whether a site is to host a treatment or a disposal facility. Stakeholder input should now be solicited to help guide the final selection. This input will include (a) siting issues not already identified in the siting, work to date, and (b) relative importances of the individual siting criteria. Final site selection will not be completed until stakeholder input (from the State of Idaho, regulatory agencies, the public, etc.) in the above areas has been obtained and a strategy has been developed to make a composite ranking of all candidate sites that accounts for all the siting criteria.

  1. Preliminary siting activities for new waste handling facilities at the Idaho National Engineering Laboratory

    The Idaho Waste Processing Facility, the Mixed and Low-Level Waste Treatment Facility, and the Mixed and Low-Level Waste Disposal Facility are new waste treatment, storage, and disposal facilities that have been proposed at the Idaho National Engineering Laboratory (INEL). A prime consideration in planning for such facilities is the selection of a site. Since spring of 1992, waste management personnel at the INEL have been involved in activities directed to this end. These activities have resulted in the (a) identification of generic siting criteria, considered applicable to either treatment or disposal facilities for the purpose of preliminary site evaluations and comparisons, (b) selection of six candidate locations for siting,and (c) site-specific characterization of candidate sites relative to selected siting criteria. This report describes the information gathered in the above three categories for the six candidate sites. However, a single, preferred site has not yet been identified. Such a determination requires an overall, composite ranking of the candidate sites, which accounts for the fact that the sites under consideration have different advantages and disadvantages, that no single site is superior to all the others in all the siting criteria, and that the criteria should be assigned different weighing factors depending on whether a site is to host a treatment or a disposal facility. Stakeholder input should now be solicited to help guide the final selection. This input will include (a) siting issues not already identified in the siting, work to date, and (b) relative importances of the individual siting criteria. Final site selection will not be completed until stakeholder input (from the State of Idaho, regulatory agencies, the public, etc.) in the above areas has been obtained and a strategy has been developed to make a composite ranking of all candidate sites that accounts for all the siting criteria

  2. Are nest sites actively chosen? Testing a common assumption for three non-resource limited birds

    Goodenough, A. E.; Elliot, S. L.; Hart, A. G.

    2009-09-01

    Many widely-accepted ecological concepts are simplified assumptions about complex situations that remain largely untested. One example is the assumption that nest-building species choose nest sites actively when they are not resource limited. This assumption has seen little direct empirical testing: most studies on nest-site selection simply assume that sites are chosen actively (and seek explanations for such behaviour) without considering that sites may be selected randomly. We used 15 years of data from a nestbox scheme in the UK to test the assumption of active nest-site choice in three cavity-nesting bird species that differ in breeding and migratory strategy: blue tit ( Cyanistes caeruleus), great tit ( Parus major) and pied flycatcher ( Ficedula hypoleuca). Nest-site selection was non-random (implying active nest-site choice) for blue and great tits, but not for pied flycatchers. We also considered the relative importance of year-specific and site-specific factors in determining occupation of nest sites. Site-specific factors were more important than year-specific factors for the tit species, while the reverse was true for pied flycatchers. Our results show that nest-site selection, in birds at least, is not always the result of active choice, such that choice should not be assumed automatically in studies of nesting behaviour. We use this example to highlight the need to test key ecological assumptions empirically, and the importance of doing so across taxa rather than for single "model" species.

  3. Structure and cytotoxic activity of sesquiterpene glycoside esters from Calendula officinalis L.: Studies on the conformation of viridiflorol.

    D'Ambrosio, Michele; Ciocarlan, Alexandru; Colombo, Elisa; Guerriero, Antonio; Pizza, Cosimo; Sangiovanni, Enrico; Dell'Agli, Mario

    2015-09-01

    Topic applications of Calendula officinalis L. lipophilic extracts are used in phytotherapy to relieve skin inflammatory conditions whereas infusions are used as a remedy for gastric complaints. Such a different usage might be explained by some cytotoxicity of lipophilic extracts at gastric level but little is known about this. Therefore, we screened the CH2Cl2 extract from the flowers of C. officinalis by MTT and LDH assays in human epithelial gastric cells AGS. This bioassay-oriented approach led to the isolation of several sesquiterpene glycosides which were structurally characterized by spectroscopic measurements, chemical reactions and MM calculations. The conformational preferences of viridiflorol fucoside were established and a previously assigned stereochemistry was revised. The compounds 1a, 2a and 3f showed comparably high cytotoxicity in the MTT assays, whereas the effect on LDH release was lower. Our study provides new insights on the composition of C. officinalis extracts of medium polarity and identifies the main compounds that could be responsible for cytotoxic effects at gastric level. PMID:26057223

  4. Applications of hydrogen deuterium exchange (HDX for the characterization of conformational dynamics in light-activated photoreceptors

    Robert eLindner

    2015-06-01

    Full Text Available Rational design of optogenetic tools is inherently linked to the understanding of photoreceptor function. Structural analysis of elements involved in signal integration in individual sensor domains provides an initial idea of their mode of operation, but understanding how local structural rearrangements eventually affect signal transmission to output domains requires inclusion of the effector regions in the characterization. However, the dynamic nature of these assemblies renders their structural analysis challenging and therefore a combination of high- and low-resolution techniques is required to appreciate functional aspects of photoreceptors.This review focuses on the potential of Hydrogen-Deuterium exchange coupled to mass spectrometry (HDX-MS for complementing the structural characterization of photoreceptors. In this respect, the ability of HDX-MS to provide information on the conformational dynamics and the possibility to address multiple functionally relevant states in solution render this methodology ideally suitable. We highlight recent examples demonstrating the potential of HDX-MS and discuss how these results can help to improve existing optogenetic systems or guide the design of novel optogenetic tools.

  5. Active-Site Hydration and Water Diffusion in Cytochrome P450cam: A Highly Dynamic Process

    Miao, Yinglong [ORNL; Baudry, Jerome Y [ORNL

    2011-01-01

    Long-timescale molecular dynamics simulations (300 ns) are performed on both the apo- (i.e., camphor-free) and camphor-bound cytochrome P450cam (CYP101). Water diffusion into and out of the protein active site is observed without biased sampling methods. During the course of the molecular dynamics simulation, an average of 6.4 water molecules is observed in the camphor-binding site of the apo form, compared to zero water molecules in the binding site of the substrate-bound form, in agreement with the number of water molecules observed in crystal structures of the same species. However, as many as 12 water molecules can be present at a given time in the camphor-binding region of the active site in the case of apo-P450cam, revealing a highly dynamic process for hydration of the protein active site, with water molecules exchanging rapidly with the bulk solvent. Water molecules are also found to exchange locations frequently inside the active site, preferentially clustering in regions surrounding the water molecules observed in the crystal structure. Potential-of-mean-force calculations identify thermodynamically favored trans-protein pathways for the diffusion of water molecules between the protein active site and the bulk solvent. Binding of camphor in the active site modifies the free-energy landscape of P450cam channels toward favoring the diffusion of water molecules out of the protein active site.

  6. Human population and activities at Simpevarp. Site description

    The Swedish Nuclear Fuel and Waste Management Co (SKB) is in the process of selecting a safe and environmentally acceptable location for a deep repository of radioactive waste. Two alternative locations are under investigation. These are Forsmark, Oesthammars kommun (kommun = municipality) and Simpevarp/Laxemar, Oskarshamns kommun. SKB has expressed the importance of describing the humans and their activities in these areas and therefore has this synthesis concerning the human population in Forsmark been produced. The description is a statistical synthesis, mainly based upon statistical data from SCB (Statistics Sweden) that has been collected, processed and analysed. The statistical data has not been verified through site inspections and interviews. When using statistical data, it is advisable to note that the data becomes more unreliable if the areas are small, with small populations. The data in this description is essential for future evaluations of the impact on the environment and its human population (environmental impacts assessments). The data is also important when modelling the potential flows of radio nuclides and calculating the risk of exposure in future safety assessments. The actual area for the study is in this report called 'the Simpevarp area', an area of 127.0 km2 near Oskarshamn nuclear power plant. The land use in Simpevarp area differs notably from the land use in Kalmar laen. The forest area is far more dominating in Simpevarp area than in Kalmar laen and it represents as much as 89% compared to 63% of the total area. Only 4.4% of the area is arable land compared to 11.6% in Kalmar laen and only 0.3% is of other type (wetlands, bare rock, quarries, pites etc) compared to 15.6% in the county. The main observation is that Simpevarp area is a sparsely populated area located in a relatively lightly populated county. In 2002, the population density was 7.4 inhabitants/km2, three times lower than in Kalmar laen. The demography statistics show no

  7. Human population and activities at Simpevarp. Site description

    Miliander, Sofia; Punakivi, Mari; Kylaekorpi, Lasse; Rydgren, Bernt [SwedPower AB, Stockholm (Sweden)

    2004-12-01

    The Swedish Nuclear Fuel and Waste Management Co (SKB) is in the process of selecting a safe and environmentally acceptable location for a deep repository of radioactive waste. Two alternative locations are under investigation. These are Forsmark, Oesthammars kommun (kommun = municipality) and Simpevarp/Laxemar, Oskarshamns kommun. SKB has expressed the importance of describing the humans and their activities in these areas and therefore has this synthesis concerning the human population in Forsmark been produced. The description is a statistical synthesis, mainly based upon statistical data from SCB (Statistics Sweden) that has been collected, processed and analysed. The statistical data has not been verified through site inspections and interviews. When using statistical data, it is advisable to note that the data becomes more unreliable if the areas are small, with small populations. The data in this description is essential for future evaluations of the impact on the environment and its human population (environmental impacts assessments). The data is also important when modelling the potential flows of radio nuclides and calculating the risk of exposure in future safety assessments. The actual area for the study is in this report called 'the Simpevarp area', an area of 127.0 km{sup 2} near Oskarshamn nuclear power plant. The land use in Simpevarp area differs notably from the land use in Kalmar laen. The forest area is far more dominating in Simpevarp area than in Kalmar laen and it represents as much as 89% compared to 63% of the total area. Only 4.4% of the area is arable land compared to 11.6% in Kalmar laen and only 0.3% is of other type (wetlands, bare rock, quarries, pites etc) compared to 15.6% in the county. The main observation is that Simpevarp area is a sparsely populated area located in a relatively lightly populated county. In 2002, the population density was 7.4 inhabitants/km{sup 2}, three times lower than in Kalmar laen. The

  8. Human population and activities at Simpevarp. Site description

    Miliander, Sofia; Punakivi, Mari; Kylaekorpi, Lasse; Rydgren, Bernt [SwedPower AB, Stockholm (Sweden)

    2004-12-01

    The Swedish Nuclear Fuel and Waste Management Co (SKB) is in the process of selecting a safe and environmentally acceptable location for a deep repository of radioactive waste. Two alternative locations are under investigation. These are Forsmark, Oesthammars kommun (kommun = municipality) and Simpevarp/Laxemar, Oskarshamns kommun. SKB has expressed the importance of describing the humans and their activities in these areas and therefore has this synthesis concerning the human population in Forsmark been produced. The description is a statistical synthesis, mainly based upon statistical data from SCB (Statistics Sweden) that has been collected, processed and analysed. The statistical data has not been verified through site inspections and interviews. When using statistical data, it is advisable to note that the data becomes more unreliable if the areas are small, with small populations. The data in this description is essential for future evaluations of the impact on the environment and its human population (environmental impacts assessments). The data is also important when modelling the potential flows of radio nuclides and calculating the risk of exposure in future safety assessments. The actual area for the study is in this report called 'the Simpevarp area', an area of 127.0 km{sup 2} near Oskarshamn nuclear power plant. The land use in Simpevarp area differs notably from the land use in Kalmar laen. The forest area is far more dominating in Simpevarp area than in Kalmar laen and it represents as much as 89% compared to 63% of the total area. Only 4.4% of the area is arable land compared to 11.6% in Kalmar laen and only 0.3% is of other type (wetlands, bare rock, quarries, pites etc) compared to 15.6% in the county. The main observation is that Simpevarp area is a sparsely populated area located in a relatively lightly populated county. In 2002, the population density was 7.4 inhabitants/km{sup 2}, three times lower than in Kalmar laen. The

  9. Human Activities in Natura 2000 Sites: A Highly Diversified Conservation Network

    Tsiafouli, Maria A.; Apostolopoulou, Evangelia; Mazaris, Antonios D.; Kallimanis, Athanasios S; Drakou, Evangelia G.; Pantis, John D.

    2013-01-01

    The Natura 2000 network was established across the European Union’s (EU) Member States with the aim to conserve biodiversity, while ensuring the sustainability of human activities. However, to what kind and to what extent Natura 2000 sites are subject to human activities and how this varies across Member States remains unspecified. Here, we analyzed 111,269 human activity records from 14,727 protected sites in 20 Member States. The frequency of occurrence of activities differs among countries...

  10. Improved affinity of engineered streptavidin for the Strep-tag II peptide is due to a fixed open conformation of the lid-like loop at the binding site

    Korndörfer, Ingo P.; Skerra, Arne

    2002-01-01

    The Strep-tag II is a nine-amino acid peptide that was developed as an affinity tool for the purification of corresponding fusion proteins on streptavidin columns. The peptide recognizes the same pocket of streptavidin where the natural ligand is normally bound so that biotin or its chemical derivatives can be used for competitive elution. We report here the crystal structures of the streptavidin mutants `1' and `2,' which had been engineered for 10-fold higher affinity towards the Strep-tag II. Both streptavidin mutants carry mutations at positions 44, 45, and 47, that is, in a flexible loop region close to the binding site. The crystal structures of the two apo-proteins and their complexes with the Strep-tag II peptide were refined at resolutions below 2 Å. Both in the presence and absence of the peptide, the lid-like loop next to the ligand pocket—comprising residues 45 through 52—adopts an `open' conformation in all four subunits within the asymmetric unit. The same loop was previously described to be disordered in the wild-type apo-streptavidin and to close over the pocket upon complexation of the natural ligand biotin. Our findings suggest that stabilization of the `open' loop conformation in the absence of a ligand abolishes the need for conformational rearrangement prior to the docking of the voluminous peptide. Because no direct contacts between the flexible part of the loop and the peptide ligand were detected, it seems likely that the higher affinity of the two streptavidin mutants for the Strep-tag II is caused by a predominantly entropic mechanism. PMID:11910031

  11. 76 FR 24871 - Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    2011-05-03

    ... Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites AGENCY: Department of... from eligible active uranium and thorium processing site licensees for reimbursement under Title X of the Energy Policy Act of 1992. DATES: In our Federal Register Notice of November 24, 2010, (75...

  12. Early Site Permit Demonstration Program: Recommendations for communication activities and public participation in the Early Site Permit Demonstration Program

    1993-01-27

    On October 24, 1992, President Bush signed into law the National Energy Policy Act of 1992. The bill is a sweeping, comprehensive overhaul of the Nation`s energy laws, the first in more than a decade. Among other provisions, the National Energy Policy Act reforms the licensing process for new nuclear power plants by adopting a new approach developed by the US Nuclear Regulatory Commission (NRC) in 1989, and upheld in court in 1992. The NRC 10 CFR Part 52 rule is a three-step process that guarantees public participation at each step. The steps are: early site permit approval; standard design certifications; and, combined construction/operating licenses for nuclear power reactors. Licensing reform increases an organization`s ability to respond to future baseload electricity generation needs with less financial risk for ratepayers and the organization. Costly delays can be avoided because design, safety and siting issues will be resolved before a company starts to build a plant. Specifically, early site permit approval allows for site suitability and acceptability issues to be addressed prior to an organization`s commitment to build a plant. Responsibility for site-specific activities, including communications and public participation, rests with those organizations selected to try out early site approval. This plan has been prepared to assist those companies (referred to as sponsoring organizations) in planning their communications and public involvement programs. It provides research findings, information and recommendations to be used by organizations as a resource and starting point in developing their own plans.

  13. Early Site Permit Demonstration Program: Recommendations for communication activities and public participation in the Early Site Permit Demonstration Program

    On October 24, 1992, President Bush signed into law the National Energy Policy Act of 1992. The bill is a sweeping, comprehensive overhaul of the Nation's energy laws, the first in more than a decade. Among other provisions, the National Energy Policy Act reforms the licensing process for new nuclear power plants by adopting a new approach developed by the US Nuclear Regulatory Commission (NRC) in 1989, and upheld in court in 1992. The NRC 10 CFR Part 52 rule is a three-step process that guarantees public participation at each step. The steps are: early site permit approval; standard design certifications; and, combined construction/operating licenses for nuclear power reactors. Licensing reform increases an organization's ability to respond to future baseload electricity generation needs with less financial risk for ratepayers and the organization. Costly delays can be avoided because design, safety and siting issues will be resolved before a company starts to build a plant. Specifically, early site permit approval allows for site suitability and acceptability issues to be addressed prior to an organization's commitment to build a plant. Responsibility for site-specific activities, including communications and public participation, rests with those organizations selected to try out early site approval. This plan has been prepared to assist those companies (referred to as sponsoring organizations) in planning their communications and public involvement programs. It provides research findings, information and recommendations to be used by organizations as a resource and starting point in developing their own plans

  14. Flap Conformations in HIV-1 Protease are Altered by Mutations

    Fanucci, Gail; Blackburn, Mandy; Veloro, Angelo; Galiano, Luis; Fangu, Ding; Simmerling, Carlos

    2009-03-01

    HIV-1 protease (PR) is an enzyme that is a major drug target in the treatment of AIDS. Although the structure and function of HIV-1 PR have been studied for over 20 years, questions remain regarding the conformations and dynamics of the β-hairpin turns (flaps) that cover the active site cavity. Distance measurements with pulsed EPR spectroscopy of spin labeled constructs of HIV-1 PR have been used to characterize the flap conformations in the apo and inhibitor bound states. From the most probably distances and the breadth of the distance distribution profiles from analysis of the EPR data, insights regarding the flap conformations and flexibility are gained. The EPR results clearly show how drug pressure selected mutations alter the average conformation of the flaps and the degree of opening of the flaps. Molecular dynamics simulations successfully regenerate the experimentally determined distance distribution profiles, and more importantly, provide structural models for full interpretation of the EPR results. By combining experiment and theory to understand the role that altered flap flexibility/conformations play in the mechanism of drug resistance, key insights are gained toward the rational development of new inhibitors of this important enzyme.

  15. Isolation of active site and antibody-binding fragments of human erythrocyte transglutaminase

    Catalytically active human erythrocyte transglutaminase (TGase) was purified using an immunoaffinity column prepared from a monoclonal antibody to guinea pig liver TGase. The enzyme activity was completely inhibited by incorporation of iodo[14C]acetamide to the level of 1 mole per 1 mole of TGase. The 14C-labeled TGase was digested with cyanogen bromide, subjected to HPLC, and four pure peptides were isolated with molecular weights ranging from 3-22 KDa. Only one of the peptides was radiolabeled and characterized as an active site peptide of 10 KDa. Another peptide of 18 KDa was identified as a monoclonal antibody-binding domain of TGase. Although the active site and the antibody-binding domain were present on different cyanogen bromide fragments, the mouse anti-TGase inhibited 100% of TGase activity. The results suggest that the antibody-binding site is not located on the enzyme active site sequence, but that the three dimensional space configuration of the antigen-antibody complex hinders substrate binding to the active site. The radiolabeled active site cysteine residue was not found in the N-terminal 21 amino acids of the 10 KDa peptide. Additional fragments of the active site peptide are currently being analyzed

  16. Assessment of former uranium sites and their ongoing remediation activities

    Carried out analysis on tailing's buildings operation shows that period for engineer barrier service, taking into account any catastrophic natural impacts, is too little in comparison with life-time of long-live radionuclides. Priorities should be defined by danger degree and isolation costs (protection optimization), therefore uncommon, non-traditional methods, developed taking into account natural factors for long-live waste (radionuclides) isolation are necessary. That's why, it is necessary to carry out specialized research and development, design and exploratory and other works on monitoring of social-ecological condition of these sites, as well as on demographic public diseases, living in these regions.

  17. Synthesis and characterization of 18F-labeled active site inhibited factor VII (ASIS)

    Erlandsson, Maria; Nielsen, Carsten Haagen; Jeppesen, Troels Elmer;

    2015-01-01

    Activated factor VII blocked in the active site with Phe-Phe-Arg-chloromethyl ketone (active site inhibited factor VII (ASIS)) is a 50-kDa protein that binds with high affinity to its receptor, tissue factor (TF). TF is a transmembrane glycoprotein that plays an important role in, for example......, thrombosis, metastasis, tumor growth, and tumor angiogenesis. The aim of this study was to develop an 18F-labeled ASIS derivative to assess TF expression in tumors. Active site inhibited factor VII was labeled using N-succinimidyl-4-[18F]fluorobenzoate, and the [18F]ASIS was purified on a PD-10 desalting...

  18. Full and partial agonists of thromboxane prostanoid receptor unveil fine tuning of receptor superactive conformation and G protein activation.

    Valérie Capra

    Full Text Available The intrahelical salt bridge between E/D(3.49 and R(3.50 within the E/DRY motif on helix 3 (H3 and the interhelical hydrogen bonding between the E/DRY and residues on H6 are thought to be critical in stabilizing the class A G protein-coupled receptors in their inactive state. Removal of these interactions is expected to generate constitutively active receptors. This study examines how neutralization of E(3.49/6.30 in the thromboxane prostanoid (TP receptor alters ligand binding, basal, and agonist-induced activity and investigates the molecular mechanisms of G protein activation. We demonstrate here that a panel of full and partial agonists showed an increase in affinity and potency for E129V and E240V mutants. Yet, even augmenting the sensitivity to detect constitutive activity (CA with overexpression of the receptor or the G protein revealed resistance to an increase in basal activity, while retaining fully the ability to cause agonist-induced signaling. However, direct G protein activation measured through bioluminescence resonance energy transfer (BRET indicates that these mutants more efficiently communicate and/or activate their cognate G proteins. These results suggest the existence of additional constrains governing the shift of TP receptor to its active state, together with an increase propensity of these mutants to agonist-induced signaling, corroborating their definition as superactive mutants. The particular nature of the TP receptor as somehow "resistant" to CA should be examined in the context of its pathophysiological role in the cardiovascular system. Evolutionary forces may have favored regulation mechanisms leading to low basal activity and selected against more highly active phenotypes.

  19. Multi-Institution Prospective Trial of Reduced-Dose Craniospinal Irradiation (23.4 Gy) Followed by Conformal Posterior Fossa (36 Gy) and Primary Site Irradiation (55.8 Gy) and Dose-Intensive Chemotherapy for Average-Risk Medulloblastoma

    Purpose: Limiting the neurocognitive sequelae of radiotherapy (RT) has been an objective in the treatment of medulloblastoma. Conformal RT to less than the entire posterior fossa (PF) after craniospinal irradiation might reduce neurocognitive sequelae and requires evaluation. Methods and Materials: Between October 1996 and August 2003, 86 patients, 3-21 years of age, with newly diagnosed, average-risk medulloblastoma were treated in a prospective, institutional review board-approved, multi-institution trial of risk-adapted RT and dose-intensive chemotherapy. RT began within 28 days of definitive surgery and consisted of craniospinal irradiation (23.4 Gy), conformal PF RT (36.0 Gy), and primary site RT (55.8 Gy). The planning target volume for the primary site included the postoperative tumor bed surrounded by an anatomically confined margin of 2 cm that was then expanded with a geometric margin of 0.3-0.5 cm. Chemotherapy was initiated 6 weeks after RT and included four cycles of high-dose cyclophosphamide, cisplatin, and vincristine. Results: At a median follow-up of 61.2 months (range, 5.2-115.0 months), the estimated 5-year event-free survival and cumulative incidence of PF failure rate was 83.0% ± 5.3% and 4.9% ± 2.4% (± standard error), respectively. The targeting guidelines used in this study resulted in a mean reduction of 13% in the volume of the PF receiving doses >55 Gy compared with conventionally planned RT. The reductions in the dose to the temporal lobes, cochleae, and hypothalamus were statistically significant. Conclusion: This prospective trial has demonstrated that irradiation of less than the entire PF after 23.4 Gy craniospinal irradiation for average-risk medulloblastoma results in disease control comparable to that after treatment of the entire PF

  20. Molecular Basis for Enzymatic Sulfite Oxidation -- HOW THREE CONSERVED ACTIVE SITE RESIDUES SHAPE ENZYME ACTIVITY

    Bailey, Susan; Rapson, Trevor; Johnson-Winters, Kayunta; Astashkin, Andrei; Enemark, John; Kappler, Ulrike

    2008-11-10

    Sulfite dehydrogenases (SDHs) catalyze the oxidation and detoxification of sulfite to sulfate, a reaction critical to all forms of life. Sulfite-oxidizing enzymes contain three conserved active site amino acids (Arg-55, His-57, and Tyr-236) that are crucial for catalytic competency. Here we have studied the kinetic and structural effects of two novel and one previously reported substitution (R55M, H57A, Y236F) in these residues on SDH catalysis. Both Arg-55 and His-57 were found to have key roles in substrate binding. An R55M substitution increased Km(sulfite)(app) by 2-3 orders of magnitude, whereas His-57 was required for maintaining a high substrate affinity at low pH when the imidazole ring is fully protonated. This effect may be mediated by interactions of His-57 with Arg-55 that stabilize the position of the Arg-55 side chain or, alternatively, may reflect changes in the protonation state of sulfite. Unlike what is seen for SDHWT and SDHY236F, the catalytic turnover rates of SDHR55M and SDHH57A are relatively insensitive to pH (~;;60 and 200 s-1, respectively). On the structural level, striking kinetic effects appeared to correlate with disorder (in SDHH57A and SDHY236F) or absence of Arg-55 (SDHR55M), suggesting that Arg-55 and the hydrogen bonding interactions it engages in are crucial for substrate binding and catalysis. The structure of SDHR55M has sulfate bound at the active site, a fact that coincides with a significant increase in the inhibitory effect of sulfate in SDHR55M. Thus, Arg-55 also appears to be involved in enabling discrimination between the substrate and product in SDH.

  1. Assessment of activation products in the Savannah River Site environment

    This document assesses the impact of radioactive activation products released from SRS facilities since the first reactor became operational late in 1953. The isotopes reported here are those whose release resulted in the highest dose to people living near SRS: 32P, 51Cr, 60C, and 65Zn. Release pathways, emission control features, and annual releases to the aqueous and atmospheric environments are discussed. No single incident has resulted in a major acute release of activation products to the environment. The releases were the result of normal operations of the reactors and separations facilities. Releases declined over the years as better controls were established and production was reduced. The overall radiological impact of SRS activation product atmospheric releases from 1954 through 1994 on the offsite maximally exposed individual can be characterized by a total dose of 0.76 mrem. During the same period, such an individual received a total dose of 14,400 mrem from non-SRS sources of ionizing radiation present in the environment. SRS activation product aqueous releases between 1954 and 1994 resulted in a total dose of 54 mrem to the offsite maximally exposed individual. The impact of SRS activation product releases on offsite populations also has been evaluated

  2. Assessment of activation products in the Savannah River Site environment

    Carlton, W.H.; Denham, M.

    1996-07-01

    This document assesses the impact of radioactive activation products released from SRS facilities since the first reactor became operational late in 1953. The isotopes reported here are those whose release resulted in the highest dose to people living near SRS: {sup 32}P, {sup 51}Cr, {sup 60}C, and {sup 65}Zn. Release pathways, emission control features, and annual releases to the aqueous and atmospheric environments are discussed. No single incident has resulted in a major acute release of activation products to the environment. The releases were the result of normal operations of the reactors and separations facilities. Releases declined over the years as better controls were established and production was reduced. The overall radiological impact of SRS activation product atmospheric releases from 1954 through 1994 on the offsite maximally exposed individual can be characterized by a total dose of 0.76 mrem. During the same period, such an individual received a total dose of 14,400 mrem from non-SRS sources of ionizing radiation present in the environment. SRS activation product aqueous releases between 1954 and 1994 resulted in a total dose of 54 mrem to the offsite maximally exposed individual. The impact of SRS activation product releases on offsite populations also has been evaluated.

  3. Stimulatory effects of opioid neuropeptides on locomotory activity and conformational changes in invertebrate and human immunocytes: evidence for a subtype of delta receptor.

    Stefano, G B; Cadet, P; Scharrer, B

    1989-08-01

    The presence of opioid neuropeptides was shown to stimulate conformational changes and locomotory activity in immunocytes of two representatives of invertebrates as well as in human leukocytes. Cells were examined by use of phase-contrast and Nomarski optics coupled with a Zeiss Axiophot microscope, and of the Zeiss Videoplan/Vidas Image Analysis system. Immunocompetent blood cells, activated by exogenous opioids or stressful stimuli presumed to engage endogenous opioids, showed flattening, elongation, and formation of pseudopodia. In the mollusc Mytilus edulis, ameboid movements resulted in the formation of cell clusters, an activity not observed in untreated controls, or in immunocytes simultaneously exposed to opioid and naloxone. Tests with nine immunoreactive substances revealed immunocyte stimulation by delta, mu-, kappa-, and epsilon(?)-selective ligands. One of these, [D-Ala2,D-Met5]enkephalinamide (DAMA), active at a concentration of 10 pM, proved to be considerably more effective than the rest. The high pharmacological potency of DAMA, observed in both human and invertebrate immunocytes, sets this opioid apart from the closely related [D-Ala2,D-Leu5]enkephalin, a discrepancy not occurring in the mammalian nervous system. This suggests a specific function for [Met]enkephalin in immunoregulation, mediated perhaps by a special subtype of delta receptor. PMID:2548205

  4. Active site mutants of Escherichia coli dethiobiotin synthetase: effects of mutations on enzyme catalytic and structural properties.

    Yang, G; Sandalova, T; Lohman, K; Lindqvist, Y; Rendina, A R

    1997-04-22

    , showed that the crystals were essentially isomorphous to that of the wild-type DTBS. The models of these mutant enzymes were well refined (1.9 -2.6 A) and showed good similarity to the wild-type enzyme (rmsd of C alpha atoms: 0.16-0.24 A). The crystal structure of S41C complexed with DAPA, Mn2+/Mg2+, and AMPPCP revealed a localized conformational change (rotations of side chains of Cys41 and Thr11) which can account for the changes in the kinetic parameters observed for S41C. The crystal structures of the Lys37 mutant enzymes showed that the positive charge of the side chain of Lys37 is indispensable. Mutations of Lys37 to either glutamine or leucine resulted in a shift of the metal ion (up to 0.5 A) together with side chains of other active site residues which could disrupt the subtle balance between the positive and negative charges in the active site. The conformational change of the phosphate binding loop (Gly8-X-X-X-X-X-Gly14-Lys15-Thr16) upon nucleotide binding observed previously [Huang, W., Jia, J., Gibson, K. J., Taylor, W. S., Rendina, A. R., Schneider, G., & Lindqvist, Y. (1995) Biochemistry 34, 10985] appears to be important to attain the proper active site scaffold. PMID:9125495

  5. The landscape degradation in the mining sites with suspended activity

    Anca IONCE

    2009-01-01

    The extracting industry, through its extraction activities, of shipping the ores, of breaking the ores, of preparing the practical substances, of stowing the useless rock, of transporting the practical substances, etc. might modify the area’s relief and the quality of ground, of thesurface waters and of the air. Suceava County has an old tradition of mining, where the results of this activity are visible, especially the visual point of view, and where not taking certain measures of ecological...

  6. Probing the putative active site of YjdL

    Jensen, Johanne Mørch; Ismat, Fouzia; Szakonyi, Gerda;

    2012-01-01

    with Glu388, a preliminary orientation model of a dipeptide in the YjdL cavity is presented. Single site mutations of particularly Ala281 and Trp278 support the presented orientation. A dipeptide bound in the cavity of YjdL appears to be oriented such that the N-terminal side chain protrudes into a sub...... pocket that opens towards the extracellular space. The C-terminal side chain faces in the opposite direction into a sub pocket that faces the cytoplasm. These data indicated a stabilizing effect on a bulky N-terminal residue by an Ala281Phe variant and on the dipeptide backbone by Trp278. In the...... presented orientation model, Tyr25 and Tyr58 both appear to be in proximity of the dipeptide backbone while Lys117 appears to be in proximity of the peptide C-terminus. Mutational studies of these conserved residues highlight their functional importance....

  7. Active site proton delivery and the lyase activity of human CYP17A1

    Khatri, Yogan; Gregory, Michael C.; Grinkova, Yelena V.; Denisov, Ilia G.; Sligar, Stephen G., E-mail: s-sligar@illinois.edu

    2014-01-03

    equivalents and protons are funneled into non-productive pathways. This is similar to previous work with other P450 catalyzed hydroxylation. However, catalysis of carbon–carbon bond scission by the T306A mutant was largely unimpeded by disruption of the CYP17A1 acid-alcohol pair. The unique response of CYP17A1 lyase activity to mutation of Thr306 is consistent with a reactive intermediate formed independently of proton delivery in the active site, and supports involvement of a nucleophilic peroxo-anion rather than the traditional Compound I in catalysis.

  8. Active site proton delivery and the lyase activity of human CYP17A1

    equivalents and protons are funneled into non-productive pathways. This is similar to previous work with other P450 catalyzed hydroxylation. However, catalysis of carbon–carbon bond scission by the T306A mutant was largely unimpeded by disruption of the CYP17A1 acid-alcohol pair. The unique response of CYP17A1 lyase activity to mutation of Thr306 is consistent with a reactive intermediate formed independently of proton delivery in the active site, and supports involvement of a nucleophilic peroxo-anion rather than the traditional Compound I in catalysis

  9. Off-site medical activities, Nevada Test Site and the medical liaison officer network: a historical review

    The ''off-site'' was originally defined as ''that area surrounding the Nevada Test Site (NTS) for a radius of about 300 miles.'' Prior to 1954, the off-site radiological safety activities were conducted by the Atomic Energy Commission. In 1954, the Public Health Service was given the responsibility for off-site monitoring, and, in addition, a physician was also on temporary assignment. This physician, in addition to functioning as a monitor, also functioned part time as a physical liaison in regard to possible or alleged radiation injury. Medical concern was based upon two crude guidelines: possible radiation ''overexposure'' based upon extrapolation from surface and air radiological monitoring; and determination of actual radiation injury based upon signs and symptoms among people alleging radiation injury. The area of concern expanded to 13 areas surrounding the Nevada Test Site, and in 1956, the first Medical Liaison Officer Network (MLON) was initiated. Over the years, MLON increased to a point where there was a representative from every state in the Union; the area of concern expanded to include the entire United States, parts of the South Pacific, Hawaii, and Alaska; and sophisticated methods of evaluation were added--urine sampling, thyroid scanning, blood counts, and whole-body counting. Epidemiological studies were initiated on body burdens of radionuclides and certain disease clusters

  10. The landscape degradation in the mining sites with suspended activity

    Anca IONCE

    2009-08-01

    Full Text Available The extracting industry, through its extraction activities, of shipping the ores, of breaking the ores, of preparing the practical substances, of stowing the useless rock, of transporting the practical substances, etc. might modify the area’s relief and the quality of ground, of thesurface waters and of the air. Suceava County has an old tradition of mining, where the results of this activity are visible, especially the visual point of view, and where not taking certain measures of ecological remediation will emphasize the disappointing image of the landscape within the areas of mining activity performing.The predominant mountainous landscape, in which mining activities have been held, is being affected also by the abandoned industrial and administrative buildings, in an advanced degradation state.The hydrographic system, very rich in mining areas, has its water quality affected by the acid rock drainage- phenomenon which appeared in many mining waste deposits.

  11. Alternative conformal quantum mechanics

    Ho, Shih-Hao

    2011-01-01

    We investigate a one dimensional quantum mechanical model, which is invariant under translations and dilations but does not respect the conventional conformal invariance. We describe the possibility of modifying the conventional conformal transformation such that a scale invariant theory is also invariant under this new conformal transformation.

  12. Superspace conformal field theory

    Quella, Thomas [Koeln Univ. (Germany). Inst. fuer Theoretische Physik; Schomerus, Volker [Deutsches Elektronen-Synchrotron (DESY), Hamburg (Germany)

    2013-07-15

    Conformal sigma models and WZW models on coset superspaces provide important examples of logarithmic conformal field theories. They possess many applications to problems in string and condensed matter theory. We review recent results and developments, including the general construction of WZW models on type I supergroups, the classification of conformal sigma models and their embedding into string theory.

  13. Superspace conformal field theory

    Conformal sigma models and WZW models on coset superspaces provide important examples of logarithmic conformal field theories. They possess many applications to problems in string and condensed matter theory. We review recent results and developments, including the general construction of WZW models on type I supergroups, the classification of conformal sigma models and their embedding into string theory.

  14. Conformal and non Conformal Dilaton Gravity

    Alvarez, Enrique; Mart'\\in, C P

    2014-01-01

    The quantum dynamics of the gravitational field non-minimally coupled to an (also dynamical) scalar field is studied in the broken phase. For a particular value of the coupling the system is classically conformal, and can actually be understood as the group averaging of Einstein-Hilbert's action under conformal transformations. Contradicting cherished beliefs, a conformal anomaly is found in the trace of the equations of motion. To one loop order, this anomaly vanishes on shell. Arguments are given supporting the fact that this does not happen to two loop order, where the anomaly is argued to be a real physical effect.

  15. Activation of brown adipose tissue mitochondrial GDP binding sites

    The primary function of brown adipose tissue (BAT) is heat production. This ability is attributed to the existence of a unique inner mitochondrial membrane protein termed the uncoupling protein or thermogenin. This protein is permeable to H+ and thus allows respiration (and therefore thermogenesis) to proceed at a rapid rate, independent of ADP phosphorylation. Proton conductance can be inhibited by the binding of purine nucleotides to the uncoupling protein. The binding of [3H]-GDP to BAT mitochondria is frequently used as a measure of BAT thermogenic activity. Rats fed a diet that was low but adequate in protein exhibited a decrease in feed efficiency. In addition, BAT thermogenesis was activated as indicated by an elevation in the level of GDP binding to BAT mitochondria. This phenomena occurred in older rats and persisted over time

  16. Activation of brown adipose tissue mitochondrial GDP binding sites

    Swick, A.G.

    1987-01-01

    The primary function of brown adipose tissue (BAT) is heat production. This ability is attributed to the existence of a unique inner mitochondrial membrane protein termed the uncoupling protein or thermogenin. This protein is permeable to H+ and thus allows respiration (and therefore thermogenesis) to proceed at a rapid rate, independent of ADP phosphorylation. Proton conductance can be inhibited by the binding of purine nucleotides to the uncoupling protein. The binding of (/sup 3/H)-GDP to BAT mitochondria is frequently used as a measure of BAT thermogenic activity. Rats fed a diet that was low but adequate in protein exhibited a decrease in feed efficiency. In addition, BAT thermogenesis was activated as indicated by an elevation in the level of GDP binding to BAT mitochondria. This phenomena occurred in older rats and persisted over time.

  17. Activation of Mitogen-Activated Protein Kinase Is Required for Migration and Invasion of Placental Site Trophoblastic Tumor

    Köbel, Martin; Pohl, Gudrun; Schmitt, Wolfgang D.; Hauptmann, Steffen; Wang, Tian-Li; Shih, Ie-Ming

    2005-01-01

    Placental site trophoblastic tumor (PSTT) is a gestational neoplasm derived from the extravillous (intermediate) trophoblast of the implantation site. PSTT is characterized by a highly invasive phenotype, but the molecular mechanisms are poorly understood. In this report, we demonstrate that PSTTs expressed the activated (phosphorylated) form of mitogen-activated protein kinase (MAPK) in 84% of cases, whereas the normal extravillous trophoblastic cells did not. To characterize the role of MAP...

  18. Experience in decommissioning activities at the BR3 site

    Klein, Michel E-mail: mklein@sckcen.be; Dadoumont, Jerome; Demeulemeester, Yves; Massaut, Vincent

    2001-04-01

    We give an overview of the experience, SCK-CEN acquired during 11 years of decommissioning activities of a PWR fission reactor. Experience has been gained in decontamination and dismantling technologies and in waste management. Dismantling an old PWR is quite different from the dismantling of the future fusion reactor. However, we can try to find out some common generic features and draw some lessons. Our experience shows that it is important to take the decommissioning aspects into account as soon as possible.

  19. Lipolytic activity from bacteria prospected in polluted portuary sites

    Kaori Levy Fonseca

    2014-06-01

    This study demonstrates that these TBT resistant isolates have, at the same time, the capacity to produce enzymes with a large biotechnological potential but, nevertheless, their relationship is not well understood, representing a novel approach. It is expected for these organisms to produce highly biotechnological relevant biocatalysts, due to their severe adaptations (Suehiro et al., 2007. The fully characterization of these lipases, mostly for F3 with elevated lipolytic activity exhibited, presents also a future challenge.

  20. The evolutionary conformation from traditional lecture to active learning in an undergraduate biology course and its effects on student achievement

    Diederich, Kirsten Bakke

    In response to the declining number of students in the United States entering into the STEM (science, technology, engineering, and math) disciplines, there has been an attempt to retain student interest in the sciences through the implementation of more active learning in the classroom. Active learning is defined as any instructional method that requires students do something in the classroom rather than simply listen to a lecture (Herreid, 2006). These student centered approaches provide the students with the opportunity to work cooperatively while developing the skills required for critical inquiry. They also help the students make the connections between what is being taught and how it can be applied in a real world setting. Science education researchers have attempted to analyze the efficacy of active learning. Although they find it difficult to compare the data, they state unequivocally that "Active learning is a better strategy for learning than the traditional didactic lecture format" (Prince, 2004). However, even though research supports the efficacy of active learning, instructors find it difficult to adopt this pedagogy into their classrooms due to concerns such as loss of content knowledge and student resistance. This three year qualitative and quantitative study addressed the level of student learning and satisfaction in an introductory vertebrate biology class at a small liberal arts college. The courses were taught by the same instructor using three pedagogical methods; traditional lecture (TL), problem-based learning (PBL), and case-based learning (CBL). Student grades and levels of assessment were compared between the TL and PBL, while student attrition rates, course satisfaction and views of active and group learning were analyzed across all three sections. The evolutionary confirmations from TL to PBL and ultimately the adoption of CBL as the method of choice are discussed from the view of both the faculty member and the students.

  1. Small Angle Neutron Scattering Reveals pH-dependent Conformational Changes in Trichoderma reesei Cellobiohydrolase I: IMPLICATIONS FOR ENZYMATIC ACTIVITY*

    Pingali, Sai Venkatesh; O'Neill, Hugh M.; McGaughey, Joseph; Urban, Volker S.; Rempe, Caroline S.; Petridis, Loukas; Jeremy C Smith; Evans, Barbara R.; Heller, William T.

    2011-01-01

    Cellobiohydrolase I (Cel7A) of the fungus Trichoderma reesei (now classified as an anamorph of Hypocrea jecorina) hydrolyzes crystalline cellulose to soluble sugars, making it of key interest for producing fermentable sugars from biomass for biofuel production. The activity of the enzyme is pH-dependent, with its highest activity occurring at pH 4–5. To probe the response of the solution structure of Cel7A to changes in pH, we measured small angle neutron scattering of it in a series of solut...

  2. Current disposal planning for dry active wastes at Rokkasho Site

    Takeuchi, Mitsuo [Japan Nuclear Fuel Ltd., Aomori (Japan)

    1997-02-01

    In nuclear power stations, two kinds of low level radioactive wastes are generated: `uniform solidified waste` in which waste liquid, spent resin and so on are uniformly solidified and `solid waste` in which metals, lagging materials, plastics and others are solidified. In Rokkasho Low Level Radioactive Waste Burying Center, the burying facility for the first period for the uniform solidified waste started the operation in December, 1992, and this time as the second period plan, it has been planned to increase No. 2 waste burying facility for the solid waste. The kinds of the radioactive waste solidified in containers to be buried are the solid state radioactive waste generated by the operation of nuclear power stations and that generated accompanying the operation of this facility. The wastes are classified, cut, pressed and melted as occasion demands so that cement filling material is easily filled in containers, and solidified in the containers. As for the waste to be buried, at the time of its acceptance, 6 months or longer have elapsed since its generation in nuclear power stations, and the surface dose equivalent rate does not exceed 10 mSv/h. The acceptance plan and the expected number of burying, the total radioactivity of buried waste, and the location, geological and hydraulic features, the structure and facilities of waste burying facilities, the method of burying, the management of waste burying site and the evaluation of dose equivalent are reported. (K.I.)

  3. Current disposal planning for dry active wastes at Rokkasho Site

    In nuclear power stations, two kinds of low level radioactive wastes are generated: 'uniform solidified waste' in which waste liquid, spent resin and so on are uniformly solidified and 'solid waste' in which metals, lagging materials, plastics and others are solidified. In Rokkasho Low Level Radioactive Waste Burying Center, the burying facility for the first period for the uniform solidified waste started the operation in December, 1992, and this time as the second period plan, it has been planned to increase No. 2 waste burying facility for the solid waste. The kinds of the radioactive waste solidified in containers to be buried are the solid state radioactive waste generated by the operation of nuclear power stations and that generated accompanying the operation of this facility. The wastes are classified, cut, pressed and melted as occasion demands so that cement filling material is easily filled in containers, and solidified in the containers. As for the waste to be buried, at the time of its acceptance, 6 months or longer have elapsed since its generation in nuclear power stations, and the surface dose equivalent rate does not exceed 10 mSv/h. The acceptance plan and the expected number of burying, the total radioactivity of buried waste, and the location, geological and hydraulic features, the structure and facilities of waste burying facilities, the method of burying, the management of waste burying site and the evaluation of dose equivalent are reported. (K.I.)

  4. Identifying high dose activities in industrial site radiography

    Although the radiation doses received by industrial radiographers in the UK have progressively fallen over the last few years, with most now receiving less than 1 mSv/y, a few still receive, relative to the rest, much higher doses. As a percentage of all radiographers the number stays surprisingly constant from year to year. This paper describes a survey to identify the work causing these doses and suggest possible solutions. The UK Central Index of Dose Information was interrogated to identify the industrial radiography companies having staff (not necessarily the same person) with doses of greater than 5mSv/y in the last three years for which information was available. This was 15 in total. The people on the staff receiving these doses were identified and a questionnaire sent to the companies concerned requesting information about their work. A general questionnaire about the operation of the company was also included. With the agreement of the company these questionnaires were followed up by a visit to the company to interviews a number of the management and the radiographers if available. Both groups were generally very open about their problems and every discussion had a positive outcome. Several areas of work/reasons for the doses have been identified. These are: pipeline radiography, ultra sound radiographers working on nuclear reactors, complex plant work often with several teams in the area, inability to retreat from the wind out equipment due to height or access problems, site pressure to not follow the best practices and a lack of appreciation when a dose was being received or, alternatively, carelessness. Some o these problem areas are very difficult to resolve. However ways in which the Health and Safety can help influence the doses have been identified together with practical suggestions radiographers could adopt. These will be reported. (author)

  5. School Pharmacist/School Environmental Hygienic Activities at School Site.

    Muramatsu, Akiyoshi

    2016-01-01

    The "School Health and Safety Act" was enforced in April 2009 in Japan, and "school environmental health standards" were established by the Minister of Education, Culture, Sports, Science and Technology. In Article 24 of the Enforcement Regulations, the duties of the school pharmacist have been clarified; school pharmacists have charged with promoting health activities in schools and carrying out complete and regular checks based on the "school environmental health standards" in order to protect the health of students and staff. In supported of this, the school pharmacist group of Japan Pharmaceutical Association has created and distributed digital video discs (DVDs) on "check methods of school environmental health standards" as support material. We use the DVD to ensure the basic issues that school pharmacists deal with, such as objectives, criteria, and methods for each item to be checked, advice, and post-measures. We conduct various workshops and classes, and set up Q&A committees so that inquiries from members are answered with the help of such activities. In addition, school pharmacists try to improve the knowledge of the school staff on environmental hygiene during their in-service training. They also conduct "drug abuse prevention classes" at school and seek to improve knowledge and recognition of drugs, including "dangerous drugs". PMID:27252053

  6. Catalytic water co-existing with a product peptide in the active site of HIV-1 protease revealed by X-ray structure analysis.

    Vishal Prashar

    Full Text Available BACKGROUND: It is known that HIV-1 protease is an important target for design of antiviral compounds in the treatment of Acquired Immuno Deficiency Syndrome (AIDS. In this context, understanding the catalytic mechanism of the enzyme is of crucial importance as transition state structure directs inhibitor design. Most mechanistic proposals invoke nucleophilic attack on the scissile peptide bond by a water molecule. But such a water molecule coexisting with any ligand in the active site has not been found so far in the crystal structures. PRINCIPAL FINDINGS: We report here the first observation of the coexistence in the active site, of a water molecule WAT1, along with the carboxyl terminal product (Q product peptide. The product peptide has been generated in situ through cleavage of the full-length substrate. The N-terminal product (P product has diffused out and is replaced by a set of water molecules while the Q product is still held in the active site through hydrogen bonds. The position of WAT1, which hydrogen bonds to both the catalytic aspartates, is different from when there is no substrate bound in the active site. We propose WAT1 to be the position from where catalytic water attacks the scissile peptide bond. Comparison of structures of HIV-1 protease complexed with the same oligopeptide substrate, but at pH 2.0 and at pH 7.0 shows interesting changes in the conformation and hydrogen bonding interactions from the catalytic aspartates. CONCLUSIONS/SIGNIFICANCE: The structure is suggestive of the repositioning, during substrate binding, of the catalytic water for activation and subsequent nucleophilic attack. The structure could be a snap shot of the enzyme active site primed for the next round of catalysis. This structure further suggests that to achieve the goal of designing inhibitors mimicking the transition-state, the hydrogen-bonding pattern between WAT1 and the enzyme should be replicated.

  7. Isolated metal active site concentration and stability control catalytic CO2 reduction selectivity.

    Matsubu, John C; Yang, Vanessa N; Christopher, Phillip

    2015-03-01

    CO2 reduction by H2 on heterogeneous catalysts is an important class of reactions that has been studied for decades. However, atomic scale details of structure-function relationships are still poorly understood. Particularly, it has been suggested that metal particle size plays a unique role in controlling the stability of CO2 hydrogenation catalysts and the distribution of active sites, which dictates reactivity and selectivity. These studies often have not considered the possible role of isolated metal active sites in the observed dependences. Here, we utilize probe molecule diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) with known site-specific extinction coefficients to quantify the fraction of Rh sites residing as atomically dispersed isolated sites (Rhiso), as well as Rh sites on the surface of Rh nanoparticles (RhNP) for a series of TiO2 supported Rh catalysts. Strong correlations were observed between the catalytic reverse water gas shift turn over frequency (TOF) and the fraction of Rhiso sites and between catalytic methanation TOF and the fraction of RhNP sites. Furthermore, it was observed that reaction condition-induced disintegration of Rh nanoparticles, forming Rhiso active sites, controls the changing reactivity with time on stream. This work demonstrates that isolated atoms and nanoparticles of the same metal on the same support can exhibit uniquely different catalytic selectivity in competing parallel reaction pathways and that disintegration of nanoparticles under reaction conditions can play a significant role in controlling stability. PMID:25671686

  8. FRET Characterization of Complex Conformational Changes in a Large 16S Ribosomal RNA Fragment Site-Specifically Labeled Using Unnatural Base Pairs.

    Lavergne, Thomas; Lamichhane, Rajan; Malyshev, Denis A; Li, Zhengtao; Li, Lingjun; Sperling, Edit; Williamson, James R; Millar, David P; Romesberg, Floyd E

    2016-05-20

    Ribosome assembly has been studied intensively using Förster resonance energy transfer (FRET) with fluorophore-labeled fragments of RNA produced by chemical synthesis. However, these studies are limited by the size of the accessible RNA fragments. We have developed a replicable unnatural base pair (UBP) formed between (d)5SICS and (d)MMO2 or (d)NaM, which efficiently directs the transcription of RNA containing unnatural nucleotides. We now report the synthesis and evaluation of several of the corresponding ribotriphosphates bearing linkers that enable the chemoselective attachment of different functionalities. We found that the RNA polymerase from T7 bacteriophage does not incorporate NaM derivatives but does efficiently incorporate 5SICS(CO), whose linker enables functional group conjugation via Click chemistry, and when combined with the previously identified MMO2(A), whose amine side chains permits conjugation via NHS coupling chemistry, enables site-specific double labeling of transcribed RNA. To study ribosome assembly, we transcribed RNA corresponding to a 243-nt fragment of the central domain of Thermus thermophilus 16S rRNA containing 5SICS(CO) and MMO2(A) at defined locations and then site-specifically attached the fluorophores Cy3 and Cy5. FRET was characterized using single-molecule total internal reflection fluorescence (smTIRF) microscopy in the presence of various combinations of added ribosomal proteins. We demonstrate that each of the fragment's two three-helix junctions exist in open and closed states, with the latter favored by sequential protein binding. These results elucidate early and previously uncharacterized folding events underlying ribosome assembly and demonstrate the applicability of UBPs for biochemical, structural, and functional studies of RNAs. PMID:26942998

  9. Conformational Heterogeneity of Bax Helix 9 Dimer for Apoptotic Pore Formation

    Liao, Chenyi; Zhang, Zhi; Kale, Justin; Andrews, David W.; Lin, Jialing; Li, Jianing

    2016-07-01

    Helix α9 of Bax protein can dimerize in the mitochondrial outer membrane (MOM) and lead to apoptotic pores. However, it remains unclear how different conformations of the dimer contribute to the pore formation on the molecular level. Thus we have investigated various conformational states of the α9 dimer in a MOM model — using computer simulations supplemented with site-specific mutagenesis and crosslinking of the α9 helices. Our data not only confirmed the critical membrane environment for the α9 stability and dimerization, but also revealed the distinct lipid-binding preference of the dimer in different conformational states. In our proposed pathway, a crucial iso-parallel dimer that mediates the conformational transition was discovered computationally and validated experimentally. The corroborating evidence from simulations and experiments suggests that, helix α9 assists Bax activation via the dimer heterogeneity and interactions with specific MOM lipids, which eventually facilitate proteolipidic pore formation in apoptosis regulation.

  10. Conformational Heterogeneity of Bax Helix 9 Dimer for Apoptotic Pore Formation.

    Liao, Chenyi; Zhang, Zhi; Kale, Justin; Andrews, David W; Lin, Jialing; Li, Jianing

    2016-01-01

    Helix α9 of Bax protein can dimerize in the mitochondrial outer membrane (MOM) and lead to apoptotic pores. However, it remains unclear how different conformations of the dimer contribute to the pore formation on the molecular level. Thus we have investigated various conformational states of the α9 dimer in a MOM model - using computer simulations supplemented with site-specific mutagenesis and crosslinking of the α9 helices. Our data not only confirmed the critical membrane environment for the α9 stability and dimerization, but also revealed the distinct lipid-binding preference of the dimer in different conformational states. In our proposed pathway, a crucial iso-parallel dimer that mediates the conformational transition was discovered computationally and validated experimentally. The corroborating evidence from simulations and experiments suggests that, helix α9 assists Bax activation via the dimer heterogeneity and interactions with specific MOM lipids, which eventually facilitate proteolipidic pore formation in apoptosis regulation. PMID:27381287

  11. 77 FR 74218 - Commercial Wind Leasing and Site Assessment Activities on the Atlantic Outer Continental Shelf...

    2012-12-13

    ... published a Notice of Availability (NOA) in the Federal Register (72 FR 62,672) of the Programmatic EIS for... Federal Register (77 FR 5560) of the Final EA for Commercial Wind Lease Issuance and Site Assessment... Bureau of Ocean Energy Management Commercial Wind Leasing and Site Assessment Activities on the...

  12. 75 FR 71677 - Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    2010-11-24

    ... Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites AGENCY: Department of... uranium and thorium processing site licensees for reimbursement under Title X of the Energy Policy Act of... published a final rule under 10 CFR Part 765 in the Federal Register on May 23, 1994, (59 FR 26714) to...

  13. Poisoning Experiments Aimed at Discriminating Active and Less-Active Sites of Silica-Supported Tantalum Hydride for Alkane Metathesis

    Saggio, Guillaume

    2010-10-04

    Only 50% of the silica-supported tantalum hydride sites are active in the metathesis of propane. Indeed, more than 45% of the tantalum hydride can be eliminated by a selective oxygen poisoning of inactive sites with no significant decrease in the global turnover. Conversely, cyclopentane induces no such selective poisoning. Hence, the active tantalum hydride sites that show greater resistance to oxygen poisoning correspond to the νTa-H bands of higher wavenumbers, particularly that at 1860cm-1. These active tantalum hydride sites should correspond to tris- or monohydride species relatively far from silica surface oxygen atoms. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Recent progress in volcanism studies: Site characterization activities for the Yucca Mountain site characterization project

    Significant progress has been made on volcanism studies over the past calendar year. There are a number of major highlights from this work. Geochronology data have been obtained for the Lathrop Wells center using a range of isotopic, radiogenic, and age-calibrated methods. Initial work is encouraging but still insufficient to resolve the age of the center with confidence. Geologic mapping of the Sleeping Butte volcanic centers was completed and a report issued on the geology and chronology data. Twenty shallow trenches have been constructed in volcanic units of the Lathrop Wells volcanic center. Results of detailed studies of the trenches support a polycyclic eruptive history. New soil data from the trenches continue to support a late Pleistocene or Holocene age for many of the volcanic units at the center. Geochemical data (trace element and isotopic analysis) show that the volcanic units of the Lathrop Wells center cannot be related to one another by fractional crystallization of a single magma batch, supporting a polycyclic model of volcanism. Structural models using existing data are used to evaluate the probability of magmatic disruption of a potential repository. Several permissive models have been developed but none lead to significant differences in calculating the disruption ratio. Work was initiated on the eruptive and subsurface effects of magmatic activity on a repository. (author)

  15. Site specific rationale for technical impracticability of active groundwater restoration at a former manufactured gas plant site

    The National Contingency Plan (40 CFR Part 300 ) requires that remedial strategies must, at minimum, protect human health and the environment and meet applicable and relevant or appropriate requirements (ARARs). Where groundwater is impacted, maximum contaminant levels (MCLs) and maximum contaminant level goals (MCLGs) set under the Safe Drinking Water Act are often used as ARARs, whether or not the aquifer is a reasonably anticipated future source of drinking water. The US Environmental Protection Agency now recognizes the difficulty of groundwater restoration at sites where dense nonaqueous phase liquids are present, particularly in certain complex hydrogeological settings (EPA 1993). However, demonstration of impracticability generally does not occur until active remediation (e.g., pump and treat) has been shown to be ineffective. A case study of a former manufactured gas plant (MGP) is used to demonstrate how physical and chemical properties of the aquifer and coal tar, the major waste product from MGP sites, influence the feasibility of active restoration. Field characterization investigations, laboratory studies, and groundwater modeling are integrated into a demonstration following EPA guidelines. Laboratory studies included microbiological characterization and natural biodegradation and suggest that intrinsic bioremediation is occurring at this site. This work will be useful as EPA continues to develop presumptive remedies for cleanup under Superfund

  16. 1993 annual report of hazardous waste activities for the Oak Ridge K-25 site

    This report is a detailed listing of all of the Hazardous Waste activities occurring at Martin Marietta's K-25 site. Contained herein are hazardous waste notification forms, waste stream reports, generator fee forms and various TSDR reports

  17. The alloantigenic sites of alpha3alpha4alpha5(IV) collagen: pathogenic X-linked alport alloantibodies target two accessible conformational epitopes in the alpha5NC1 domain.

    Kang, Jeong Suk; Kashtan, Clifford E; Turner, A Neil; Heidet, Laurence; Hudson, Billy G; Borza, Dorin-Bogdan

    2007-04-01

    Anti-glomerular basement membrane (GBM) antibody nephritis is caused by an autoimmune or alloimmune reaction to the NC1 domains of alpha3alpha4alpha5(IV) collagen. Some patients with X-linked Alport syndrome (XLAS) develop post-transplant nephritis mediated by pathogenic anti-GBM alloantibodies to collagen IV chains present in the renal allograft but absent from the tissues of the patient. In this work, the epitopes targeted by alloantibodies from these patients were identified and characterized. All XLAS alloantibodies recognized conformational epitopes in the NC1 domain of alpha5(IV) collagen, which were mapped using chimeric alpha1/alpha5 NC1 domains expressed in mammalian cells. Allograft-eluted alloantibodies mainly targeted two conformational alloepitopes mapping to alpha5NC1 residues 1-45 and 114-168. These regions also encompassed the major epitopes of circulating XLAS alloantibodies, which in some patients additionally targeted alpha5NC1 residues 169-229. Both kidney-eluted and circulating alloantibodies to alpha5NC1 distinctively targeted epitopes accessible in the alpha3alpha4alpha5NC1 hexamers of human GBM, unlike anti-GBM autoantibodies, which targeted sequestered alpha3NC1 epitopes. The results identify two immunodominant alpha5NC1 epitopes as major alloantigenic sites of alpha3alpha4alpha5(IV) collagen specifically implicated in the pathogenesis of post-transplant nephritis in XLAS patients. The contrast between the accessibility of these alloepitopes and the crypticity of autoepitopes indicates that distinct molecular forms of antigen may initiate the immunopathogenic processes in the two forms of anti-GBM disease. PMID:17293596

  18. Coordination environment of the active-site metal ion of liver alcohol dehydrogenase.

    Makinen, M W; Yim, M B

    1981-01-01

    The coordination environment of the catalytically active metal ion of horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) has been investigated by electron paramagnetic resonance (EPR) methods with use of the active-site-specific Co2+-reconstituted enzyme. The EPR absorption spectrum of the metal-substituted enzyme is characteristic of a rhombically distorted environment. The spectrum of the enzyme--NAD+ complex shows approximate axial symmetry of the metal ion site, i...

  19. Molecular dynamics explorations of active site structure in designed and evolved enzymes

    Osuna Oliveras, Sílvia; Jiménez-Osés, Gonzalo; Noey, Elizabeth L.; Houk, Kendall N.

    2015-01-01

    This Account describes the use of molecular dynamics (MD) simulations to reveal how mutations alter the structure and organization of enzyme active sites. As proposed by Pauling about 70 years ago and elaborated by many others since then, biocatalysis is efficient when functional groups in the active site of an enzyme are in optimal positions for transition state stabilization. Changes in mechanism and covalent interactions are often critical parts of enzyme catalysis. We describe our explora...

  20. Active site dynamics of toluene hydroxylation by cytochrome P-450

    Rat liver cytochrome P-450 hydroxylates toluene to benzyl alcohol plus o-, m-, and p-cresol. Deuterated toluenes were incubated under saturating conditions with liver microsomes from phenobarbital-pretreated rats, and product yields and ratios were measured. Stepwise deuteration of the methyl leads to stepwise decreases in the alcohol/cresol ratio without changing the cresol isomer ratios. Extensive deuterium retention in the benzyl alcohols from PhCH2D and PhCHD2 suggests there is a large intrinsic isotope effect for benzylic hydroxylation. After replacement of the third benzylic H by D, the drop in the alcohol/cresol ratio was particularly acute, suggsting that metabolic switching from D to H within the methyl group was easier than switching from the methyl to the ring. Comparison of the alcohol/cresol ratio for PhCH3 vs PhCD3 indicated a net isotope effect of 6.9 for benzylic hydroxylation. From product yield data for PhCH3 and PhCD3, DV for benzyl alcohol formation is only 1.92, whereas DV for total product formation is 0.67 (i.e., inverse). From competitive incubations of PhCH3/PhCD3 mixtures D(V/K) isotope effects on benzyl alcohol formation and total product formation (3.6 and 1.23, respectively) are greatly reduced, implying strong commitment to catalysis. In contrast, D(V/K) for the alcohol/cresol ratio is 6.3, indicating that the majority of the intrinsic isotope effect is expressed through metabolic switching. Overall, these data are consistent with reversible formation of a complex between toluene and the active oxygen form of cytochrome P-450, which rearranges internally and reacts to form products faster than it dissociates back to release substrate

  1. 'Unconventional' coordination chemistry by metal chelating fragments in a metalloprotein active site.

    Martin, David P; Blachly, Patrick G; Marts, Amy R; Woodruff, Tessa M; de Oliveira, César A F; McCammon, J Andrew; Tierney, David L; Cohen, Seth M

    2014-04-01

    The binding of three closely related chelators: 5-hydroxy-2-methyl-4H-pyran-4-thione (allothiomaltol, ATM), 3-hydroxy-2-methyl-4H-pyran-4-thione (thiomaltol, TM), and 3-hydroxy-4H-pyran-4-thione (thiopyromeconic acid, TPMA) to the active site of human carbonic anhydrase II (hCAII) has been investigated. Two of these ligands display a monodentate mode of coordination to the active site Zn(2+) ion in hCAII that is not recapitulated in model complexes of the enzyme active site. This unprecedented binding mode in the hCAII-thiomaltol complex has been characterized by both X-ray crystallography and X-ray spectroscopy. In addition, the steric restrictions of the active site force the ligands into a 'flattened' mode of coordination compared with inorganic model complexes. This change in geometry has been shown by density functional computations to significantly decrease the strength of the metal-ligand binding. Collectively, these data demonstrate that the mode of binding by small metal-binding groups can be significantly influenced by the protein active site. Diminishing the strength of the metal-ligand bond results in unconventional modes of metal coordination not found in typical coordination compounds or even carefully engineered active site models, and understanding these effects is critical to the rational design of inhibitors that target clinically relevant metalloproteins. PMID:24635441

  2. Limited proteolysis combined with isotope labeling and quantitative LC-MALDI MS for monitoring protein conformational changes: a study on calcium-binding sites of cardiac Troponin C

    Studies of protein-protein and protein-ligand interactions are important for understanding biological functions of proteins. A new technique based on the partial proteolysis of proteins combined with quantitative mass spectrometry is developed as a means of tracking structural changes after the formation of a protein-ligand complex. In this technique, a protein of interest with and without the binding of a ligand is digested with an enzyme to generate a set of peptides, followed by separation of the peptides by liquid chromatography. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) is used to identify chromatographically separated peptides, and locate their sequence alignments in the parent protein. Using an isotopically labeled protein as a sample against an unlabeled protein standard, quantitative information can be gathered. This overcomes the inherent lack of quantitative capability of MALDI MS. The utility of the technique to investigate protein-ligand interactions is demonstrated in a model system involving calcium binding to cardiac Troponin C (cTnC). Using this technique, the general location of the three calcium-binding sites of cTnC can be determined by using several different enzymes to generate overlapping peptide maps of cTnC

  3. Conformational, spectroscopic and nonlinear optical properties of biologically active N,N-dimethyltryptamine molecule: A theoretical study

    Öner, Nazmiye; Tamer, Ömer; Avcı, Davut; Atalay, Yusuf

    2014-12-01

    The effective psychoactive properties of N,N-dimethyltryptamine (DMT) known as the near-death molecule have encouraged the imagination of many research disciplines for several decades. Although there is no theoretical study, a number of paper composed by experimental techniques have been reported for DMT molecule. In this study, the molecular modeling of DMT was carried out using B3LYP and HSEh1PBE levels of density functional theory (DFT). Our calculations showed that the energy gap between HOMO and LUMO is low, demonstrating that DMT is a biologically active molecule. Large hyperconjugation interaction energies imply that molecular charge transfer occurs in DMT. Moreover, NLO analysis indicates that DMT can be used an effective NLO material.

  4. XAFS Study of the Photo-Active Site of Mo/MCM-41

    An Mo/MCM-41 catalyst was prepared and used for study of propene and 1-butene photo-metathesis reactions. XAFS analysis revealed that hydrogen reduction leads to a decreased role for the Mo=O site. The Mo-O site plays an important role for the olefin photo-metathesis reaction on the H2 reduced Mo/MCM-41. From EXAFS analysis, the active site of photo-metathesis reaction is the Mo=O part for oxidized Mo/MCM-41, whereas it is the Mo-O site for reduced Mo/MCM-41

  5. Active sites for NO reduction over Fe-ZSM-5 catalysts.

    Schwidder, M; Santhosh Kumar, M; Brückner, A; Grünert, W

    2005-02-14

    A study of Fe-ZSM-5 catalysts with variable amounts of isolated, oligomeric and heavily aggregated Fe3+ oxo sites (as evidenced by UV-Vis and EPR spectroscopic data) and their catalytic properties in the selective catalytic reduction of NO by isobutane or by NH3 is presented, which allows development of a unified concept of the active Fe sites in these reactions, according to which isolated Fe sites catalyse both SCR reactions while oligomeric sites, though also involved in the selective reduction path, limit the catalyst performance by causing the total oxidation of the reductant. PMID:15685345

  6. Benzene Hydroxylation over FeZSM-5 Catalysts: Which Fe-sites Are Active?

    Yuranov, I.; Bulushev, D. A.; Renken, A.; Kiwi-Minsker, L.

    2004-01-01

    FeZSM-5 with a wide range of Fe content (0.015–2.1 wt%) were studied in the benzene hydroxylation to phenol with nitrous oxide (C6H6:N2O = 1:5) at low temperatures (98%) was obtained within 3 h without any deactivation of the catalyst. Three types of Fe(II) sites were formed in the zeolites extraframework due to activation and are attributed to: (1) Fe(II) sites in mononuclear species, (2) oligonuclear species with at least two oxygen-bridged Fe(II) sites, and (3) Fe(II) sites within Fe2O3 na...

  7. Activator anion binding site in pyridoxal phosphorylase b: the binding of phosphite, phosphate, and fluorophosphate in the crystal.

    Oikonomakos, N G; Zographos, S E; Tsitsanou, K E; Johnson, L N; Acharya, K R

    1996-12-01

    It has been established that phosphate analogues can activate glycogen phosphorylase reconstituted with pyridoxal in place of the natural cofactor pyridoxal 5'-phosphate (Change YC. McCalmont T, Graves DJ. 1983. Biochemistry 22:4987-4993). Pyridoxal phosphorylase b has been studied by kinetic, ultracentrifugation, and X-ray crystallographic experiments. In solution, the catalytically active species of pyridoxal phosphorylase b adopts a conformation that is more R-state-like than that of native phosphorylase b, but an inactive dimeric species of the enzyme can be stabilized by activator phosphite in combination with the T-state inhibitor glucose. Co-crystals of pyridoxal phosphorylase b complexed with either phosphite, phosphate, or fluorophosphate, the inhibitor glucose, and the weak activator IMP were grown in space group P4(3)2(1)2, with native-like unit cell dimensions, and the structures of the complexes have been refined to give crystallographic R factors of 18.5-19.2%, for data between 8 and 2.4 A resolution. The anions bind tightly at the catalytic site in a similar but not identical position to that occupied by the cofactor 5'-phosphate group in the native enzyme (phosphorus to phosphorus atoms distance = 1.2 A). The structural results show that the structures of the pyridoxal phosphorylase b-anion-glucose-IMP complexes are overall similar to the glucose complex of native T-state phosphorylase b. Structural comparisons suggest that the bound anions, in the position observed in the crystal, might have a structural role for effective catalysis. PMID:8976550

  8. The Clusters-in-a-Liquid Approach for Solvation: New Insights from the Conformer Specific Gas Phase Spectroscopy and Vibrational Optical Activity Spectroscopy.

    Perera, Angelo S; Thomas, Javix; Poopari, Mohammad R; Xu, Yunjie

    2016-01-01

    Vibrational optical activity spectroscopies, namely vibrational circular dichroism (VCD) and Raman optical activity (ROA), have been emerged in the past decade as powerful spectroscopic tools for stereochemical information of a wide range of chiral compounds in solution directly. More recently, their applications in unveiling solvent effects, especially those associated with water solvent, have been explored. In this review article, we first select a few examples to demonstrate the unique sensitivity of VCD spectral signatures to both bulk solvent effects and explicit hydrogen-bonding interactions in solution. Second, we discuss the induced solvent chirality, or chiral transfer, VCD spectral features observed in the water bending band region in detail. From these chirality transfer spectral data, the related conformer specific gas phase spectroscopic studies of small chiral hydration clusters, and the associated matrix isolation VCD experiments of hydrogen-bonded complexes in cold rare gas matrices, a general picture of solvation in aqueous solution emerges. In such an aqueous solution, some small chiral hydration clusters, rather than the chiral solutes themselves, are the dominant species and are the ones that contribute mainly to the experimentally observed VCD features. We then review a series of VCD studies of amino acids and their derivatives in aqueous solution under different pHs to emphasize the importance of the inclusion of the bulk solvent effects. These experimental data and the associated theoretical analyses are the foundation for the proposed "clusters-in-a-liquid" approach to account for solvent effects effectively. We present several approaches to identify and build such representative chiral hydration clusters. Recent studies which applied molecular dynamics simulations and the subsequent snapshot averaging approach to generate the ROA, VCD, electronic CD, and optical rotatory dispersion spectra are also reviewed. Challenges associated with the

  9. Design and synthesis of side-chain conformationally restricted phenylalanines and their use for structure-activity studies on tachykinin NK-1 receptor.

    Josien, H; Lavielle, S; Brunissen, A; Saffroy, M; Torrens, Y; Beaujouan, J C; Glowinski, J; Chassaing, G

    1994-05-27

    Constrained analogues of phenylalanine have been conceptually designed for analyzing the binding pockets of Phe7 (S7) and Phe8 (S8), two aromatic residues important for the pharmacological properties of SP, i.e., L-tetrahydroisoquinoleic acid, L-diphenylalanine, L-9-fluorenylglycine (Flg), 2-indanylglycine, the diastereomers of L-1-indanylglycine (Ing) and L-1-benz[f]indanylglycine (Bfi), and the Z and E isomers of dehydrophenylalanine (delta ZPhe, delta EPhe). Binding studies were performed with appropriate ligands and tissue preparations allowing the discrimination of the three tachykinin binding sites, NK-1, NK-2, and NK-3. The potencies of these agonists were evaluated in the guinea pig ileum bioassay. According to the binding data, we can conclude that the S7 subsite is small, only the gauche (-) probe [(2S,3S)-Ing7]SP presents a high affinity for specific NK-1 binding sites. Surprisingly, the [delta EPhe7]SP analogue, which projects the aromatic ring toward the trans orientation, is over 40-fold more potent than the Z isomer, [delta ZPhe7]SP. A plausible explanation of these conflictual results is that either the binding protein quenches the minor trans rotamer of [(2S,3S)-Ing7]SP in solution or this constrained amino acid side chain rotates when inserted in the protein. In position 8, the high binding affinities of [Flg8]SP and [(2S,3S)-Bfi8]SP suggest that the S8 subsite is large enough to accept two aromatic rings in the gauche (-) and one aromatic ring in the trans direction. Peptides bearing two conformational probes in positions 7, 8, or 9 led to postulate that S7, S8, and S9 subsites are independent from each other. The volumes available for side chains 7 and 8 can be estimated to be close to 110 and 240 A3, respectively. The large volume of the S8 subsite raises question on the localization of the SP-binding site in the NK-1 receptor. If SP were to bind in the transmembrane domains, the cleft defined by the seven transmembrane segments must rearrange

  10. Concept for calculating dose rates from activated groundwater at accelerator sites

    Prolingheuer, N; Vanderborght, J; Schlögl, B; Nabbi, R; Moormann, R

    Licensing of particle accelerators requires the proof that the groundwater outside of the site will not be significantly contaminated by activation products formed below accelerator and target. In order to reduce the effort for this proof, a site independent simplified but conservative method is under development. The conventional approach for calculation of activation of soil and groundwater is shortly described on example of a site close to Forschungszentrum Juelich, Germany. Additionally an updated overview of a data library for partition coefficients for relevant nuclides transported in the aquifer at the site is presented. The approximate model for transport of nuclides with ground water including exemplary results on nuclide concentrations outside of the site boundary and of resulting effective doses is described. Further applications and developments are finally outlined.

  11. Functional characterization of autophosphorylation sites of the activated insulin receptor-tyrosine kinase

    Insulin receptor, solubilized from 3T3-L1 cellular membranes and then purified, was autophosphorylated with [γ-32P]ATP in the absence or presence of insulin. Specific phosphopeptides generated by trypsin digestion of the 32P-labeled β-subunit were identified and separated by reverse phase HPLC. In the absence of insulin, radioactivity of the phosphopeptides is evenly distributed among four major peaks designated as sites I, II, III and IV, according to their order of elution. This pattern is maintained for at least the first 30 min of autophosphorylation. When the reaction is carried out in the presence of insulin, > 50% of the total 32P radioactivity is found in site I and the rate of 32P incorporation into this site is markedly higher than into sites II, III and IV. Maximal activation of tyrosine kinase activity, as estimated by substrate phosphorylation, is coincident with the nearly complete phosphorylation of site I. Delayed activation of previously autophosphorylated receptor by insulin, but not by EGF or IGF-I, produced a similar pattern where phosphorylated site I predominates. These observations indicate that one major insulin-regulated autophosphorylation site in the β-subunit is responsible for activation of the insulin receptor tyrosine kinase. The isolation of this phosphopeptide on a preparative scale and its characterization are now in progress

  12. Functional characterization of autophosphorylation sites of the activated insulin receptor-tyrosine kinase

    Flores-Riveros, J.R.; Lane, M.D.

    1987-05-01

    Insulin receptor, solubilized from 3T3-L1 cellular membranes and then purified, was autophosphorylated with (..gamma..-/sup 32/P)ATP in the absence or presence of insulin. Specific phosphopeptides generated by trypsin digestion of the /sup 32/P-labeled ..beta..-subunit were identified and separated by reverse phase HPLC. In the absence of insulin, radioactivity of the phosphopeptides is evenly distributed among four major peaks designated as sites I, II, III and IV, according to their order of elution. This pattern is maintained for at least the first 30 min of autophosphorylation. When the reaction is carried out in the presence of insulin, > 50% of the total /sup 32/P radioactivity is found in site I and the rate of /sup 32/P incorporation into this site is markedly higher than into sites II, III and IV. Maximal activation of tyrosine kinase activity, as estimated by substrate phosphorylation, is coincident with the nearly complete phosphorylation of site I. Delayed activation of previously autophosphorylated receptor by insulin, but not by EGF or IGF-I, produced a similar pattern where phosphorylated site I predominates. These observations indicate that one major insulin-regulated autophosphorylation site in the ..beta..-subunit is responsible for activation of the insulin receptor tyrosine kinase. The isolation of this phosphopeptide on a preparative scale and its characterization are now in progress.

  13. Site-specific targeting of antibody activity in vivo mediated by disease-associated proteases

    Erster, Oran; Thomas, Jerry M; Hamzah, Juliana; Jabaiah, Abeer M.; Getz, Jennifer A.; Schoep, Tobias; Hall, Sejal S.; Ruoslahti, Erkki; Daugherty, Patrick S.

    2012-01-01

    As a general strategy to selectively target antibody activity in vivo, a molecular architecture was designed to render binding activity dependent upon proteases in disease tissues. A protease-activated antibody (pro-antibody) targeting vascular cell adhesion molecule 1 (VCAM-1), a marker of atherosclerotic plaques, was constructed by tethering a binding site-masking peptide to the antibody via a matrix metalloprotease (MMP) susceptible linker. Pro-antibody activation in vitro by MMP-1 yielded...

  14. Calculation of Relative Binding Free Energy in the Water-Filled Active Site of Oligopeptide-Binding Protein A.

    Maurer, Manuela; de Beer, Stephanie B A; Oostenbrink, Chris

    2016-01-01

    The periplasmic oligopeptide binding protein A (OppA) represents a well-known example of water-mediated protein-ligand interactions. Here, we perform free-energy calculations for three different ligands binding to OppA, using a thermodynamic integration approach. The tripeptide ligands share a high structural similarity (all have the sequence KXK), but their experimentally-determined binding free energies differ remarkably. Thermodynamic cycles were constructed for the ligands, and simulations conducted in the bound and (freely solvated) unbound states. In the unbound state, it was observed that the difference in conformational freedom between alanine and glycine leads to a surprisingly slow convergence, despite their chemical similarity. This could be overcome by increasing the softness parameter during alchemical transformations. Discrepancies remained in the bound state however, when comparing independent simulations of the three ligands. These difficulties could be traced to a slow relaxation of the water network within the active site. Fluctuations in the number of water molecules residing in the binding cavity occur mostly on a timescale larger than the simulation time along the alchemical path. After extensive simulations, relative binding free energies that were converged to within thermal noise could be obtained, which agree well with available experimental data. PMID:27092480

  15. Structural and Kinetic Analyses of Macrophage Migration Inhibitory Factor Active Site Interactions

    Crichlow, G.; Lubetsky, J; Leng, L; Bucala, R; Lolis, E

    2009-01-01

    Macrophage migration inhibitory factor (MIF) is a secreted protein expressed in numerous cell types that counters the antiinflammatory effects of glucocorticoids and has been implicated in sepsis, cancer, and certain autoimmune diseases. Interestingly, the structure of MIF contains a catalytic site resembling the tautomerase/isomerase sites of microbial enzymes. While bona fide physiological substrates remain unknown, model substrates have been identified. Selected compounds that bind in the tautomerase active site also inhibit biological functions of MIF. It had previously been shown that the acetaminophen metabolite, N-acetyl-p-benzoquinone imine (NAPQI), covalently binds to the active site of MIF. In this study, kinetic data indicate that NAPQI inhibits MIF both covalently and noncovalently. The structure of MIF cocrystallized with NAPQI reveals that the NAPQI has undergone a chemical alteration forming an acetaminophen dimer (bi-APAP) and binds noncovalently to MIF at the mouth of the active site. We also find that the commonly used protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), forms a covalent complex with MIF and inhibits the tautomerase activity. Crystallographic analysis reveals the formation of a stable, novel covalent bond for PMSF between the catalytic nitrogen of the N-terminal proline and the sulfur of PMSF with complete, well-defined electron density in all three active sites of the MIF homotrimer. Conclusions are drawn from the structures of these two MIF-inhibitor complexes regarding the design of novel compounds that may provide more potent reversible and irreversible inhibition of MIF.

  16. Conformal symmetries of spacetimes

    In this paper, we give a unified and global new approach to the study of the conformal structure of the three classical Riemannian spaces as well as of the six relativistic and non-relativistic spacetimes (Minkowskian, de Sitter, anti-de Sitter, and both Newton-Hooke and Galilean). We obtain general expressions within a Cayley-Klein framework, holding simultaneously for all these nine spaces, whose cycles (including geodesics and circles) are explicitly characterized in a new way. The corresponding cycle-preserving symmetries, which give rise to (Moebius-like) conformal Lie algebras, together with their differential realizations are then deduced without having to resort to solving the conformal Killing equations. We show that each set of three spaces with the same signature type and any curvature have isomorphic conformal algebras; these are related through an apparently new conformal duality. Laplace and wave-type differential equations with conformal algebra symmetry are finally constructed. (author)

  17. Conformal Anomalies in Hydrodynamics

    Eling, Christopher; Theisen, Stefan; Yankielowicz, Shimon

    2013-01-01

    We study the effect of conformal anomalies on the hydrodynamic description of conformal field theories in four spacetime dimensions. We consider equilibrium curved backgrounds characterized by a time-like Killing vector and construct a local low energy effective action that captures the conformal anomalies. Using as a special background the Rindler spacetime we derive a formula for the effect of the anomaly on the hydrodynamic pressure.

  18. Galilean Conformal Electrodynamics

    Bagchi, Arjun; Basu, Rudranil; Mehra, Aditya

    2014-01-01

    Maxwell’s Electrodynamics admits two distinct Galilean limits called the Electric and Magnetic limits. We show that the equations of motion in both these limits are invariant under the Galilean Conformal Algebra in D = 4, thereby exhibiting non-relativistic conformal symmetries. Remarkably, the symmetries are infinite dimensional and thus Galilean Electrodynamics give us the first example of an infinitely extended Galilean Conformal Field Theory in D > 2. We examine details of the theory by l...

  19. Structure of product-bound SMG1 lipase: active site gating implications.

    Guo, Shaohua; Xu, Jinxin; Pavlidis, Ioannis V; Lan, Dongming; Bornscheuer, Uwe T; Liu, Jinsong; Wang, Yonghua

    2015-12-01

    Monoacylglycerol and diacylglycerol lipases are industrially interesting enzymes, due to the health benefits that arise from the consumption of diglycerides compared to the traditional triglyceride oils. Most lipases possess an α-helix (lid) directly over the catalytic pocket which regulates the activity of the enzyme. Generally, lipases exist in active and inactive conformations, depending on the positioning of this lid subdomain. However, lipase SMG1, a monoacylglycerol and diacylglycerol specific lipase, has an atypical activation mechanism. In the present study we were able to prove by crystallography, in silico analysis and activity tests that only two positions, residues 102 and 278, are responsible for a gating mechanism that regulates the active and inactive states of the lipase, and that no significant structural changes take place during activation except for oxyanion hole formation. The elucidation of the gating effect provided data enabling the rational design of improved lipases with 6-fold increase in the hydrolytic activity toward diacylglycerols, just by providing additional substrate stabilization with a single mutation (F278N or F278T). Due to the conservation of F278 among the monoacylglycerol and diacylglycerol lipases in the Rhizomucor miehei lipase-like family, the gating mechanism described herein might represent a general mechanism applicable to other monoacylglycerol and diacylglycerol lipases as well. Database: Structural data are available in the Protein Data Bank under the accession numbers 4ZRE (F278D mutant) and 4ZRD (F278N mutant). PMID:26365206

  20. Galilean Conformal Electrodynamics

    Bagchi, Arjun; Mehra, Aditya

    2014-01-01

    Maxwell's Electrodynamics admits two distinct Galilean limits called the Electric and Magnetic limits. We show that the equations of motion in both these limits are invariant under the Galilean Conformal Algebra in D=4, thereby exhibiting non-relativistic conformal symmetries. Remarkably, the symmetries are infinite dimensional and thus Galilean Electrodynamics give us the first example of an infinitely extended Galilean Conformal Field Theory in D>2. We examine details of the theory by looking at purely non-relativistic conformal methods and also use input from the limit of the relativistic theory.

  1. Galilean conformal electrodynamics

    Bagchi, Arjun; Basu, Rudranil; Mehra, Aditya

    2014-11-01

    Maxwell's Electrodynamics admits two distinct Galilean limits called the Electric and Magnetic limits. We show that the equations of motion in both these limits are invariant under the Galilean Conformal Algebra in D = 4, thereby exhibiting non-relativistic conformal symmetries. Remarkably, the symmetries are infinite dimensional and thus Galilean Electrodynamics give us the first example of an infinitely extended Galilean Conformal Field Theory in D > 2. We examine details of the theory by looking at purely non-relativistic conformal methods and also use input from the limit of the relativistic theory.

  2. Pyrone-based Cu(II) complexes, their characterization, DFT based conformational drift from square planar to square pyramidal geometry and biological activities

    Pradeep Kumar Vishwakarma; Jan Mohammad Mir; Ram Charitra Maurya

    2016-04-01

    This work deals with the synthesis and characterization of a series of three -Dehydroacetic acid- 4-phenyl-3-thiosemicarbazide (H2dha-ptsc) Schiff base Cu(II) complexes based on combined experimental and theoretical approach, having the general composition formula as [Cu(dha-ptsc)(L-L)], where L-L is H2O, 2,2-bipyridine (bipy) or 1,10-phenanthroline (phen). H2O containing complex acts as origin for the latter two complexes and in due course, the geometry of the complex changes from square planar to square pyramidal. DFT calculations were carried out for both the geometrical forms. B3LYP/LANL2DZ level of theory was used to carry out the required computations. From the overall DFT computations, square pyramidal geometry was found to be more stable as compared to the square planar conformation for the complexes under investigation. Super oxide dismutation, thermal behaviour and electrochemical activity were also studied. The results have shown satisfactory super oxide scavenging potential, high degree of thermal resistance and efficient redox properties for the title complexes. Moreover, charge analysis and nonlinear optical properties were computed to establish a comprehensive note of atomic constituents differing in nature of charge delocalization.

  3. Design, synthesis and biological activity of new neurohypophyseal hormones analogues conformationally restricted in the N-terminal part of the molecule. Highly potent OT receptor antagonists.

    Kwiatkowska, Anna; Ptach, Monika; Borovičková, Lenka; Slaninová, Jiřina; Lammek, Bernard; Prahl, Adam

    2012-08-01

    In this study we present the synthesis and some pharmacological properties of fourteen new analogues of neurohypophyseal hormones conformationally restricted in the N-terminal part of the molecule. All new peptides were substituted at position 2 with cis-1-amino-4-phenylcyclohexane-1-carboxylic acid (cis-Apc). Moreover, one of the new analogues: [cis-Apc(2), Val(4)]AVP was also prepared in N-acylated forms with various bulky acyl groups. All the peptides were tested for pressor, antidiuretic, and in vitro uterotonic activities. We also determined the binding affinity of the selected compounds to human OT receptor. Our results showed that introduction of cis -Apc(2) in position 2 of either AVP or OT resulted in analogues with high antioxytocin potency. Two of the new compounds, [Mpa(1),cis-Apc(2)]AVP and [Mpa(1),cis-Apc(2),Val(4)]AVP, were exceptionally potent antiuterotonic agents (pA(2) = 8.46 and 8.40, respectively) and exhibited higher affinities for the human OT receptor than Atosiban (K (i) values 5.4 and 9.1 nM). Moreover, we have demonstrated for the first time that N -terminal acylation of AVP analogue can improve its selectivity. Using this approach, we obtained compound Aba[cis-Apc(2),Val(4)]AVP (XI) which turned out to be a moderately potent and exceptionally selective OT antagonist (pA(2) = 7.26). PMID:22038179

  4. Modeling Steroid 5alpha-reductase and Characterizing Its Potential Active Sites

    OU Min-Rui; LI Jun-Qian

    2012-01-01

    Steroid 5alpha-reductase of human is an enzyme in the biosynthetic pathway from testosterone (T) to dihydrotestosterone (DHT). Up to now, no crystal structure of this enzyme has been reported. However, knowledge of the tertiary structure and possible active sites is essential for understanding the catalysis mechanism and for the design of inhibitors. A model with putative active sites has been created and evaluated by using homology modeling and molecular docking techniques based on the bioinformatics knowledge. The homology model is optimized in Swiss PDB Viewer with MM method and substrate structures before docking are also optimized on HF/6-31G. The active site for the docking of NADP, T, DHT and Finasteride is located near the N-terminus of enzyme. Four active amino acids in the active site are identified as Ala26, Arg53, Arg176 and Lys177. Reaction procedure, binding pattern of active sites, the types of weak interaction and so on are also discussed.

  5. Effects of resource activities upon repository siting and waste containment with reference to bedded salt

    The primary consideration for the suitability of a nuclear waste repository site is the overall ability of the repository to safely contain radioactive waste. This report is a discussion of the past, present, and future effects of resource activities on waste containment. Past and present resource activities which provide release pathways (i.e., leaky boreholes, adjacent mines) will receive initial evaluation during the early stages of any repository site study. However, other resource activities which may have subtle effects on containment (e.g., long-term pumping causing increased groundwater gradients, invasion of saline water causing lower retardation) and all potential future resource activities must also be considered during the site evaluation process. Resource activities will affect both the siting and the designing of repositories. Ideally, sites should be located in areas of low resource activity and low potential for future activity, and repository design should seek to eliminate or minimize the adverse effects of any resource activity. Buffer zones should be created to provide areas in which resource activities that might adversely affect containment can be restricted or curtailed. This could mean removing large areas of land from resource development. The impact of these frozen assets should be assessed in terms of their economic value and of their effect upon resource reserves. This step could require a major effort in data acquisition and analysis followed by extensive numerical modeling of regional fluid flow and mass transport. Numerical models should be used to assess the effects of resource activity upon containment and should include the cumulative effects of different resource activities. Analysis by other methods is probably not possible except for relatively simple cases

  6. A caspase active site probe reveals high fractional inhibition needed to block DNA fragmentation.

    Méthot, Nathalie; Vaillancourt, John P; Huang, JingQi; Colucci, John; Han, Yongxin; Ménard, Stéphane; Zamboni, Robert; Toulmond, Sylvie; Nicholson, Donald W; Roy, Sophie

    2004-07-01

    Apoptotic markers consist of either caspase substrate cleavage products or phenotypic changes that manifest themselves as a consequence of caspase-mediated substrate cleavage. We have shown recently that pharmacological inhibitors of caspase activity prevent the appearance of two such apoptotic manifestations, alphaII-spectrin cleavage and DNA fragmentation, but that blockade of the latter required a significantly higher concentration of inhibitor. We investigated this phenomenon through the use of a novel radiolabeled caspase inhibitor, [(125)I]M808, which acts as a caspase active site probe. [(125)I]M808 bound to active caspases irreversibly and with high sensitivity in apoptotic cell extracts, in tissue extracts from several commonly used animal models of cellular injury, and in living cells. Moreover, [(125)I]M808 detected active caspases in septic mice when injected intravenously. Using this caspase probe, an active site occupancy assay was developed and used to measure the fractional inhibition required to block apoptosis-induced DNA fragmentation. In thymocytes, occupancy of up to 40% of caspase active sites had no effect on DNA fragmentation, whereas inhibition of half of the DNA cleaving activity required between 65 and 75% of active site occupancy. These results suggest that a high and persistent fractional inhibition will be required for successful caspase inhibition-based therapies. PMID:15067000

  7. Metal ion site engineering indicates a global toggle switch model for seven-transmembrane receptor activation

    Elling, Christian E; Frimurer, Thomas M; Gerlach, Lars-Ole;

    2006-01-01

    to form high affinity metal ion sites in both the bidentate and potential tridentate settings. This indicates that the residues involved in the main ligand-binding pocket will have to move closer to each other during receptor activation. On the basis of the distance constraints from these activating metal...

  8. 77 FR 39508 - Commercial Wind Lease Issuance and Site Assessment Activities on the Atlantic Outer Continental...

    2012-07-03

    ... specific project proposals on those leases) in an identified Wind Energy Area (WEA) on the OCS offshore... Activities on the Atlantic OCS Offshore RI and MA'' to: Program Manager, Office of Renewable Energy Programs... Bureau of Ocean Energy Management Commercial Wind Lease Issuance and Site Assessment Activities on...

  9. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand

    Parashar, Abhinav [Center for Biomedical Research, VIT University, Vellore, Tamil Nadu, 632014 India (India); Venkatachalam, Avanthika [REDOx Lab, PSG Institute of Advanced Studies, Avinashi Road, Peelamedu, Coimbatore, Tamil Nadu, 641004 (India); Gideon, Daniel Andrew [Center for Biomedical Research, VIT University, Vellore, Tamil Nadu, 632014 India (India); Manoj, Kelath Murali, E-mail: satyamjayatu@yahoo.com [REDOx Lab, PSG Institute of Advanced Studies, Avinashi Road, Peelamedu, Coimbatore, Tamil Nadu, 641004 (India)

    2014-12-12

    Highlights: • Cyanide (CN) is a well-studied toxic principle, known to inhibit heme-enzymes. • Inhibition is supposed to result from CN binding at the active site as a ligand. • Diverse heme enzymes’ CN inhibition profiles challenge prevailing mechanism. • Poor binding efficiency of CN at low enzyme concentrations and ligand pressures. • CN-based diffusible radicals cause ‘non-productive electron transfers’ (inhibition). - Abstract: The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins’ active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes.

  10. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand

    Highlights: • Cyanide (CN) is a well-studied toxic principle, known to inhibit heme-enzymes. • Inhibition is supposed to result from CN binding at the active site as a ligand. • Diverse heme enzymes’ CN inhibition profiles challenge prevailing mechanism. • Poor binding efficiency of CN at low enzyme concentrations and ligand pressures. • CN-based diffusible radicals cause ‘non-productive electron transfers’ (inhibition). - Abstract: The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins’ active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes

  11. Structure of Human Pancreatic Lipase-Related Protein 2 with the Lid in an Open Conformation

    Eydoux, Cecilia; Spinelli, Silvia; Davis, Tara L.; Walker, John R.; Seitova, Alma; Dhe-Paganon, Sirano; De Caro, Alain; Cambillau, Christian; Carriere, Frederic (CNRS-UMR); (Toronto)

    2008-10-02

    Access to the active site of pancreatic lipase (PL) is controlled by a surface loop, the lid, which normally undergoes conformational changes only upon addition of lipids or amphiphiles. Structures of PL with their lids in the open and functional conformation have required cocrystallization with amphiphiles. Here we report two crystal structures of wild-type and unglycosylated human pancreatic lipase-related protein 2 (HPLRP2) with the lid in an open conformation in the absence of amphiphiles. These structures solved independently are strikingly similar, with some residues of the lid being poorly defined in the electron-density map. The open conformation of the lid is however different from that previously observed in classical liganded PL, suggesting different kinetic properties for HPLRP2. Here we show that the HPLRP2 is directly inhibited by E600, does not present interfacial activation, and acts preferentially on substrates forming monomers or small aggregates (micelles) dispersed in solution like monoglycerides, phospholipids and galactolipids, whereas classical PL displays reverse properties and a high specificity for unsoluble substrates like triglycerides and diglycerides forming oil-in-water interfaces. These biochemical properties imply that the lid of HPLRP2 is likely to spontaneously adopt in solution the open conformation observed in the crystal structure. This open conformation generates a large cavity capable of accommodating the digalactose polar head of galactolipids, similar to that previously observed in the active site of the guinea pig PLRP2, but absent from the classical PL. Most of the structural and kinetic properties of HPLRP2 were found to be different from those of rat PLRP2, the structure of which was previously obtained with the lid in a closed conformation. Our findings illustrate the essential role of the lid in determining the substrate specificity and the mechanism of action of lipases.

  12. Insight into the mechanism of phosphoenolpyruvate mutase catalysis derived from site-directed mutagenesis studies of active site residues.

    Jia, Y; Lu, Z; Huang, K; Herzberg, O; Dunaway-Mariano, D

    1999-10-26

    PEP mutase catalyzes the conversion of phosphoenolpyruvate (PEP) to phosphonopyruvate in biosynthetic pathways leading to phosphonate secondary metabolites. A recent X-ray structure [Huang, K., Li, Z., Jia, Y., Dunaway-Mariano, D., and Herzberg, O. (1999) Structure (in press)] of the Mytilus edulis enzyme complexed with the Mg(II) cofactor and oxalate inhibitor reveals an alpha/beta-barrel backbone-fold housing an active site in which Mg(II) is bound by the two carboxylate groups of the oxalate ligand and the side chain of D85 and, via bridging water molecules, by the side chains of D58, D85, D87, and E114. The oxalate ligand, in turn, interacts with the side chains of R159, W44, and S46 and the backbone amide NHs of G47 and L48. Modeling studies identified two feasible PEP binding modes: model A in which PEP replaces oxalate with its carboxylate group interacting with R159 and its phosphoryl group positioned close to D58 and Mg(II) shifting slightly from its original position in the crystal structure, and model B in which PEP replaces oxalate with its phosphoryl group interacting with R159 and Mg(II) retaining its original position. Site-directed mutagenesis studies of the key mutase active site residues (R159, D58, D85, D87, and E114) were carried out in order to evaluate the catalytic roles predicted by the two models. The observed retention of low catalytic activity in the mutants R159A, D85A, D87A, and E114A, coupled with the absence of detectable catalytic activity in D58A, was interpreted as evidence for model A in which D58 functions in nucleophilic catalysis (phosphoryl transfer), R159 functions in PEP carboxylate group binding, and the carboxylates of D85, D87 and E114 function in Mg(II) binding. These results also provide evidence against model B in which R159 serves to mediate the phosphoryl transfer. A catalytic motif, which could serve both the phosphoryl transfer and the C-C cleavage enzymes of the PEP mutase superfamily, is proposed. PMID:10571990

  13. Conformal Bootstrap in Embedding Space

    Fortin, Jean-François

    2016-01-01

    It is shown how to obtain conformal blocks from embedding space with the help of the operator product expansion. The minimal conformal block originates from scalar exchange in a four-point correlation functions of four scalars. All remaining conformal blocks are simple derivatives of the minimal conformal block. With the help of the orthogonality properties of the conformal blocks, the analytic conformal bootstrap can be implemented directly in embedding space, leading to a Jacobi-like definition of conformal field theories.

  14. Conformal bootstrap in embedding space

    Fortin, Jean-François; Skiba, Witold

    2016-05-01

    It is shown how to obtain conformal blocks from embedding space with the help of the operator product expansion. The minimal conformal block originates from scalar exchange in a four-point correlation function of four scalars. All remaining conformal blocks are simple derivatives of the minimal conformal block. With the help of the orthogonality properties of the conformal blocks, the analytic conformal bootstrap can be implemented directly in embedding space, leading to a Jacobi-like definition of conformal field theories.

  15. Crystal structure of an avian influenza polymerase PA[subscript N] reveals an endonuclease active site

    Yuan, Puwei; Bartlam, Mark; Lou, Zhiyong; Chen, Shoudeng; Zhou, Jie; He, Xiaojing; Lv, Zongyang; Ge, Ruowen; Li, Xuemei; Deng, Tao; Fodor, Ervin; Rao, Zihe; Liu, Yingfang; (NU Sinapore); (Nankai); (Oxford); (Chinese Aca. Sci.); (Tsinghua)

    2009-11-10

    The heterotrimeric influenza virus polymerase, containing the PA, PB1 and PB2 proteins, catalyses viral RNA replication and transcription in the nucleus of infected cells. PB1 holds the polymerase active site and reportedly harbours endonuclease activity, whereas PB2 is responsible for cap binding. The PA amino terminus is understood to be the major functional part of the PA protein and has been implicated in several roles, including endonuclease and protease activities as well as viral RNA/complementary RNA promoter binding. Here we report the 2.2 angstrom (A) crystal structure of the N-terminal 197 residues of PA, termed PA(N), from an avian influenza H5N1 virus. The PA(N) structure has an alpha/beta architecture and reveals a bound magnesium ion coordinated by a motif similar to the (P)DX(N)(D/E)XK motif characteristic of many endonucleases. Structural comparisons and mutagenesis analysis of the motif identified in PA(N) provide further evidence that PA(N) holds an endonuclease active site. Furthermore, functional analysis with in vivo ribonucleoprotein reconstitution and direct in vitro endonuclease assays strongly suggest that PA(N) holds the endonuclease active site and has critical roles in endonuclease activity of the influenza virus polymerase, rather than PB1. The high conservation of this endonuclease active site among influenza strains indicates that PA(N) is an important target for the design of new anti-influenza therapeutics.

  16. In silico analysis of Pycnoporus cinnabarinus laccase active site with toxic industrial dyes.

    Prasad, Nirmal K; Vindal, Vaibhav; Narayana, Siva Lakshmi; Ramakrishna, V; Kunal, Swaraj Priyaranjan; Srinivas, M

    2012-05-01

    Laccases belong to multicopper oxidases, a widespread class of enzymes implicated in many oxidative functions in various industrial oxidative processes like production of fine chemicals to bioremediation of contaminated soil and water. In order to understand the mechanisms of substrate binding and interaction between substrates and Pycnoporus cinnabarinus laccase, a homology model was generated. The resulted model was further validated and used for docking studies with toxic industrial dyes- acid blue 74, reactive black 5 and reactive blue 19. Interactions of chemical mediators with the laccase was also examined. The docking analysis showed that the active site always cannot accommodate the dye molecules, due to constricted nature of the active site pocket and steric hindrance of the residues whereas mediators are relatively small and can easily be accommodated into the active site pocket, which, thereafter leads to the productive binding. The binding properties of these compounds along with identification of critical active site residues can be used for further site-directed mutagenesis experiments in order to identify their role in activity and substrate specificity, ultimately leading to improved mutants for degradation of these toxic compounds. PMID:21877154

  17. Locomotor activity influences muscle architecture and bone growth but not muscle attachment site morphology

    Rabey, Karyne N.; Green, David J; Taylor, Andrea B.; Begun, David R.; Richmond, Brian G.; McFarlin, Shannon C.

    2014-01-01

    The ability to make behavioural inferences from skeletal remains is critical to understanding the lifestyles and activities of past human populations and extinct animals. Muscle attachment site (enthesis) morphology has long been assumed to reflect muscle strength and activity during life, but little experimental evidence exists to directly link activity patterns with muscle development and the morphology of their attachments to the skeleton. We used a mouse model to experimentally test how t...

  18. Nuclear waste: Issues concerning DOE's postponement of second repository siting activities

    In May 1986 the Department of Energy announced it was postponing site-specific work for a second nuclear waste repository because of progress made in siting the first repository and the uncertainty about when a second repository might be needed. A DOE draft report, which tentatively identified potential sites in seven different states, received considerable comment and objections by concerned states, Indian tribes, and individuals. Following the postponement decision, DOE initiated actions to redirect and restructure the second repository program, concentrating on technical issues rather than site-specific work. This reduction includes a phase-down of siting activities and the formulation of a Technology Development Program with an emphasis on cooperative efforts in international research

  19. Large zinc cation occupancy of octahedral sites in mechanically activated zinc ferrite powders

    The cation site occupancy of a mechanically activated nanocrystalline zinc ferrite powder was determined as (Zn0.552+Fe0.183+)tet[Zr0.452+Fe1.823+]octO4 through analysis of extended x-ray absorption fine structure measurements, showing a large redistribution of cations between sites compared to normal zinc ferrite samples. The overpopulation of cations in the octahedral sites was attributed to the ascendance in importance of the ionic radii over the crystal energy and bonding coordination in determining which interstitial sites are occupied in this structurally disordered powder. Slight changes are observed in the local atomic environment about the zinc cations, but not the iron cations, with respect to the spinel structure. The presence of Fe3+ on both sites is consistent with the measured room temperature magnetic properties. (c) 2000 American Institute of Physics

  20. The toponymy of communal activity: Anglo-Saxon assembly sites and their functions

    Baker, John

    2014-01-01

    The paper builds on earlier discussion of the multiple functions of medieval judicial assembly sites, providing a comprehensive evaluation of relevant English hundred-names, and making reference to associated microtoponymy. While religious, military, commercial, and recreational activities may all have occurred at assembly-sites, it can be hard to delineate the evidence so clearly along these lines, and attempts to do so may be anachronistic in some instances; nevertheless, the analysis of di...

  1. Changes to gonadotropin-releasing hormone (GnRH) receptor extracellular loops differentially affect GnRH analog binding and activation: evidence for distinct ligand-stabilized receptor conformations.

    Pfleger, Kevin D G; Pawson, Adam J; Millar, Robert P

    2008-06-01

    GnRH and its structural variants bind to GnRH receptors from different species with different affinities and specificities. By investigating chimeric receptors that combine regions of mammalian and nonmammalian GnRH receptors, a greater understanding of how different domains influence ligand binding and receptor activation can be achieved. Using human-catfish and human-chicken chimeric receptors, we demonstrate the importance of extracellular loop conformation for ligand binding and agonist potency, providing further evidence for GnRH and GnRH II stabilization of distinct active receptor conformations. We demonstrate examples of GnRH receptor gain-of-function mutations that apparently improve agonist potency independently of affinity, implicating a role for extracellular loops in stabilizing the inactive receptor conformation. We also show that entire extracellular loop substitution can overcome the detrimental effects of localized mutations, thereby demonstrating the importance of considering the conformation of entire domains when drawing conclusions from point-mutation studies. Finally, we present evidence implicating the configuration of extracellular loops 2 and 3 in combination differentiating GnRH analog binding modes. Because there are two endogenous forms of GnRH ligand but only one functional form of full-length GnRH receptor in humans, understanding how GnRH and GnRH II can elicit distinct functional effects through the same receptor is likely to provide important insights into how these ligands can have differential effects in both physiological and pathological situations. PMID:18356273

  2. Targeting the active site of the placental isozyme of alkaline phosphatase by phage-displayed scFv antibodies selected by a specific uncompetitive inhibitor

    Kala Mrinalini

    2005-12-01

    Full Text Available Abstract Background The isozymes of alkaline phosphatase, the tissue non-specific, intestinal and placental, have similar properties and a high degree of identity. The placental isozyme (PLAP is an oncofetal antigen expressed in several malignancies including choriocarcinoma, seminoma and ovarian carcinoma. We had earlier attempted to isolate PLAP-specific scFv from a synthetic human immunoglobulin library but were unable to do so, presumably because of the similarity between the isozymes. In this work, we have employed a PLAP-specific uncompetitive inhibitor, L-Phe-Gly-Gly, to select isozyme specific scFvs. An uncompetitive inhibitor binds to the enzyme in the presence of substrate and stabilizes the enzyme-substrate complex. Several uncompetitive inhibitors have varying degrees of isozyme specificity for human alkaline phosphatase isozymes. A specific uncompetitive inhibitor would be able to unmask conformational differences between the otherwise very similar molecules. Also, such inhibitors would be directed to regions at/close to the active site of the enzyme. In this work, the library was first incubated with PLAP and the bound clones then eluted by incubation with L-Phe-Gly-Gly along with the substrate, para-nitro phenyl phosphate (pNPP. The scFvs were then studied with regard to the biochemical modulation of their binding, isozyme specificity and effect on enzyme activity. Results Of 13 clones studied initially, the binding of 9 was inhibited by L-Phe-Gly-Gly (with pNPP and 2 clones were inhibited by pNPP alone. Two clones had absolute and 2 clones had partial specificity to PLAP. Two clones were cross-reactive with only one other isozyme. Three scFv clones, having an accessible His6-tag, were purified and studied for their modulation of enzyme activity. All the three scFvs inhibited PLAP activity with the kinetics of competitive inhibition. Cell ELISA could demonstrate binding of the specific scFvs to the cell surface expressed PLAP

  3. Counting Active Sites on Titanium Oxide-Silica Catalysts for Hydrogen Peroxide Activation through In Situ Poisoning with Phenylphosphonic Acid

    Eaton, Todd R.; Boston, Andrew M.; Thompson, Anthony B.; Gray, Kimberly A.; Notestein, Justin M. [NWU

    2015-06-04

    Quantifying specific active sites in supported catalysts improves our understanding and assists in rational design. Supported oxides can undergo significant structural changes as surface densities increase from site-isolated cations to monolayers and crystallites, which changes the number of kinetically relevant sites. Herein, TiOx domains are titrated on TiOx–SiO2 selectively with phenylphosphonic acid (PPA). An ex situ method quantifies all fluid-accessible TiOx, whereas an in situ titration during cis-cyclooctene epoxidation provides previously unavailable values for the number of tetrahedral Ti sites on which H2O2 activation occurs. We use this method to determine the active site densities of 22 different catalysts with different synthesis methods, loadings, and characteristic spectra and find a single intrinsic turnover frequency for cis-cyclooctene epoxidation of (40±7) h-1. This simple method gives molecular-level insight into catalyst structure that is otherwise hidden when bulk techniques are used.

  4. Mutations Closer to the Active Site Improve the Promiscuous Aldolase Activity of 4-Oxalocrotonate Tautomerase More Effectively than Distant Mutations.

    Rahimi, Mehran; van der Meer, Jan-Ytzen; Geertsema, Edzard M; Poddar, Harshwardhan; Baas, Bert-Jan; Poelarends, Gerrit J

    2016-07-01

    The enzyme 4-oxalocrotonate tautomerase (4-OT), which catalyzes enol-keto tautomerization as part of a degradative pathway for aromatic hydrocarbons, promiscuously catalyzes various carbon-carbon bond-forming reactions. These include the aldol condensation of acetaldehyde with benzaldehyde to yield cinnamaldehyde. Here, we demonstrate that 4-OT can be engineered into a more efficient aldolase for this condensation reaction, with a >5000-fold improvement in catalytic efficiency (kcat /Km ) and a >10(7) -fold change in reaction specificity, by exploring small libraries in which only "hotspots" are varied. The hotspots were identified by systematic mutagenesis (covering each residue), followed by a screen for single mutations that give a strong improvement in the desired aldolase activity. All beneficial mutations were near the active site of 4-OT, thus underpinning the notion that new catalytic activities of a promiscuous enzyme are more effectively enhanced by mutations close to the active site. PMID:27238293

  5. Stringency of the 2-His-1-Asp active-site motif in prolyl 4-hydroxylase.

    Kelly L Gorres

    Full Text Available The non-heme iron(II dioxygenase family of enzymes contain a common 2-His-1-carboxylate iron-binding motif. These enzymes catalyze a wide variety of oxidative reactions, such as the hydroxylation of aliphatic C-H bonds. Prolyl 4-hydroxylase (P4H is an alpha-ketoglutarate-dependent iron(II dioxygenase that catalyzes the post-translational hydroxylation of proline residues in protocollagen strands, stabilizing the ensuing triple helix. Human P4H residues His412, Asp414, and His483 have been identified as an iron-coordinating 2-His-1-carboxylate motif. Enzymes that catalyze oxidative halogenation do so by a mechanism similar to that of P4H. These halogenases retain the active-site histidine residues, but the carboxylate ligand is replaced with a halide ion. We replaced Asp414 of P4H with alanine (to mimic the active site of a halogenase and with glycine. These substitutions do not, however, convert P4H into a halogenase. Moreover, the hydroxylase activity of D414A P4H cannot be rescued with small molecules. In addition, rearranging the two His and one Asp residues in the active site eliminates hydroxylase activity. Our results demonstrate a high stringency for the iron-binding residues in the P4H active site. We conclude that P4H, which catalyzes an especially demanding chemical transformation, is recalcitrant to change.

  6. Quantum delocalization of protons in the hydrogen bond network of an enzyme active site

    Wang, Lu; Boxer, Steven G; Markland, Thomas E

    2015-01-01

    Enzymes utilize protein architectures to create highly specialized structural motifs that can greatly enhance the rates of complex chemical transformations. Here we use experiments, combined with ab initio simulations that exactly include nuclear quantum effects, to show that a triad of strongly hydrogen bonded tyrosine residues within the active site of the enzyme ketosteroid isomerase (KSI) facilitates quantum proton delocalization. This delocalization dramatically stabilizes the deprotonation of an active site tyrosine residue, resulting in a very large isotope effect on its acidity. When an intermediate analog is docked, it is incorporated into the hydrogen bond network, giving rise to extended quantum proton delocalization in the active site. These results shed light on the role of nuclear quantum effects in the hydrogen bond network that stabilizes the reactive intermediate of KSI, and the behavior of protons in biological systems containing strong hydrogen bonds.

  7. Conformal invariance in supergravity

    In this thesis the author explains the role of conformal invariance in supergravity. He presents the complete structure of extended conformal supergravity for N <= 4. The outline of this work is as follows. In chapter 2 he briefly summarizes the essential properties of supersymmetry and supergravity and indicates the use of conformal invariance in supergravity. The idea that the introduction of additional symmetry transformations can make clear the structure of a field theory is not reserved to supergravity only. By means of some simple examples it is shown in chapter 3 how one can always introduce additional gauge transformations in a theory of massive vector fields. Moreover it is shown how the gauge invariant formulation sometimes explains the quantum mechanical properties of the theory. In chapter 4 the author defines the conformal transformations and summarizes their main properties. He explains how these conformal transformations can be used to analyse the structure of gravity. The supersymmetric extension of these results is discussed in chapter 5. Here he describes as an example how N=1 supergravity can be reformulated in a conformally-invariant way. He also shows that beyond N=1 the gauge fields of the superconformal symmetries do not constitute an off-shell field representation of extended conformal supergravity. Therefore, in chapter 6, a systematic method to construct the off-shell formulation of all extended conformal supergravity theories with N <= 4 is developed. As an example he uses this method to construct N=1 conformal supergravity. Finally, in chapter 7 N=4 conformal supergravity is discussed. (Auth.)

  8. Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity.

    Hartmann, Jens; Linke, Dennis; Bönniger, Christine; Tholey, Andreas; Sauter, Margret

    2015-12-15

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser(696) and Ser(698) in the JM (juxtamembrane) region and probably Ser(886) and/or Ser(893) in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser(717) in the JM, and at Ser(733), Thr(752), Ser(783), Ser(864), Ser(911), Ser(958) and Thr(998) in the kinase domain. The LC-ESI-MS/MS spectra provided support that up to three sites (Thr(890), Ser(893) and Thr(894)) in the AL were likely to be phosphorylated in vitro. These sites are evolutionarily highly conserved in PSK receptors, indicative of a conserved function. Site-directed mutagenesis of the four conserved residues in the activation segment, Thr(890), Ser(893), Thr(894) and Thr(899), differentially altered kinase activity in vitro and growth-promoting activity in planta. The T899A and the quadruple-mutated TSTT-A (T890A/S893A/T894A/T899A) mutants were both kinase-inactive, but PSKR1(T899A) retained growth-promoting activity. The T890A and S893A/T894A substitutions diminished kinase activity and growth promotion. We hypothesize that phosphorylation within the AL activates kinase activity and receptor function in a gradual and distinctive manner that may be a means to modulate the PSK response. PMID:26472115

  9. Testing the applicability of rapid on-site enzymatic activity detection for surface water monitoring

    Stadler, Philipp; Vogl, Wolfgang; Juri, Koschelnik; Markus, Epp; Maximilian, Lackner; Markus, Oismüller; Monika, Kumpan; Peter, Strauss; Regina, Sommer; Gabriela, Ryzinska-Paier; Farnleitner Andreas, H.; Matthias, Zessner

    2015-04-01

    On-site detection of enzymatic activities has been suggested as a rapid surrogate for microbiological pollution monitoring of water resources (e.g. using glucuronidases, galactosidases, esterases). Due to the possible short measuring intervals enzymatic methods have high potential as near-real time water quality monitoring tools. This presentation describes results from a long termed field test. For twelve months, two ColiMinder devices (Vienna Water Monitoring, Austria) for on-site determination of enzymatic activity were tested for stream water monitoring at the experimental catchment HOAL (Hydrological Open Air Laboratory, Center for Water Resource Systems, Vienna University of Technology). The devices were overall able to follow and reflect the diverse hydrological and microbiological conditions of the monitored stream during the test period. Continuous data in high temporal resolution captured the course of enzymatic activity in stream water during diverse rainfall events. The method also proofed sensitive enough to determine diurnal fluctuations of enzymatic activity in stream water during dry periods. The method was able to capture a seasonal trend of enzymatic activity in stream water that matches the results gained from Colilert18 analysis for E. coli and coliform bacteria of monthly grab samples. Furthermore the comparison of ColiMinder data with measurements gained at the same test site with devices using the same method but having different construction design (BACTcontrol, microLAN) showed consistent measuring results. Comparative analysis showed significant differences between measured enzymatic activity (modified fishman units and pmol/min/100ml) and cultivation based analyses (most probable number, colony forming unit). Methods of enzymatic activity measures are capable to detect ideally the enzymatic activity caused by all active target bacteria members, including VBNC (viable but nonculturable) while cultivation based methods cannot detect VBNC

  10. Preliminary examination of the impacts of repository site characterization activities and facility construction and operation activities on Hanford air quality

    Glantz, C.S.; Ramsdell, J.V.

    1986-04-01

    Air quality impacts that would result from site characterization activities and from the construction and operation of a high-level nuclear wste repository at Hanford are estimated using two simple atmospheric dispersion models, HANCHI and CHISHORT. Model results indicate that pollutant concentrations would not exceed ambient air quality standards at any point outside the Hanford fenceline or at any publicly accessible location within the Hanford Site. The increase in pollutant concentrations in nearby communities due to site activities would be minimal. HANCHI and CHISHORT are documented in the appendices of this document. Further study of the repository's impact on air quality will be conducted when more detailed project plans and work schedules are available.

  11. Preliminary examination of the impacts of repository site characterization activities and facility construction and operation activities on Hanford air quality

    Air quality impacts that would result from site characterization activities and from the construction and operation of a high-level nuclear wste repository at Hanford are estimated using two simple atmospheric dispersion models, HANCHI and CHISHORT. Model results indicate that pollutant concentrations would not exceed ambient air quality standards at any point outside the Hanford fenceline or at any publicly accessible location within the Hanford Site. The increase in pollutant concentrations in nearby communities due to site activities would be minimal. HANCHI and CHISHORT are documented in the appendices of this document. Further study of the repository's impact on air quality will be conducted when more detailed project plans and work schedules are available

  12. Activator anion binding site in pyridoxal phosphorylase b: the binding of phosphite, phosphate, and fluorophosphate in the crystal.

    Oikonomakos, Nikos G.; Zographos, Spyros E.; Tsitsanou, K. E.; Johnson, L N; Acharya, K. R.

    1996-01-01

    It has been established that phosphate analogues can activate glycogen phosphorylase reconstituted with pyridoxal in place of the natural cofactor pyridoxal 5'-phosphate (Change YC. McCalmont T, Graves DJ. 1983. Biochemistry 22:4987-4993). Pyridoxal phosphorylase b has been studied by kinetic, ultracentrifugation, and X-ray crystallographic experiments. In solution, the catalytically active species of pyridoxal phosphorylase b adopts a conformation that is more R-state-like than that of nativ...

  13. Threatened and endangered wildlife species of the Hanford Site related to CERCLA characterization activities

    Fitzner, R.E. [Pacific Northwest Lab., Richland, WA (United States); Weiss, S.G.; Stegen, J.A. [Westinghouse Hanford Co., Richland, WA (United States)

    1994-06-01

    The US Department of Energy`s (DOE) Hanford Site has been placed on the National Priorities List, which requires that it be remediated under the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) or Superfund. Potentially contaminated areas of the Hanford Site were grouped into operable units, and detailed characterization and investigation plans were formulated. The DOE Richland Operations Office requested Westinghouse Hanford Company (WHC) to conduct a biological assessment of the potential impact of these characterization activities on the threatened, endangered, and sensitive wildlife species of the Hanford Site. Additional direction for WHC compliances with wildlife protection can be found in the Environmental Compliance Manual. This document is intended to meet these requirements, in part, for the CERCLA characterization activities, as well as for other work comparable in scope. This report documents the biological assessment and describes the pertinent components of the Hanford Site as well as the planned characterization activities. Also provided are accounts of endangered, threatened, and federal candidate wildlife species on the Hanford Site and information as to how human disturbances can affect these species. Potential effects of the characterization activities are described with recommendations for mitigation measures.

  14. The active site of low-temperature methane hydroxylation in iron-containing zeolites.

    Snyder, Benjamin E R; Vanelderen, Pieter; Bols, Max L; Hallaert, Simon D; Böttger, Lars H; Ungur, Liviu; Pierloot, Kristine; Schoonheydt, Robert A; Sels, Bert F; Solomon, Edward I

    2016-08-18

    An efficient catalytic process for converting methane into methanol could have far-reaching economic implications. Iron-containing zeolites (microporous aluminosilicate minerals) are noteworthy in this regard, having an outstanding ability to hydroxylate methane rapidly at room temperature to form methanol. Reactivity occurs at an extra-lattice active site called α-Fe(ii), which is activated by nitrous oxide to form the reactive intermediate α-O; however, despite nearly three decades of research, the nature of the active site and the factors determining its exceptional reactivity are unclear. The main difficulty is that the reactive species-α-Fe(ii) and α-O-are challenging to probe spectroscopically: data from bulk techniques such as X-ray absorption spectroscopy and magnetic susceptibility are complicated by contributions from inactive 'spectator' iron. Here we show that a site-selective spectroscopic method regularly used in bioinorganic chemistry can overcome this problem. Magnetic circular dichroism reveals α-Fe(ii) to be a mononuclear, high-spin, square planar Fe(ii) site, while the reactive intermediate, α-O, is a mononuclear, high-spin Fe(iv)=O species, whose exceptional reactivity derives from a constrained coordination geometry enforced by the zeolite lattice. These findings illustrate the value of our approach to exploring active sites in heterogeneous systems. The results also suggest that using matrix constraints to activate metal sites for function-producing what is known in the context of metalloenzymes as an 'entatic' state-might be a useful way to tune the activity of heterogeneous catalysts. PMID:27535535

  15. Utility experiences in redevelopment of formerly used sites -- Wisconsin Electric's risk management and economic development activities

    Wisconsin Electric Power Company, which recently celebrated its 100th anniversary, has actively promoted the redevelopment of its former sites as well as those of its customers. Serving Milwaukee and southeast Wisconsin, Wisconsin Electric's (WE) sites include former power plants, landfills, right-of-ways, and manufactured gas plant sites. In setting an example for others, as well as seeking to maximize the economic value of these sites, WE has either redeveloped or promoted the redevelopment of these sites by others. Examples include the East Wells Power Plant (now home of the Milwaukee Repertory Theater), the Lakeside Power Plant Site (now the home of Harnischfeger Corporation's headquarters), and the Commerce Street Power Plant located on the Milwaukee River near downtown Milwaukee. In each case the company evaluated the potential environmental liabilities against the unrealized asset value derived from facility location, site size, architectural uniqueness, or other characteristics. At the Commerce Street Power Plant, walking distance to the downtown Milwaukee business district combined with river frontage, were significant site values leveraged against a $5 million asbestos and lead-based paint removal project done to prepare the plant for marketing. More recently, WE has used its experience in promoting the redevelopment of the Menomonee River Valley, the original core of Milwaukee's industrial community, and in advancing a more practical regulatory approach to redeveloping older sites. Finally, the company is working with a non-profit community health clinic, community groups and local foundations in linking these redevelopment activities with the economic and physical health of inner city residents

  16. Crystal Structures of a Multidrug-Resistant Human Immunodeficiency Virus Type 1 Protease Reveal an Expanded Active-Site Cavity

    Logsdon, Bradley C.; Vickrey, John F.; Martin, Philip; Proteasa, Gheorghe; Koepke, Jay I.; Terlecky, Stanley R.; Wawrzak, Zdzislaw; Winters, Mark A.; Merigan, Thomas C.; Kovari, Ladislau C. (Stanford); (WSU-MED); (NWU); (Stanford-MED)

    2010-03-08

    The goal of this study was to use X-ray crystallography to investigate the structural basis of resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors. We overexpressed, purified, and crystallized a multidrug-resistant (MDR) HIV-1 protease enzyme derived from a patient failing on several protease inhibitor-containing regimens. This HIV-1 variant contained codon mutations at positions 10, 36, 46, 54, 63, 71, 82, 84, and 90 that confer drug resistance to protease inhibitors. The 1.8-{angstrom} crystal structure of this MDR patient isolate reveals an expanded active-site cavity. The active-site expansion includes position 82 and 84 mutations due to the alterations in the amino acid side chains from longer to shorter (e.g., V82A and I84V). The MDR isolate 769 protease 'flaps' stay open wider, and the difference in the flap tip distances in the MDR 769 variant is 12 {angstrom}. The MDR 769 protease crystal complexes with lopinavir and DMP450 reveal completely different binding modes. The network of interactions between the ligands and the MDR 769 protease is completely different from that seen with the wild-type protease-ligand complexes. The water molecule-forming hydrogen bonds bridging between the two flaps and either the substrate or the peptide-based inhibitor are lacking in the MDR 769 clinical isolate. The S1, S1', S3, and S3' pockets show expansion and conformational change. Surface plasmon resonance measurements with the MDR 769 protease indicate higher k{sub off} rates, resulting in a change of binding affinity. Surface plasmon resonance measurements provide k{sub on} and k{sub off} data (K{sub d} = k{sub off}/k{sub on}) to measure binding of the multidrug-resistant protease to various ligands. This MDR 769 protease represents a new antiviral target, presenting the possibility of designing novel inhibitors with activity against the open and expanded protease forms.

  17. Human Activities in Natura 2000 Sites: A Highly Diversified Conservation Network

    Tsiafouli, Maria A.; Apostolopoulou, Evangelia; Mazaris, Antonios D.; Kallimanis, Athanasios S.; Drakou, Evangelia G.; Pantis, John D.

    2013-05-01

    The Natura 2000 network was established across the European Union's (EU) Member States with the aim to conserve biodiversity, while ensuring the sustainability of human activities. However, to what kind and to what extent Natura 2000 sites are subject to human activities and how this varies across Member States remains unspecified. Here, we analyzed 111,269 human activity records from 14,727 protected sites in 20 Member States. The frequency of occurrence of activities differs among countries, with more than 86 % of all sites being subjected to agriculture or forestry. Activities like hunting, fishing, urbanization, transportation, and tourism are more frequently recorded in south European sites than in northern or eastern ones. The observed variations indicate that Natura 2000 networks are highly heterogeneous among EU Member States. Our analysis highlights the importance of agriculture in European landscapes and indicates possible targets for policy interventions at national, European, or "sub-European" level. The strong human presence in the Natura 2000 network throughout Member States, shows that conservation initiatives could succeed only by combining social and ecological sustainability and by ensuring the integration of policies affecting biodiversity.

  18. Human activities in Natura 2000 sites: a highly diversified conservation network.

    Tsiafouli, Maria A; Apostolopoulou, Evangelia; Mazaris, Antonios D; Kallimanis, Athanasios S; Drakou, Evangelia G; Pantis, John D

    2013-05-01

    The Natura 2000 network was established across the European Union's (EU) Member States with the aim to conserve biodiversity, while ensuring the sustainability of human activities. However, to what kind and to what extent Natura 2000 sites are subject to human activities and how this varies across Member States remains unspecified. Here, we analyzed 111,269 human activity records from 14,727 protected sites in 20 Member States. The frequency of occurrence of activities differs among countries, with more than 86 % of all sites being subjected to agriculture or forestry. Activities like hunting, fishing, urbanization, transportation, and tourism are more frequently recorded in south European sites than in northern or eastern ones. The observed variations indicate that Natura 2000 networks are highly heterogeneous among EU Member States. Our analysis highlights the importance of agriculture in European landscapes and indicates possible targets for policy interventions at national, European, or "sub-European" level. The strong human presence in the Natura 2000 network throughout Member States, shows that conservation initiatives could succeed only by combining social and ecological sustainability and by ensuring the integration of policies affecting biodiversity. PMID:23571828

  19. A facile reflux procedure to increase active surface sites form highly active and durable supported palladium@platinum bimetallic nanodendrites

    Wang, Qin; Li, Yingjun; Liu, Baocang; Xu, Guangran; Zhang, Geng; Zhao, Qi; Zhang, Jun

    2015-11-01

    A series of well-dispersed bimetallic Pd@Pt nanodendrites uniformly supported on XC-72 carbon black are fabricated by using different capping agents. These capping agents are essential for the branched morphology control. However, the surfactant adsorbed on the nanodendrites surface blocks the access of reactant molecules to the active surface sites, and the catalytic activities of these bimetallic nanodendrites are significantly restricted. Herein, a facile reflux procedure to effectively remove the capping agent molecules without significantly affecting their sizes is reported for activating supported nanocatalysts. More significantly, the structure and morphology of the nanodendrites can also be retained, enhancing the numbers of active surface sites, catalytic activity and stability toward methanol and ethanol electro-oxidation reactions. The as-obtained hot water reflux-treated Pd@Pt/C catalyst manifests superior catalytic activity and stability both in terms of surface and mass specific activities, as compared to the untreated catalysts and the commercial Pt/C and Pd/C catalysts. We anticipate that this effective and facile removal method has more general applicability to highly active nanocatalysts prepared with various surfactants, and should lead to improvements in environmental protection and energy production.

  20. Conformational kinetics of aliphatic tails

    Ferrarini, Alberta; Moro, Giorgio; Nordio, Pier Luigi

    The master equation describing the random walk between sites identified with the stable conformers of a chain molecule, represents the extension to the time domain of the Rotational Isomeric State model. The asymptotic analysis of the multidimensional diffusion equation in the continuous torsional variables subjected to the configurational potential, provides a rigorous justification for the discrete models, and it supplies, without resorting to phenomenological parameters, molecular definitions of the kinetic rates for the conformational transitions occurring at each segment of the chain. The coupling between the torsional variables is fully taken into account, giving rise to cooperative effects. A complete calculation of the specific correlation functions which describe the time evolution of the angular functions probed by N.M.R. and dielectric relaxation measurements, has been performed for alkyl chains attached to a massive core. The resulting behaviour has been compared with the decay of trans and gauche populations of specific bonds, expressed in terms of suitable correlation functions whose time integrals lead quite naturally to the definition of effective kinetic constants for the conformational transitions.

  1. Immobilized low-activity waste site borehole 299-E17-21

    The Tank Waste Remediation System (TWRS) is the group at the Hanford Site responsible for the safe underground storage of liquid waste from previous Hanford Site operations, the storage and disposal of immobilized tank waste, and closure of underground tanks. The current plan is to dispose of immobilized low-activity tank waste (ILAW) in new facilities in the southcentral part of 200-East Area and in four existing vaults along the east side of 200-East Area. Boreholes 299-E17-21, B8501, and B8502 were drilled at the southwest corner of the ILAW site in support of the Performance Assessment activities for the disposal options. This report summarizes the initial geologic findings, field tests conducted on those boreholes, and ongoing studies. One deep (480 feet) borehole and two shallow (50 feet) boreholes were drilled at the southwest corner of the ILAW site. The primary factor dictating the location of the boreholes was their characterization function with respect to developing the geohydrologic model for the site and satisfying associated Data Quality Objectives. The deep borehole was drilled to characterize subsurface conditions beneath the ILAW site, and two shallow boreholes were drilled to support an ongoing environmental tracer study. The tracer study will supply information to the Performance Assessment. All the boreholes provide data on the vadose zone and saturated zone in a previously uncharacterized area

  2. Surface binding sites in carbohydrate active enzymes: An emerging picture of structural and functional diversity

    Svensson, Birte; Cockburn, Darrell

    2013-01-01

    universal and is in fact rare among some families of enzymes. In some cases an alternative to possessing a CBM is for the enzyme to bind to the substrate at a site on the catalytic domain, but away from the active site. Such a site is termed a surface (or secondary) binding site (SBS). SBSs have been...... identified in enzymes from a wide variety of families, though almost half are found in the α-amylase family GH13. The roles attributed to SBSs are not limited to targeting the enzyme to the substrate, but also include a variety of others such as guiding the substrate into the active site, altering enzyme...... specificity and acting as an allosteric site. Although SBSs share many roles with CBMs they may not simply be an alternative to CBMs, but rather complementary as SBSs and CBMs frequently co-occur in enzymes. Despite a relatively long history, it is only in recent years that SBSs have been studied in great...

  3. Immobilized low-activity waste site borehole 299-E17-21

    Reidel, S.P.; Reynolds, K.D.; Horton, D.G.

    1998-08-01

    The Tank Waste Remediation System (TWRS) is the group at the Hanford Site responsible for the safe underground storage of liquid waste from previous Hanford Site operations, the storage and disposal of immobilized tank waste, and closure of underground tanks. The current plan is to dispose of immobilized low-activity tank waste (ILAW) in new facilities in the southcentral part of 200-East Area and in four existing vaults along the east side of 200-East Area. Boreholes 299-E17-21, B8501, and B8502 were drilled at the southwest corner of the ILAW site in support of the Performance Assessment activities for the disposal options. This report summarizes the initial geologic findings, field tests conducted on those boreholes, and ongoing studies. One deep (480 feet) borehole and two shallow (50 feet) boreholes were drilled at the southwest corner of the ILAW site. The primary factor dictating the location of the boreholes was their characterization function with respect to developing the geohydrologic model for the site and satisfying associated Data Quality Objectives. The deep borehole was drilled to characterize subsurface conditions beneath the ILAW site, and two shallow boreholes were drilled to support an ongoing environmental tracer study. The tracer study will supply information to the Performance Assessment. All the boreholes provide data on the vadose zone and saturated zone in a previously uncharacterized area.

  4. POISONING OF ACTIVE SITES ON ZIEGLER-NATTA CATALYST FOR PROPYLENE POLYMERIZATION

    Kitti Tangjituabun; Sang Yull Kim; Yuichi Hiraoka; Toshiaki Taniike; Minoru Terano; Bunjerd Jongsomjit; Piyasan Praserthdam

    2008-01-01

    The effects of poisoning materials on catalytic activity and isospecificity of the supported Ziegler-Natta catalyst were investigated.A minor amount of simple structure of Lewis base,i.e.,methanol,acetone,ethyl acetate,was introduced into the catalyst slurry for partial poisoning catalytic active centers.It was found that the variations in deactivation power were in the order of methanol>acetone>ethyl acetate.The kinetic investigation via stopped-flow polymerization showed that poisoning compounds caused a decrease in activity through the reduction of the number of active sites whereas no effect on the degree of isotacticity was observed.

  5. Sphingosine-1-phosphate receptor 1 reporter mice reveal receptor activation sites in vivo

    Kono, Mari; Tucker, Ana E.; Tran, Jennifer; Bergner, Jennifer B.; Turner, Ewa M; Proia, Richard L.

    2014-01-01

    Activation of the GPCR sphingosine-1-phosphate receptor 1 (S1P1) by sphingosine-1-phosphate (S1P) regulates key physiological processes. S1P1 activation also has been implicated in pathologic processes, including autoimmunity and inflammation; however, the in vivo sites of S1P1 activation under normal and disease conditions are unclear. Here, we describe the development of a mouse model that allows in vivo evaluation of S1P1 activation. These mice, known as S1P1 GFP signaling mice, produce a ...

  6. Conformal Symmetry and Unification

    Pawlowski, M

    1998-01-01

    The Weyl-Weinberg-Salam model is presented. It is based on the local conformal gauge symmetry. The model identifies the Higgs scalar field in SM with the Penrose-Chernikov-Tagirov scalar field of the conformal theory of gravity. Higgs mechanism for generation of particle masses is replaced by the originated in Weyl's ideas conformal gauge scale fixing. Scalar field is no longer a dynamical field of the model and does not lead to quantum particle-like excitations that could be observed in HE experiments. Cosmological constant is naturally generated by the scalar quadric term. The model admits Weyl vector bosons that can mix with photon and weak bosons.

  7. Molecular dioxygen enters the active site of 12/15-lipoxygenase via dynamic oxygen access channels.

    Saam, Jan; Ivanov, Igor; Walther, Matthias; Holzhütter, Hermann-Georg; Kuhn, Hartmut

    2007-08-14

    Cells contain numerous enzymes that use molecular oxygen for their reactions. Often, their active sites are buried deeply inside the protein, which raises the question whether there are specific access channels guiding oxygen to the site of catalysis. Choosing 12/15-lipoxygenase as a typical example for such oxygen-dependent enzymes, we determined the oxygen distribution within the protein and defined potential routes for oxygen access. For this purpose, we have applied an integrated strategy of structural modeling, molecular dynamics simulations, site-directed mutagenesis, and kinetic measurements. First, we computed the 3D free-energy distribution for oxygen, which led to identification of four oxygen channels in the protein. All channels connect the protein surface with a region of high oxygen affinity at the active site. This region is localized opposite to the nonheme iron providing a structural explanation for the reaction specificity of this lipoxygenase isoform. The catalytically most relevant path can be obstructed by L367F exchange, which leads to a strongly increased Michaelis constant for oxygen. The blocking mechanism is explained in detail by reordering the hydrogen-bonding network of water molecules. Our results provide strong evidence that the main route for oxygen access to the active site of the enzyme follows a channel formed by transiently interconnected cavities whereby the opening and closure are governed by side chain dynamics. PMID:17675410

  8. PubChem3D: Conformer generation

    Bolton Evan E

    2011-01-01

    Full Text Available Abstract Background PubChem, an open archive for the biological activities of small molecules, provides search and analysis tools to assist users in locating desired information. Many of these tools focus on the notion of chemical structure similarity at some level. PubChem3D enables similarity of chemical structure 3-D conformers to augment the existing similarity of 2-D chemical structure graphs. It is also desirable to relate theoretical 3-D descriptions of chemical structures to experimental biological activity. As such, it is important to be assured that the theoretical conformer models can reproduce experimentally determined bioactive conformations. In the present study, we investigate the effects of three primary conformer generation parameters (the fragment sampling rate, the energy window size, and force field variant upon the accuracy of theoretical conformer models, and determined optimal settings for PubChem3D conformer model generation and conformer sampling. Results Using the software package OMEGA from OpenEye Scientific Software, Inc., theoretical 3-D conformer models were generated for 25,972 small-molecule ligands, whose 3-D structures were experimentally determined. Different values for primary conformer generation parameters were systematically tested to find optimal settings. Employing a greater fragment sampling rate than the default did not improve the accuracy of the theoretical conformer model ensembles. An ever increasing energy window did increase the overall average accuracy, with rapid convergence observed at 10 kcal/mol and 15 kcal/mol for model building and torsion search, respectively; however, subsequent study showed that an energy threshold of 25 kcal/mol for torsion search resulted in slightly improved results for larger and more flexible structures. Exclusion of coulomb terms from the 94s variant of the Merck molecular force field (MMFF94s in the torsion search stage gave more accurate conformer models at

  9. Active catalytic sites in the ammoxidation of propane and propene over V-Sb-O catalysts

    Buchholz, S.A.; Zanthoff, H.W. [Bochum Univ. (Germany). Lehrstuhl fuer Technische Chemie

    1998-12-31

    The ammoxidation of propane over VSb{sub y}O{sub x} catalysts (y=1, 2, 5) was investigated with respect to the role of different oxygen species in the selective and non selective reaction steps using transient experiments in the Temporal Analysis of Products (TAP) reactor. Only lattice oxygen is involved in the oxidation reactions. Using isotopic labelled oxygen it is shown that two different active sites exist on the surface. On site A, which can be reoxidized faster by gas phase oxygen compared to site B, mainly CO is formed. On site B CO{sub 2} and acrolein as well as NO and N{sub 2}O in the presence of ammonia in the feed gas are formed and reoxidation mainly occurs with bulk lattice oxygen. (orig.)

  10. A three-dimensional model of mammalian tyrosinase active site accounting for loss of function mutations.

    Schweikardt, Thorsten; Olivares, Concepción; Solano, Francisco; Jaenicke, Elmar; García-Borrón, José Carlos; Decker, Heinz

    2007-10-01

    Tyrosinases are the first and rate-limiting enzymes in the synthesis of melanin pigments responsible for colouring hair, skin and eyes. Mutation of tyrosinases often decreases melanin production resulting in albinism, but the effects are not always understood at the molecular level. Homology modelling of mouse tyrosinase based on recently published crystal structures of non-mammalian tyrosinases provides an active site model accounting for loss-of-function mutations. According to the model, the copper-binding histidines are located in a helix bundle comprising four densely packed helices. A loop containing residues M374, S375 and V377 connects the CuA and CuB centres, with the peptide oxygens of M374 and V377 serving as hydrogen acceptors for the NH-groups of the imidazole rings of the copper-binding His367 and His180. Therefore, this loop is essential for the stability of the active site architecture. A double substitution (374)MS(375) --> (374)GG(375) or a single M374G mutation lead to a local perturbation of the protein matrix at the active site affecting the orientation of the H367 side chain, that may be unable to bind CuB reliably, resulting in loss of activity. The model also accounts for loss of function in two naturally occurring albino mutations, S380P and V393F. The hydroxyl group in S380 contributes to the correct orientation of M374, and the substitution of V393 for a bulkier phenylalanine sterically impedes correct side chain packing at the active site. Therefore, our model explains the mechanistic necessity for conservation of not only active site histidines but also adjacent amino acids in tyrosinase. PMID:17850513

  11. Recent Experience Using Active Love Wave Techniques to Characterize Seismographic Station Sites

    Martin, A. J.; Yong, A.; Salomone, L.

    2014-12-01

    Active-source Love waves recorded by the multi-channel analysis of surface wave (MASLW) technique were recently analyzed in two site characterization projects. Between 2010 and 2011, the 2009 American Recovery and Reinvestment Act (ARRA) funded GEOVision to conduct geophysical investigations at 189 seismographic stations—185 in California and 4 in the Central Eastern U.S. (CEUS). The original project plan was to utilize active and passive Rayleigh wave-based techniques to obtain shear-wave velocity (VS) profiles to a minimum depth of 30 m and the time-averaged VS of the upper 30 meters (VS30). Early in the investigation it became evident that Rayleigh wave techniques, such as multi-channel analysis of surface waves (MASRW), were not effective at characterizing all sites. Shear-wave seismic refraction and MASLW techniques were therefore applied. The MASLW technique was deployed at a total of 38 sites, in addition to other methods, and used as the primary technique to characterize 22 sites, 5 of which were also characterized using Rayleigh wave techniques. In 2012, the Electric Power Research Institute funded characterization of 33 CEUS station sites. Based on experience from the ARRA investigation, both MASRW and MASLW data were acquired by GEOVision at 24 CEUS sites—the remaining 9 sites and 2 overlapping sites were characterized by University of Texas, Austin. Of the 24 sites characterized by GEOVision, 16 were characterized using MASLW data, 4 using both MASLW and MASRW data and 4 using MASRW data. Love wave techniques were often found to perform better, or at least yield phase velocity data that could be more readily modeled using the fundamental mode assumption, at shallow rock sites, sites with steep velocity gradients, and, sites with a thin, low velocity, surficial soil layer overlying stiffer sediments. These types of velocity structure often excite dominant higher modes in Rayleigh wave data, but not in Love wave data. At such sites, it may be possible

  12. Dimer-dimer interaction of the bacterial selenocysteine synthase SelA promotes functional active-site formation and catalytic specificity.

    Itoh, Yuzuru; Bröcker, Markus J; Sekine, Shun-ichi; Söll, Dieter; Yokoyama, Shigeyuki

    2014-04-17

    The 21st amino acid, selenocysteine (Sec), is incorporated translationally into proteins and is synthesized on its specific tRNA (tRNA(Sec)). In Bacteria, the selenocysteine synthase SelA converts Ser-tRNA(Sec), formed by seryl-tRNA synthetase, to Sec-tRNA(Sec). SelA, a member of the fold-type-I pyridoxal 5'-phosphate-dependent enzyme superfamily, has an exceptional homodecameric quaternary structure with a molecular mass of about 500kDa. Our previously determined crystal structures of Aquifex aeolicus SelA complexed with tRNA(Sec) revealed that the ring-shaped decamer is composed of pentamerized SelA dimers, with two SelA dimers arranged to collaboratively interact with one Ser-tRNA(Sec). The SelA catalytic site is close to the dimer-dimer interface, but the significance of the dimer pentamerization in the catalytic site formation remained elusive. In the present study, we examined the quaternary interactions and demonstrated their importance for SelA activity by systematic mutagenesis. Furthermore, we determined the crystal structures of "depentamerized" SelA variants with mutations at the dimer-dimer interface that prevent pentamerization. These dimeric SelA variants formed a distorted and inactivated catalytic site and confirmed that the pentamer interactions are essential for productive catalytic site formation. Intriguingly, the conformation of the non-functional active site of dimeric SelA shares structural features with other fold-type-I pyridoxal 5'-phosphate-dependent enzymes with native dimer or tetramer (dimer-of-dimers) quaternary structures. PMID:24456689

  13. Variants of the cell recognition site of fibronectin that retain attachment-promoting activity

    1985-01-01

    A tetrapeptide sequence, Arg-Gly-Asp-Ser, is the minimal structure recognized by cells in the large, adhesive glycoprotein fibronectin. We now have defined the structural requirements for this cell recognition site by testing several synthetic variants of the active tetrapeptide sequence. The conservative substitutions of lysine for arginine, alanine for glycine, or glutamic acid for aspartic acid each resulted in abrogation of the cell attachment-promoting activity characteristic of the natu...

  14. Screening Approach to the Activation of Soil and Contamination of Groundwater at Linear Proton Accelerator Sites

    Otto, Thomas

    The activation of soil and the contamination of groundwater at proton accelerator sites with the radionuclides 3H and 22Na are estimated with a Monte-Carlo calculation and a conservative soil- and ground water model. The obtained radionuclide concentrations show that the underground environment of future accelerators must be adequately protected against a migration of activation products. This study is of particular importance for the proton driver accelerator in the planned EURISOL facility.

  15. Study on the active sites of Cu-ZSM-5 in trichloroethylene catalytic combustion with air

    Cheng Hua Xu; Chuan Qi Liu; Yan Zhong; Xiu Zhou Yang; Jian Ying Liu; Ying Chun Yang; Zhi Xiang Ye

    2008-01-01

    The catalytic activity of Cu-ZSM-5 in trichloroethylene (TCE) combustion increases with the increasing skeletal Cu amount and however decreases with the increase of surface amorphous CuO,which is detected by infrared spectroscopy (IR) and diffuse reflectance ultraviolet-visible spectroscopy (DRS-UV-vis),therefore the skeletal Cu species are concluded to be the active sites for the TCE combustion.

  16. Remediation of uranium contaminated sites: clean-up activities in Serbia

    One of the serious environmental problems in Serbia represent sites contaminated with depleted uranium (DU) during past war activities. According to UNEP reports and our findings there are two types of contamination: (i) localized points of high, concentrated contamination where DU penetrators enter the soil, and (ii) low level of widespread DU contamination, which indicates that during the conflict DU dust was dispersed into the environment. Remediation of these sites is an urgent need because they represent a permanent threat to the population living in this area. Here we give a brief description of approaches commonly used in remediation of DU contaminated sites, and an overview of current clean-up activities performed in Serbia. (author)

  17. Identification and characterization of radioactively contaminated sites in Ukraine and planning for environmental restoration activities

    In the Pridniprovsk-Krivoy Rog region uranium, titanium, iron and manganese ores were mined and milled beginning in the 1950s. These activities have caused radioactive contamination of the environment at some sites. In recent times intensive works concerning the surveying of contaminated areas and substantiating the need for remediation have been initiated. The research methodologies applied and the results from radiation surveys are presented for the site of the first uranium mine in the Ukraine, for tailings originating from the Pridniprovsk Chemical Plant (PChP), for the recultivated dump-site of the former 'O'-mine, as well as for the wastes, raw materials and production of the Nicopol Ferro-Alloy Plant. The planning procedure for the remediation activities at the town of Zhovty Vody is described. (author)

  18. Active sites residues of beef liver carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT-II).

    Nic a'Bháird, N; Yankovskaya, V; Ramsay, R R

    1998-01-01

    The carnitine acyltransferases which catalyse the reversible transfer of fatty acyl groups between carnitine and coenzyme A have been proposed to contain a catalytic histidine. Here, the chemical reactivity of active site groups has been used to demonstrate differences between the active sites of beef liver carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase-II (CPT-II). Treatment of CPT-II with the histidine-selective reagent, diethyl pyrocarbonate (DEPC), resulted in simple linear pseudo-first-order kinetics. The reversal of the inhibition by hydroxylamine and the pKa (7.1) of the modified residue indicated that the residue was a histidine. The order of the inactivation kinetics showed that 1mol of histidine was modified per mol of CPT-II.When COT was treated with DEPC the kinetics of inhibition were biphasic with an initial rapid loss of activity followed by a slower loss of activity. The residue reacting in the faster phase of inhibition was not a histidine but possibly a serine. The modification of this residue did not lead to complete loss of activity suggesting that a direct role in catalysis is unlikely. It was deduced that the residue modified by DEPC in the slower phase was a lysine and indeed fluorodinitrobenzene (FDNB) inactivated COT with linear pseudo-first-order kinetics. The COT peptide containing the FDNB-labelled lysine was isolated and sequenced. Alignment of this sequence placed it 10 amino acids downstream of the putative active-site histidine. PMID:9480926

  19. Molecular Interactions Between the Active Sites of RGD (Arg-Gly-Asp with its Receptor (Integrine

    E. Jauregui

    2000-03-01

    Full Text Available A study of the molecular interactions between the active sites of RGD (Arg-Gly-Asp with it Receptor using simultaions is reported. Our calculations indicate that the guanidine-carboxylate complex is energetically favourd with respect to the guanidine-methyl tetrazole complex.

  20. 78 FR 8190 - Commercial Wind Leasing and Site Assessment Activities on the Atlantic Outer Continental Shelf...

    2013-02-05

    ... published the Notice in the Federal Register (77 FR 74218) inviting Federal, state, local government... Bureau of Ocean Energy Management Commercial Wind Leasing and Site Assessment Activities on the Atlantic... Notice of Intent to Prepare an Environmental Assessment (EA) for Commercial Wind Leasing and...

  1. 77 FR 5830 - Commercial Wind Leasing and Site Assessment Activities on the Atlantic Outer Continental Shelf...

    2012-02-06

    ... (NOA) in the Federal Register (72 FR 62,672) of the Programmatic EIS for Alternative Energy Development... FR 30,616) of the EA for Issuance of Leases for Wind Resource Data Collection on the Outer... Bureau of Ocean Energy Management Commercial Wind Leasing and Site Assessment Activities on the...

  2. 76 FR 40925 - Commercial Wind Lease Issuance and Site Characterization Activities on the Atlantic Outer...

    2011-07-12

    ... renewable energy leases (which includes reasonably foreseeable site characterization activities--geophysical...-Atlantic WEAs (76 FR 7226), which requested public input with regard to the identification of the important... Bureau of Ocean Energy Management, Regulation and Enforcement Commercial Wind Lease Issuance and...

  3. Dynamics of water molecules in the active-site cavity of human cytochromes P450

    Rydberg, Patrik; Rod, Thomas Holm; Olsen, Lars;

    2007-01-01

    We have studied the dynamics of water molecules in six crystal structures of four human cytochromes P450, 2A6, 2C8, 2C9, and 3A4, with molecular dynamics simulations. In the crystal structures, only a few water molecules are seen and the reported sizes of the active-site cavity vary a lot. In the...

  4. Organized Agents: Canadian Teacher Unions as Alternative Sites for Social Justice Activism

    Rottmann, Cindy

    2008-01-01

    Historically teachers' federations have been some of the major organizational sites for social justice leadership in K-12 public education. Despite this history of activism, social justice teacher unionism remains a relatively underdeveloped concept. This article merges four philosophical conceptions of social justice in education: liberal…

  5. Aberration-corrected imaging of active sites on industrial catalyst nanoparticles

    Gontard, Lionel Cervera; Chang, L-Y; Hetherington, CJD;

    2007-01-01

    Picture perfect: Information about the local topologies of active sites on commercial nanoparticles can be gained with atomic resolution through spherical-aberration-corrected transmission electron microscopy (TEM). A powder of Pt nanoparticles on carbon black was examined with two advanced TEM...

  6. Biosynthesis of selenosubtilisin: A novel way to target selenium into the active site of subtilisin

    LI Jing; LIU XiaoMan; JI YueTong; QI ZhenHui; GE Yan; XU JiaYun; LIU JunQiu; LUO GuiMin; SHEN JiaCong

    2008-01-01

    Glutathione peroxidase (GPx,EC1.11.1.9),an important anti-oxidative selenoenzyme,can catalyze the reduction of harmful hydroperoxides with concomitant glutathione,thereby protecting cells and other biological issues against oxidative damage.It captures considerable interest in redesign of its function for either the mechanism study or the pharmacological development as an antioxidant.In order to de-velop a general strategy for specifically targeting and operating selenium in active sites of enzymes,the catalytically essential residue selenocysteine (Sec) was first successfully bioincorporated into the catalytic center of subtilisin by using an auxotrophic expression system.The studies of the catalytic activity and the steady-state kinetics demonstrated that selenosubtilisin is an excellent GPx-like bio-catalyst.In comparison with the chemically modified method,biosynthesis exhibits obvious advan-tages:Sec could be site-directly incorporated into active sites of enzymes to overcome the non-speci-ficity generated by chemical modification.This study provides an important strategy for specifically targeting and operating selenium in the active site of an enzyme.

  7. 77 FR 3460 - Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    2012-01-24

    ...This Notice announces the Department of Energy's (DOE) acceptance of claims in FY 2012 from eligible active uranium and thorium processing site licensees for reimbursement under Title X of the Energy Policy Act of 1992. In FY 2009, Congress appropriated $70 million for Title X in the American Recovery and Reinvestment Act of 2009 (Recovery Act). Also in FY 2009, Congress provided $10 million......

  8. 78 FR 33908 - Commercial Wind Lease Issuance and Site Assessment Activities on the Atlantic Outer Continental...

    2013-06-05

    ... identified Wind Energy Area (WEA) on the OCS offshore Rhode Island (RI) and Massachusetts (MA). The revised... Bureau of Ocean Energy Management Commercial Wind Lease Issuance and Site Assessment Activities on the.... BOEM may issue one or more commercial wind energy leases in the WEA. The competitive lease process...

  9. 40 CFR 35.6260 - Combining Cooperative Agreement sites and activities.

    2010-07-01

    ... 40 Protection of Environment 1 2010-07-01 2010-07-01 false Combining Cooperative Agreement sites and activities. 35.6260 Section 35.6260 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Contracts for Superfund Response Actions Combining Cooperative Agreements § 35.6260 Combining...

  10. The role of the catalysts with highly dispersed and isolated active sites in the selective oxidation of light hydrocarbons

    WANG Hongxuan; ZHAO Zhen

    2005-01-01

    This review summarizes the role of catalysts with highly dispersed and isolated active sites (active sites: supported atoms f≤0.5 % ) in the selective oxidation of light hydrocarbons, such as methane, ethane and propane, into oxygenatesand the epoxidation of olefins. The plausible structures of the highly dispersed and isolated active species, as well as their effects on the catalytic performances are discussed. The special physico-chemical properties and the functional mechanism of the catalysts with highly dispersed and isolated active sites, as well as the preparation, characterization of the catalysts with highly dispersed and isolated active sites and their applications in other types of reactions of lower hydrocarbons are summarized.

  11. Conformal Invariant Teleparallel Cosmology

    Momeni, Davood

    2014-01-01

    Teleparallel gravities revisited under conformal transformations. We find several kinds of the Lagrangians, all invariant under conformal transformation. Motivated by observational data,we investigate FRW cosmological solutions in the vacuum. To include the matter fields,we mention that we have few possibilities for our matter Lagrangian to respect the conformal symmetry. FRW equations,have been derived in terms of the effective energy and pressure components. In vacuum we find an exact solution for Hubble parameter which is compatible with the observational data but it is valid only in the range of $z\\ge 0.07$. Scalar torsion models in which we have the extra scalar field is examined under FRW spacetime. We introduce the potential term $\\frac{1}{4!}\\mu\\phi^4$ as the minimal self interaction with conformal symmetry.

  12. [Conformers of carnosine].

    Kliuev, S A

    2006-01-01

    The geometric and energetic parameters of most stable conformations of carnosine were calculated by the semiempirical guantum-chemical method PM3. The carnosine-water-zinc (II) clusters were simulated. PMID:16909845

  13. On the conformational state of photoinactivated tyrosinase

    Ultraviolet irradiation of tyrosinase rapidly decreased the dopa oxidase activity of the enzyme. Hydrodynamic, kinetic and thermodynamic parameters revealed gross differences in the native and photoinactivated states of the enzyme. The native state of tyrosinase was characterized as a tetramer with a compact, globular and rigid conformation. However, the photoinactivated state of tyrosinase was thermodynamically less stable and unusually sensitive to temperatures as low as 35 0C. From the dose dependent loss in conformational integrity, thermodynamic stability and catalytic activity of tyrosinase, it is speculated that there are various structural segments distributed throughout the enzyme molecule. These structural segments act as centres of major molecular forces which hold the tetrameric enzyme into a compact and globular conformation. UV modification of these segments triggers a series of conformational changes leading to formation of a partially unfolded and catalytically inactive form of tyrosinase. (author)

  14. Quantum conformal mechanics

    Andrzejewski, K

    2015-01-01

    The quantum mechanics of one degree of freedom exhibiting the exact conformal SL(2,R) symmetry is presented. The starting point is the classification of the unitary irreducible representations of the SL(2,R) group (or, to some extent, its universal covering). The coordinate representation is defined as the basis diagonalizing the special conformal generator K. It is indicated how the resulting theory emerges from the canonical/geometric quantization of the Hamiltonian dynamics on the relevant coadjoint orbits.

  15. Quantum massive conformal gravity

    Faria, F. F.

    2016-01-01

    We first find the linear approximation of the second plus fourth order derivative massive conformal gravity action. Then we reduce the linearized action to separated second order derivative terms, which allows us to quantize the theory by using the standard first order canonical quantization method. It is shown that quantum massive conformal gravity is renormalizable but has ghost states. A possible decoupling of these ghost states at high energies is discussed.

  16. Quantum massive conformal gravity

    Faria, F. F.

    2016-04-01

    We first find the linear approximation of the second plus fourth order derivative massive conformal gravity action. Then we reduce the linearized action to separated second order derivative terms, which allows us to quantize the theory by using the standard first order canonical quantization method. It is shown that quantum massive conformal gravity is renormalizable but has ghost states. A possible decoupling of these ghost states at high energies is discussed.

  17. Quantum massive conformal gravity

    We first find the linear approximation of the second plus fourth order derivative massive conformal gravity action. Then we reduce the linearized action to separated second order derivative terms, which allows us to quantize the theory by using the standard first order canonical quantization method. It is shown that quantum massive conformal gravity is renormalizable but has ghost states. A possible decoupling of these ghost states at high energies is discussed. (orig.)

  18. Delineating the conformal window

    Frandsen, Mads Toudal; Pickup, Thomas; Teper, Michael

    We identify and characterise the conformal window in gauge theories relevant for beyond the standard model building, e.g. Technicolour, using the criteria of metric confinement and causal analytic couplings, which are known to be consistent with the phase diagram of supersymmetric QCD from Seiberg...... duality. Using these criteria we find perturbation theory to be consistent throughout the predicted conformal window for several of these gauge theories and we discuss recent lattice results in the light of our findings....

  19. Characterization and sequencing of the active site of 1-aminocyclopropane-1-carboxylate synthase

    The pyridoxal phosphate (PLP)-dependent 1-aminocyclopropane-1-carboxylic acid (ACC) synthase the key enzyme in ethylene biosynthesis, is inactivated by its substrate S-adenosylmethionine (AdoMet). Apple ACC synthase was purified with an immunoaffinity gel, and its active site was probed with NaB3H4 or Ado[14C]Met. Peptide sequencing of both 3H- and 14C-labeled peptides revealed a common dodecapeptide of Ser-Leu-Ser-Xaa-Asp-Leu-Gly-Leu-Pro-Gly-Phe-Arg, where Xaa was the modified, radioactive residue in each case. Acid hydrolysis of the 3H-labeled enzyme released radioactive N-pyridoxyllysine, indicating that the active-site peptide contained lysine at position 4. Mass spectrometry of the 14C-labeled peptide indicated a protonated molecular ion at m/z 1390.6, from which the mass of Xaa was calculated to be 229, a number that is equivalent to the mass of a lysine residue alkylated by the 2-aminobutyrate portion of AdoMet, as we previously proposed. These results indicate that the same active-site lysine binds the PLP and convalently links to the 2-aminobutyrate portion of AdoMet during inactivation. The active site of tomato ACC synthase was probed in the same manner with Ado [14C]Met. Sequencing of the tomato active-site peptide revealed two highly conserved dodecapeptides; the minor peptide possessed a sequence identical to that of the apple enzyme, whereas the major peptide differed from the minor peptide in that methionine replaced leucine at position 6

  20. Loop Electrostatics Asymmetry Modulates the Preexisting Conformational Equilibrium in Thrombin.

    Pozzi, Nicola; Zerbetto, Mirco; Acquasaliente, Laura; Tescari, Simone; Frezzato, Diego; Polimeno, Antonino; Gohara, David W; Di Cera, Enrico; De Filippis, Vincenzo

    2016-07-19

    Thrombin exists as an ensemble of active (E) and inactive (E*) conformations that differ in their accessibility to the active site. Here we show that redistribution of the E*-E equilibrium can be achieved by perturbing the electrostatic properties of the enzyme. Removal of the negative charge of the catalytic Asp102 or Asp189 in the primary specificity site destabilizes the E form and causes a shift in the 215-217 segment that compromises substrate entrance. Solution studies and existing structures of D102N document stabilization of the E* form. A new high-resolution structure of D189A also reveals the mutant in the collapsed E* form. These findings establish a new paradigm for the control of the E*-E equilibrium in the trypsin fold. PMID:27347732

  1. Active sites for methane activation in MgO and Li doped MgO

    Kwapień, Karolina

    2012-01-01

    Die vorliegende Dissertation präsentiert eine detaillierte quantenchemische Untersuchung der H-Abstraktion von Methan durch MgO und Li dotiertes MgO. Motiviert wurde die durch das UniCat-Excellenz-Cluster, welches sich zum Ziel gesetzt hat, die oxidative Kupplung von Methan (OCM) im Detail zu verstehen. Basierend auf der Hypothese, dass Li+O•– Spezies für den H-Abstraktion Schritt verantwortlich sind, wurden kleine kationische MgO- und Li dotierte MgO-Cluster (die O•– Sites modellieren) unt...

  2. A split active site couples cap recognition by Dcp2 to activation

    Floor, Stephen N.; Jones, Brittnee N.; Hernandez, Gail A.; Gross, John D.

    2010-01-01

    Decapping by Dcp2 is an essential step in 5′-3′ mRNA decay. In yeast, decapping requires an open-to-closed transition in Dcp2, though the link between closure and catalysis remains elusive. Here we show using NMR that cap binds conserved residues on both the catalytic and regulatory domains of Dcp2. Lesions in the cap-binding site on the regulatory domain reduce the catalytic step two orders of magnitude and block formation of the closed state whereas Dcp1 enhances the catalytic step by a fac...

  3. Social influences towards conformism in economic experiments

    Hargreaves Heap, Shaun P.

    2014-01-01

    This paper reviews some of the economic experimental evidence on conformism. There is nothing to match the early psychology experiments where subjects were often swayed by the behaviour of others to an extraordinary degree, but there is plenty of evidence of conformism. This seems built-in to our sociality either because we have preferences for conversation or status which are activated by the knowledge of what others do, or because other people face relevantly similar decisions to our own an...

  4. Conformational Mobility of GOx Coenzyme Complex on Single-Wall Carbon Nanotubes

    Yu Kang

    2008-12-01

    Full Text Available A critical issue in bioelectrochemical applications that use electrodes modified by Single Wall Carbon Nanotubes (SWCNTs is to ensure high activity of the catalytic site of an immobilized enzyme protein interacting with nanomaterials. Since Flavin Adenine Dinucleotide (FAD, a coenzyme of glucose oxidase (GOx, is the active center of the catalytic site, conformation of which could determine the activity of enzyme, it is important to understand the dynamic mechanism of its conformational mobility while GOx is adsorbed on SWCNTs with multiple orientations. However, this dynamic mechanism still remains unclear at the atomic level due to the coenzyme being embedded in the apo-GOx and the limitations of appropriate experimental methods. In this study, a molecular dynamics (MD simulation was performed to investigate the conformational mobility mechanism of the coenzyme. The trajectory and the interaction energy clearly indicate that the adsorption of GOx onto SWCNTs plays an important role in the conformational mobility of the coenzyme, and its mobility is greatly affected by the distribution of water molecules due to it being hydrophobic.

  5. Integral Public Activities as a Support to the Site Selection Process for LILW Repository

    The first site selection process for low and intermediate level radioactive waste (LILW) repository took place between 1990 and 1993 in Slovenia was stopped unsuccessfully with very strong public opposition at local level, followed by political withdrawal on national level. As one of the consequences ARAO started to develop new approach to the site selection based also on the findings from sociology, psychology and other human sciences. The recommendations on public involvement and transparency were so strong that ARAO started with first limited public relation (PR) activities which later grew to the PR process which supports all technical activities in ARAO. Presently the PR process covers communication, information and research activities and assures careful planning, prompt responds and involvement of the highest responsible persons at ARAO. Integral public relation activities are divided in several parts. Majority of activities support the on-going site selection process where activities are presently focused on functioning of local partnerships developed as a basic communication tool to involve as much citizens and public as possible on local level. Presently two local partnerships are working in Krsko and Brezice community with clear role to enhance public involvement according to Aarchus convention. Each of the partnerships is organized in a specific way adjusted to the local needs. Communication activities are organized also for different other projects and are preparing the necessary basis for the work with different groups of stake holders and in different situations. As a foundation very broad information material, such as books, leaflets, reports, magazines, video cassettes, CD and DVD on the radioactive waste management is prepared and used for different purposes. We also try to be proactive with web pages and have a well organized visitors' center. Improvement of public relation process is achieved through constant survey and feed-back information

  6. Number of active transcription factor binding sites is essential for the Hes7 oscillator

    de Angelis Martin

    2006-02-01

    Full Text Available Abstract Background It is commonly accepted that embryonic segmentation of vertebrates is regulated by a segmentation clock, which is induced by the cycling genes Hes1 and Hes7. Their products form dimers that bind to the regulatory regions and thereby repress the transcription of their own encoding genes. An increase of the half-life of Hes7 protein causes irregular somite formation. This was shown in recent experiments by Hirata et al. In the same work, numerical simulations from a delay differential equations model, originally invented by Lewis, gave additional support. For a longer half-life of the Hes7 protein, these simulations exhibited strongly damped oscillations with, after few periods, severely attenuated the amplitudes. In these simulations, the Hill coefficient, a crucial model parameter, was set to 2 indicating that Hes7 has only one binding site in its promoter. On the other hand, Bessho et al. established three regulatory elements in the promoter region. Results We show that – with the same half life – the delay system is highly sensitive to changes in the Hill coefficient. A small increase changes the qualitative behaviour of the solutions drastically. There is sustained oscillation and hence the model can no longer explain the disruption of the segmentation clock. On the other hand, the Hill coefficient is correlated with the number of active binding sites, and with the way in which dimers bind to them. In this paper, we adopt response functions in order to estimate Hill coefficients for a variable number of active binding sites. It turns out that three active transcription factor binding sites increase the Hill coefficient by at least 20% as compared to one single active site. Conclusion Our findings lead to the following crucial dichotomy: either Hirata's model is correct for the Hes7 oscillator, in which case at most two binding sites are active in its promoter region; or at least three binding sites are active, in which

  7. Transcriptional activities of mammalian genomes at sites of recombination with foreign DNA

    The nucleotide sequences of several sites of recombination between adenovirus DNA and hamster, mouse, or human cell DNAs were determined. These sites of recombination had been cloned from adenovirus type 2 (Ad2)- or type 12 (Ad12)-transformed cells, from Ad12-induced tumor cells, or from a symmetric recombinant between Ad12 DNA and human cell DNA. One important precondition for the generation of recombinants between host and foreign DNAs might be the establishment of a chromatin configuration that permits access of foreign DNA and of the recombination machinery to cellular DNA. Such favorable chromatin structures might arise during cellular DNA replication or transcription or both. As a first approach toward investigating these more complex problems of foreign DNA insertion. The authors determined transcriptional activities of cellular DNA sequences at viral junction sites. The results presented demonstrate that the cellular DNA sequences involved in linkage to viral DNA at five completely different sites in DNA from three different species are transcribed into RNAs even in cells which have not been transformed or infected by adenovirus. These results are consistent with the notion that at least some of the cellular DNA sequences at sites of insertion of adenovirus (foreign) DNA are transcriptionally active and thus provide an opportunity for recombination

  8. Role of active sites in the reaction of methanol to olefin over modified ZSM-5 zeolite

    ZSM-5 zeolites were modified with metals Mg, K, Ca, Ba and La by an incipient impregnation and characterized by X-ray diffraction, N/sub 2/ adsorption and temperature - programmed desorption of NH/sub 3/ and CO/sub 2/. The obtained samples were investigated for their selectivity and catalytic stability in the methanol-to-olefin (MTO) reaction. The catalytic performance was influenced by the properties of active sites on the catalyst, which was indicated by the changes of the light olefin selectivity and catalytic stability in the methanol conversion. Correlating the reaction evaluation with the catalyst characterization, it can be seen that the catalyst with too strong basic sites shows an inferior catalytic performance. In addition, steam treatment further increased the catalytic performance, especially for Ca modified ZSM-5. It may be concluded that basic sites must suitably match with acidic sites for a special ZSM-5 in the methanol conversion and the formed active sites were relative with the modification metal based on the obtained results. (author)

  9. Management of Ground and Groundwater Contamination on a Compact Site Constrained by Ongoing Activities

    Sellafield Site is a compact and complex site which since the 1940's has been home to a range of facilities associated with the production and reprocessing of fissile material. The site contains the UK equivalent of the Chicago Pile-1 reactor, Hanford B Reactor, Rocky Flats Buildings 771 and 774, West Valley Main Process Plant Building, Savannah River Vitrification Plant, Savannah River MOX Plant, Savannah River F Canyon, Hanford 222 Analytical Laboratory, Savannah River K-, L-, and P-Basins, and the Fort St. Vrain Reactor all in an area of approximately 1000 acres. Spent fuel reprocessing is still undertaken on site; however waste management and decommissioning activities are of increasing importance. These include the emptying and removal of fragile ponds and silos containing significant radioactive inventories, the decommissioning of reactors (including the world's first commercial reactor for power generation and the Windscale Piles, the site of a reactor fire in the late 1950's) and the construction of a new generation of vitrification and encapsulation plants. Leaks, spills and on-site disposals during the site's industrial lifetime have resulted in a legacy of fission products and other radionuclides in the ground and groundwater. Volumes of contaminated ground have been estimated as being as much as 18 million m3 and an estimated below ground inventory of approximately 1.8 E16 Bq. These have all occurred within close proximity to a range of receptors including farm land and the sea. The cramped nature of the facilities on site, overlapping source terms and ongoing decommissioning, waste management and operating activities all raise significant challenges in the management and remediation of contaminated land and groundwater. The strategy to address these challenges includes: 1. Data collection, management and interpretation. The congested nature of the site and the age of some of the monitoring facilities has resulted in particular difficulties. For example

  10. Ionizing Groups at the Active Site of the Alkaline Phosphatase from Ostrea cucullate

    CHEN Qiao; WANG Qin; LIAO Jinhua; CHEN Qingxi

    2006-01-01

    The ionizing groups at the active site of alkaline phosphatase (ALP, EC 3.1.3.1) from Ostrea cucullate were studied using kinetic methods. The ionization constant, pKe, of the ionizing groups at the active site of the enzyme was found to be 10.11 at 37.0℃ and the organic solvent, dioxin, had no effect on the pKe. The standard dissociation enthalpy (△Ho) was determined to be 10.57 kcal/mol (1 cal=4.18 J). The results show that the dissociation group of the enzyme active center is the з-NH3+ of lysine. Chemical modification of the enzyme by acetic anhydride and succinic anhydride demonstrates that the amino group is one of the enzyme's functional groups.

  11. Active space decomposition with multiple sites: Density matrix renormalization group algorithm

    Parker, Shane M

    2014-01-01

    We extend the active space decomposition method, recently developed by us, to more than two active sites using the density matrix renormalization group algorithm. The fragment wave functions are described by complete or restricted active-space wave functions. Numerical results are shown on a benzene pentamer and a perylene diimide trimer. It is found that the truncation errors in our method decrease almost exponentially with respect to the number of renormalization states M, allowing for numerically exact calculations (to a few {\\mu}Eh or less) with M = 128 in both cases, which is in contrast to conventional ab initio density matrix renormalization group.

  12. Communication: Active space decomposition with multiple sites: Density matrix renormalization group algorithm

    Parker, Shane M.; Shiozaki, Toru [Department of Chemistry, Northwestern University, 2145 Sheridan Rd., Evanston, Illinois 60208 (United States)

    2014-12-07

    We extend the active space decomposition method, recently developed by us, to more than two active sites using the density matrix renormalization group algorithm. The fragment wave functions are described by complete or restricted active-space wave functions. Numerical results are shown on a benzene pentamer and a perylene diimide trimer. It is found that the truncation errors in our method decrease almost exponentially with respect to the number of renormalization states M, allowing for numerically exact calculations (to a few μE{sub h} or less) with M = 128 in both cases. This rapid convergence is because the renormalization steps are used only for the interfragment electron correlation.

  13. Communication: Active space decomposition with multiple sites: Density matrix renormalization group algorithm

    We extend the active space decomposition method, recently developed by us, to more than two active sites using the density matrix renormalization group algorithm. The fragment wave functions are described by complete or restricted active-space wave functions. Numerical results are shown on a benzene pentamer and a perylene diimide trimer. It is found that the truncation errors in our method decrease almost exponentially with respect to the number of renormalization states M, allowing for numerically exact calculations (to a few μEh or less) with M = 128 in both cases. This rapid convergence is because the renormalization steps are used only for the interfragment electron correlation

  14. Mapping Topoisomerase IV Binding and Activity Sites on the E. coli Genome.

    El Sayyed, Hafez; Le Chat, Ludovic; Lebailly, Elise; Vickridge, Elise; Pages, Carine; Cornet, Francois; Cosentino Lagomarsino, Marco; Espéli, Olivier

    2016-05-01

    Catenation links between sister chromatids are formed progressively during DNA replication and are involved in the establishment of sister chromatid cohesion. Topo IV is a bacterial type II topoisomerase involved in the removal of catenation links both behind replication forks and after replication during the final separation of sister chromosomes. We have investigated the global DNA-binding and catalytic activity of Topo IV in E. coli using genomic and molecular biology approaches. ChIP-seq revealed that Topo IV interaction with the E. coli chromosome is controlled by DNA replication. During replication, Topo IV has access to most of the genome but only selects a few hundred specific sites for its activity. Local chromatin and gene expression context influence site selection. Moreover strong DNA-binding and catalytic activities are found at the chromosome dimer resolution site, dif, located opposite the origin of replication. We reveal a physical and functional interaction between Topo IV and the XerCD recombinases acting at the dif site. This interaction is modulated by MatP, a protein involved in the organization of the Ter macrodomain. These results show that Topo IV, XerCD/dif and MatP are part of a network dedicated to the final step of chromosome management during the cell cycle. PMID:27171414

  15. Cohomology theories on locally conformal symplectic manifolds

    Le, Hong-Van; Vanžura, Jiří

    2015-01-01

    Roč. 19, č. 1 (2015), s. 45-82. ISSN 1093-6106 Institutional support: RVO:67985840 Keywords : locally conformal symplectic manifold * Lichnerowicz-Novikov cohomology * primitive cohomology Subject RIV: BA - General Mathematics Impact factor: 0.532, year: 2014 http://www.intlpress.com/site/pub/pages/journals/items/ajm/content/vols/0019/0001/a003/

  16. Regulatory inspection activities on nuclear power plant sites during construction in the United Kingdom

    The work of regulatory inspection of the construction of the plant on the site is performed not only by the inspector who has been allocated to inspection duties for that site but also by the specialist staff who are involved with the safety assessment of the plant. The coordination of this work is described in the paper and examples are given of inspection activities associated with the enforcement requirements of license conditions as well as those related to the inspection of the plant itself

  17. Technical basis for classification of low-activity waste fraction from Hanford site tanks

    Petersen, C.A., Westinghouse Hanford

    1996-07-17

    The overall objective of this report is to provide a technical basis to support a U.S. Nuclear Regulatory Commission determination to classify the low-activity waste from the Hanford Site single-shell and double-shell tanks as `incidental` wastes after removal of additional radionuclides and immobilization.The proposed processing method, in addition to the previous radionuclide removal efforts, will remove the largest practical amount of total site radioactivity, attributable to high-level wastes, for disposal in a deep geologic repository. The remainder of the waste would be considered `incidental` waste and could be disposed onsite.

  18. Understanding modern magnets through conformal mapping

    I want to show with the help of a number of examples that conformal mapping is a unique and enormously powerful tool for thinking about, and solving, problems. Usually one has to write down only a few equations, and sometimes none at all exclamation point When I started getting involved in work for which conformal mapping seemed to be a powerful tool, I did not think that I would ever be able to use that technique successfully because it seemed to require a nearly encyclopedic memory, an impression that was strengthened when I saw K. Kober's Dictionary of Conformal Representations. This attitude changed when I started to realize that beyond the basics of the theory of a function of a complex variable, I needed to know only about a handful of conformal maps and procedures. Consequently, my second goal for this talk is to show that in most cases conformal mapping functions can be obtained by formulating the underlying physics appropriately. This means particularly that encyclopedic knowledge of conformal maps is not necessary for successful use of conformal mapping techniques. To demonstrate these facts I have chosen examples from an area of physics/engineering in which I am active, namely accelerator physics. In order to do that successfully I start with a brief introduction into high energy charged particle storage ring technology, even though not all examples used in this paper to elucidate my points come directly from this particular field of accelerator technology

  19. Kinetic model of ethopropazine interaction with horse serum butyrylcholinesterase and its docking into the active site.

    Golicnik, Marko; Sinko, Goran; Simeon-Rudolf, Vera; Grubic, Zoran; Stojan, Jure

    2002-02-01

    The action of a potent tricyclic cholinesterase inhibitor ethopropazine on the hydrolysis of acetylthiocholine and butyrylthiocholine by purified horse serum butyrylcholinesterase (EC 3.1.1.8) was investigated at 25 and 37 degrees C. The enzyme activities were measured on a stopped-flow apparatus and the analysis of experimental data was done by applying a six-parameter model for substrate hydrolysis. The model, which was introduced to explain the kinetics of Drosophila melanogaster acetylcholinesterase [Stojan et al. (1998) FEBS Lett. 440, 85-88], is defined with two dissociation constants and four rate constants and can describe both cooperative phenomena, apparent activation at low substrate concentrations and substrate inhibition by excess of substrate. For the analysis of the data in the presence of ethopropazine at two temperatures, we have enlarged the reaction scheme to allow primarily its competition with the substrate at the peripheral site, but the competition at the acylation site was not excluded. The proposed reaction scheme revealed, upon analysis, competitive effects of ethopropazine at both sites; at 25 degrees C, three enzyme-inhibitor dissociation constants could be evaluated; at 37 degrees C, only two constants could be evaluated. Although the model considers both cooperative phenomena, it appears that decreased enzyme sensitivity at higher temperature, predominantly for the ligands at the peripheral binding site, makes the determination of some expected enzyme substrate and/or inhibitor complexes technically impossible. The same reason might also account for one of the paradoxes in cholinesterases: activities at 25 degrees C at low substrate concentrations are higher than at 37 degrees C. Positioning of ethopropazine in the active-site gorge by molecular dynamics simulations shows that A328, W82, D70, and Y332 amino acid residues stabilize binding of the inhibitor. PMID:11811945

  20. Assessment of national systems for obtaining local acceptance of nuclear waste management siting activities

    On behalf of the United States Department of Energy (DOE), International Energy Associates Limited (IEAL) of Washington, D.C. has conducted surveys and analyses of fourteen countries' plans and approaches for dealing with the problems of obtaining local siting acceptance for nuclear waste management facilities. It was determined that the following elements of the formal systems generally facilitate and/or expedite waste management siting decisions: (1) a clear-cut pro-nuclear power position on the part of the government; (2) a willingness on the part of the central government to exert (with prudence and restraint) its pre-emptive rights in nuclear matters; (3) political structures in which the heads of regional or provincial governments are appointed by the central government; (4) national laws that link reactor licensing with a detailed plan for waste management; (5) an established and stable policy with regard to reprocessing. In contrast, it was determined that the following elements of the formal system generally hinder waste management siting activities: (1) historically strong local land used veto laws; (2) the use of national referenda for making nuclear decisions; (3) requirements for public hearings. The informal approaches fall into the following five categories: (1) political: e.g. assertion of will by political leaders, activities to enlist support of local politicians, activities to broaden involvement in decision-making; (2) economic: e.g. emphasis on normal benefits, provision for additional economic benefits; (3) siting: e.g. at or near existing nuclear facilities, on government or utility property, at multiple locations to spread the political burden; (4) timing: e.g. decoupling drilling activities from ultimate repository site decision, deliberate deferral to (long-range) future; (5) education: e.g. creation of special government programmes, enlisting of media support

  1. Linde FUSRAP Site Remediation: Engineering Challenges and Solutions of Remedial Activities on an Active Industrial Facility - 13506

    The Linde FUSRAP Site (Linde) is located in Tonawanda, New York at a major research and development facility for Praxair, Inc. (Praxair). Successful remediation activities at Linde combines meeting cleanup objectives of radiological contamination while minimizing impacts to Praxair business operations. The unique use of Praxair's property coupled with an array of active and abandoned utilities poses many engineering and operational challenges; each of which has been overcome during the remedial action at Linde. The U.S. Army Corps of Engineers - Buffalo District (USACE) and CABRERA SERVICES, INC. (CABRERA) have successfully faced engineering challenges such as relocation of an aboveground structure, structural protection of an active water line, and installation of active mechanical, electrical, and communication utilities to perform remediation. As remediation nears completion, continued success of engineering challenges is critical as remaining activities exist in the vicinity of infrastructure essential to business operations; an electrical substation and duct bank providing power throughout the Praxair facility. Emphasis on engineering and operations through final remediation and into site restoration will allow for the safe and successful completion of the project. (authors)

  2. Active-site titration analysis of surface influence on immobilized Candida antarctica Lipase B activity

    Matrix morphology and surface polarity effects were investigated for Candida antarctica lipase B immobilization. Measurements of the amount of lipase immobilized (bicinchoninic acid method) and the catalyst’s tributyrin hydrolysis activity, coupled with a determination of the lipase’s functional fr...

  3. Structure-guided mutagenesis of active site residues in the dengue virus two-component protease NS2B-NS3

    Salaemae Wanisa

    2010-08-01

    binding hole. Conclusions Kinetic data obtained for mutants in the NS3 protease have confirmed predictions for the conformation of the active site S1 and S2 pockets based on earlier observations. The data presented herein will be useful to further explore structure-activity relationships of the flaviviral proteases important for the structure-guided design of novel antiviral therapeutics.

  4. RCRA and CERCLA requirements affecting cleanup activities at a federal facility superfund site

    The Fernald Environmental Management Project (FEMP) achieved success on an integrated groundwater monitoring program which addressed both RCRA and CERCLA requirements. The integrated plan resulted in a cost savings of approximately $2.6 million. At present, the FEMP is also working on an integrated closure process to address Hazardous Waste Management Units (HWMUs) at the site. To date, Ohio EPA seems willing to discuss an integrated program with some stipulations. If an integrated program is implemented, a cost savings of several million dollars will be realized since the CERCLA documents can be used in place of a RCRA closure plan. The success of an integrated program at the FEMP is impossible without the support of DOE and the regulators. Since DOE is an owner/operator of the facility and Ohio EPA regulates hazardous waste management activities at the FEMP, both parties must be satisfied with the proposed integration activities. Similarly, US EPA retains CERCLA authority over the site along with a signed consent agreement with DOE, which dictates the schedule of the CERCLA activities. Another federal facility used RCRA closure plans to satisfy CERCLA activities. This federal facility was in a different US EPA Region than the FEMP. While this approach was successful for this site, an integrated approach was required at the FEMP because of the signed Consent Agreement and Consent Decree. For federal facilities which have a large number of HWMUs along with OUs, an integrated approach may result in a timely and cost-effective cleanup

  5. Structural and kinetic contributions of the oxyanion binding site to the catalytic activity of acylaminoacyl peptidase.

    Kiss, András L; Palló, Anna; Náray-Szabó, Gábor; Harmat, Veronika; Polgár, László

    2008-05-01

    It is widely accepted that the catalytic activity of serine proteases depends primarily on the Asp-His-Ser catalytic triad and other residues within the vicinity of this motif. Some of these residues form the oxyanion binding site that stabilizes the tetrahedral intermediate by hydrogen bonding to the negatively charged oxyanion. In acylaminoacyl peptidase from the thermophile Aeropyrum pernix, the main chain NH group of Gly369 is one of the hydrogen bond donors forming the oxyanion binding site. The side chain of His367, a conserved residue in acylaminoacyl peptidases across all species, fastens the loop holding Gly369. Determination of the crystal structure of the H367A mutant revealed that this loop, including Gly369, moves away considerably, accounting for the observed three orders of magnitude decrease in the specificity rate constant. For the wild-type enzyme ln(k(cat)/K(m)) vs. 1/T deviates from linearity indicating greater rate enhancement with increasing temperature for the dissociation of the enzyme-substrate complex compared with its decomposition to product. In contrast, the H367A variant provided a linear Arrhenius plot, and its reaction was associated with unfavourable entropy of activation. These results show that a residue relatively distant from the active site can significantly affect the catalytic activity of acylaminoacyl peptidase without changing the overall structure of the enzyme. PMID:18325786

  6. Progress report on decommissioning activities at the Fernald Environmental Management Project (FEMP) site

    NONE

    1998-07-01

    The Fernald Environmental Management Project (FEMP), is located about 18 miles northwest of Cincinnati, Ohio. Between 1953 and 1989, the facility, then called the Feed Material Production Center or FMPC, produced uranium metal products used in the eventual production of weapons grade material for use by other US Department of Energy (DOE) sites. In 1989, FMPC`s production was suspended by the federal government in order to focus resources on environmental restoration versus defense production. In 1992, Fluor Daniel Fernald assumed responsibility for managing all cleanup activities at the FEMP under contract to the DOE. In 1990, as part of the remediation effort, the site was divided into five operable units based on physical proximity of contaminated areas, similar amounts of types of contamination, or the potential for a similar technology to be used in cleanup activities. This report continues the outline of the decontamination and decommissioning (D and D) activities at the FEMP site Operable Unit 3 (OU3) and provides an update on the status of the decommissioning activities. OU3, the Facilities Closure and Demolition Project, involves the remediation of more than 200 uranium processing facilities. The mission of the project is to remove nuclear materials stored in these buildings, then perform the clean out of the buildings and equipment, and decontaminate and dismantle the facilities.

  7. Progress report on decommissioning activities at the Fernald Environmental Management Project (FEMP) site

    The Fernald Environmental Management Project (FEMP), is located about 18 miles northwest of Cincinnati, Ohio. Between 1953 and 1989, the facility, then called the Feed Material Production Center or FMPC, produced uranium metal products used in the eventual production of weapons grade material for use by other US Department of Energy (DOE) sites. In 1989, FMPC's production was suspended by the federal government in order to focus resources on environmental restoration versus defense production. In 1992, Fluor Daniel Fernald assumed responsibility for managing all cleanup activities at the FEMP under contract to the DOE. In 1990, as part of the remediation effort, the site was divided into five operable units based on physical proximity of contaminated areas, similar amounts of types of contamination, or the potential for a similar technology to be used in cleanup activities. This report continues the outline of the decontamination and decommissioning (D and D) activities at the FEMP site Operable Unit 3 (OU3) and provides an update on the status of the decommissioning activities. OU3, the Facilities Closure and Demolition Project, involves the remediation of more than 200 uranium processing facilities. The mission of the project is to remove nuclear materials stored in these buildings, then perform the clean out of the buildings and equipment, and decontaminate and dismantle the facilities

  8. Human glutaminyl cyclase and bacterial zinc aminopeptidase share a common fold and active site

    Misquitta Stephanie A

    2004-02-01

    Full Text Available Abstract Background Glutaminyl cyclase (QC forms the pyroglutamyl residue at the amino terminus of numerous secretory peptides and proteins. We previously proposed the mammalian QC has some features in common with zinc aminopeptidases. We now have generated a structural model for human QC based on the aminopeptidase fold (pdb code 1AMP and mutated the apparent active site residues to assess their role in QC catalysis. Results The structural model proposed here for human QC, deposited in the protein databank as 1MOI, is supported by a variety of fold prediction programs, by the circular dichroism spectrum, and by the presence of the disulfide. Mutagenesis of the six active site residues present in both 1AMP and QC reveal essential roles for the two histidines (140 and 330, QC numbering and the two glutamates (201 and 202, while the two aspartates (159 and 248 appear to play no catalytic role. ICP-MS analysis shows less than stoichiometric zinc (0.3:1 in the purified enzyme. Conclusions We conclude that human pituitary glutaminyl cyclase and bacterial zinc aminopeptidase share a common fold and active site residues. In contrast to the aminopeptidase, however, QC does not appear to require zinc for enzymatic activity.

  9. Objective interpretation as conforming interpretation

    Lidka Rodak

    2011-12-01

    Full Text Available The practical discourse willingly uses the formula of “objective interpretation”, with no regards to its controversial nature that has been discussed in literature.The main aim of the article is to investigate what “objective interpretation” could mean and how it could be understood in the practical discourse, focusing on the understanding offered by judicature.The thesis of the article is that objective interpretation, as identified with textualists’ position, is not possible to uphold, and should be rather linked with conforming interpretation. And what this actually implies is that it is not the virtue of certainty and predictability – which are usually associated with objectivity- but coherence that makes the foundation of applicability of objectivity in law.What could be observed from the analyses, is that both the phenomenon of conforming interpretation and objective interpretation play the role of arguments in the interpretive discourse, arguments that provide justification that interpretation is not arbitrary or subjective. With regards to the important part of the ideology of legal application which is the conviction that decisions should be taken on the basis of law in order to exclude arbitrariness, objective interpretation could be read as a question “what kind of authority “supports” certain interpretation”? that is almost never free of judicial creativity and judicial activism.One can say that, objective and conforming interpretation are just another arguments used in legal discourse.

  10. Multiple nucleophilic elbows leading to multiple active sites in a single module esterase from Sorangium cellulosum

    Udatha, D.B.R.K. Gupta; Madsen, Karina Marie; Panagiotou, Gianni; Olsson, Lisbeth

    2015-01-01

    The catalytic residues in carbohydrate esterase enzyme families constitute a highly conserved triad: serine, histidine and aspartic acid. This catalytic triad is generally located in a very sharp turn of the protein backbone structure, called the nucleophilic elbow and identified by the consensus...... sequence GXSXG. An esterase from Sorangium cellulosum Soce56 that contains five nucleophilic elbows was cloned and expressed in Escherichia coli and the function of each nucleophilic elbowed site was characterized. In order to elucidate the function of each nucleophilic elbow, site directed mutagenesis was...... used to generate variants with deactivated nucleophilic elbows and the functional promiscuity was analyzed. In silico analysis together with enzymological characterization interestingly showed that each nucleophilic elbow formed a local active site with varied substrate specificities and affinities. To...

  11. Soil pollution with trace elements at selected sites in Romania studied by instrumental neutron activation analysis

    Instrumental neutron activation analysis (INAA) was used to determine concentrations of 42 elements in samples of surface soil collected at seven sites polluted from various anthropogenic activities and a control site in a relatively clean area. Elements studied were Ag, Al, As, Au, Ba, Br, Ca, Cd, Ce, Co, Cr, Cs, Eu, Fe, Gd, Hf, Hg, K, La, Lu, Mg, Mn, Mo, Na, Nd, Ni, Rb, Sb, Sc, Se, Sm, Sr, Ta, Tb, Th, Ti, U, V, W, Yb, Zn, and Zr. The results are compared with data for trace elements atmospheric deposition in lichen transplants from the same sites. The most severe soil contamination was observed at Copsa Mica from non-ferrous metallurgy. Appreciable soil contamination was also indicated at Baia Mare (non-ferrous mining and metallurgy), Deva (coal-fired power plant, cement and building materials industry), Galati (ferrous metallurgy), Magurele and Afumati (general urban pollution), and Oradea (chemical and light industries). In most cases excessive levels of toxic metals in soils matched correspondingly high values in lichen transplants. Compared to Romanian norms, legal upper limits were exceeded for Zn and Cd at Copsa Mica. Also, As and Sb occurred in excessive levels at given sites. (orig.)

  12. Assessment of Tritium Activity in Groundwater at the Nuclear Objects Sites in Lithuania

    Vigilija Cidzikienė

    2014-01-01

    Full Text Available The assessment of nuclear objects sites in Lithuania, including groundwater characterization, took place in the last few years. Tritium activity in groundwater is a very useful tool for determining how groundwater systems function. Natural and artificial tritium was measured in 8 wells in different layers (from 1.5 to 15 meters depth. The results were compared with other regions of Lithuania also. The evaluated tritium activities varied from 1.8 to 6.4 Bq/L at nuclear objects sites in Lithuania and they are coming to background level (1.83 Bq/L and other places in Lithuania. The data show, that groundwater at the nuclear power objects sites is not contaminated with artificial tritium. In this work, the vertical tritium transfer from soil water to the groundwater well at nuclear objects site was estimated. The data show that the main factor for vertical tritium transfer to the well depends on the depth of wells.

  13. Structure/function correlations over binuclear non-heme iron active sites.

    Solomon, Edward I; Park, Kiyoung

    2016-09-01

    Binuclear non-heme iron enzymes activate O2 to perform diverse chemistries. Three different structural mechanisms of O2 binding to a coupled binuclear iron site have been identified utilizing variable-temperature, variable-field magnetic circular dichroism spectroscopy (VTVH MCD). For the μ-OH-bridged Fe(II)2 site in hemerythrin, O2 binds terminally to a five-coordinate Fe(II) center as hydroperoxide with the proton deriving from the μ-OH bridge and the second electron transferring through the resulting μ-oxo superexchange pathway from the second coordinatively saturated Fe(II) center in a proton-coupled electron transfer process. For carboxylate-only-bridged Fe(II)2 sites, O2 binding as a bridged peroxide requires both Fe(II) centers to be coordinatively unsaturated and has good frontier orbital overlap with the two orthogonal O2 π* orbitals to form peroxo-bridged Fe(III)2 intermediates. Alternatively, carboxylate-only-bridged Fe(II)2 sites with only a single open coordination position on an Fe(II) enable the one-electron formation of Fe(III)-O2 (-) or Fe(III)-NO(-) species. Finally, for the peroxo-bridged Fe(III)2 intermediates, further activation is necessary for their reactivities in one-electron reduction and electrophilic aromatic substitution, and a strategy consistent with existing spectral data is discussed. PMID:27369780

  14. Soil pollution with trace elements at selected sites in Romania studied by instrumental neutron activation analysis

    Pantelica, A. [Horia Hulubei National Institute for Physics and Nuclear Engineering (IFIN-HH), Magurele, Ilfov County (Romania); Carmo Freitas, M. do [Technological and Nuclear Institute (ITN), Sacavem (Portugal); Ene, A. [Dunarea de Jos Univ. of Galati (Romania). Dept. of Chemistry, Physics and Environment; Steinnes, E. [Norwegian Univ. of Science and Technology, Trondheim (Norway). Dept. of Chemistry

    2013-03-01

    Instrumental neutron activation analysis (INAA) was used to determine concentrations of 42 elements in samples of surface soil collected at seven sites polluted from various anthropogenic activities and a control site in a relatively clean area. Elements studied were Ag, Al, As, Au, Ba, Br, Ca, Cd, Ce, Co, Cr, Cs, Eu, Fe, Gd, Hf, Hg, K, La, Lu, Mg, Mn, Mo, Na, Nd, Ni, Rb, Sb, Sc, Se, Sm, Sr, Ta, Tb, Th, Ti, U, V, W, Yb, Zn, and Zr. The results are compared with data for trace elements atmospheric deposition in lichen transplants from the same sites. The most severe soil contamination was observed at Copsa Mica from non-ferrous metallurgy. Appreciable soil contamination was also indicated at Baia Mare (non-ferrous mining and metallurgy), Deva (coal-fired power plant, cement and building materials industry), Galati (ferrous metallurgy), Magurele and Afumati (general urban pollution), and Oradea (chemical and light industries). In most cases excessive levels of toxic metals in soils matched correspondingly high values in lichen transplants. Compared to Romanian norms, legal upper limits were exceeded for Zn and Cd at Copsa Mica. Also, As and Sb occurred in excessive levels at given sites. (orig.)

  15. The active sites in the heterogeneous Baeyer-Villiger oxidation of cyclopentanone by hydrotalcite catalysts

    Ueno, Shinji; Ebitani, Kohki; Ookubo, Akira; Kaneda, Kiyotomi

    1997-11-01

    The active sites of hydrotalcites, [ Mg1-x2+Alx3+( OH) 2] x+[ Ax/nn-·m H 2O ] x-, A″ n-; CO 32-, Cl -, etc., were studied in the heterogeneous Baeyer-Villiger oxidation of cyclopentanone to δ-valerolactone with a combination oxidant system of molecular oxygen and benzaldehyde (O 2/benzaldehyde) in a 1,2-dichloroethane solvent. The hydrotalcites with different basicity were prepared by changing element ratios of Al 3+ to Mg 2+ in the Brucite-like layer and by changing anionic compounds (CO 32-, Cl -, and SO 42-) in the interlayer. The basicities of hydrotalcites were evaluated by measuring the calorimetric heats of benzoic acid adsorption and the zeta-potential of potassium chloride and by the indicator titration method. Yields of δ-valerolactone were almost proportional to the basicities of hydrotalcites, i.e., the heats of benzoic acid adsorption on hydrotalcites, which suggests that basic sites of hydrotalcites are active sites for the oxidation. Yields of δ-valerolactone were also dependent on the basicities of hydrotalcites using m-chloroperbenzoic acid ( m-CPBA) as an oxidant instead of O 2/benzaldehyde. Basic sites of hydrotalcites play an important role in the oxygen transfer from perbenzoic acid to ketone.

  16. 78 FR 21352 - Update on Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    2013-04-10

    ... on Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites AGENCY... reimbursement for cleanup work performed by licensees at eligible uranium and thorium processing sites in... licensees of eligible uranium and thorium processing sites. If licensees submit claims in FY 2013,...

  17. Characterization of the interactions between the active site of a protein tyrosine kinase and a divalent metal activator

    Ayrapetov Marina K

    2005-11-01

    Full Text Available Abstract Background Protein tyrosine kinases are important enzymes for cell signalling and key targets for anticancer drug discovery. The catalytic mechanisms of protein tyrosine kinase-catalysed phosphorylation are not fully understood. Protein tyrosine kinase Csk requires two Mg2+ cations for activity: one (M1 binds to ATP, and the other (M2 acts as an essential activator. Results Experiments in this communication characterize the interaction between M2 and Csk. Csk activity is sensitive to pH in the range of 6 to 7. Kinetic characterization indicates that the sensitivity is not due to altered substrate binding, but caused by the sensitivity of M2 binding to pH. Several residues in the active site with potential of binding M2 are mutated and the effect on metal activation studied. An active mutant of Asn319 is generated, and this mutation does not alter the metal binding characteristics. Mutations of Glu236 or Asp332 abolish the kinase activity, precluding a positive or negative conclusion on their role in M2 coordination. Finally, the ability of divalent metal cations to activate Csk correlates to a combination of ionic radius and the coordination number. Conclusion These studies demonstrate that M2 binding to Csk is sensitive to pH, which is mainly responsible for Csk activity change in the acidic arm of the pH response curve. They also demonstrate critical differences in the metal activator coordination sphere in protein tyrosine kinase Csk and a protein Ser/Thr kinase, the cAMP-dependent protein kinase. They shed light on the physical interactions between a protein tyrosine kinase and a divalent metal activator.

  18. Conformational Dependence of a Protein Kinase Phosphate Transfer Reaction

    Henkelman, Graeme; Tung, Chang-Shung; Fenimore,, P W; McMahon, Benjamin H

    2004-01-01

    Atomic motions and energetics for a phosphate transfer reaction catalyzed by the cAMP-dependent protein kinase (PKA) are calculated by plane-wave density functional theory, starting from structures of proteins crystallized in both the reactant conformation (RC) and the transition-state conformation (TC). In the TC, we calculate that the reactants and products are nearly isoenergetic with a 0.2 eV barrier; while phosphate transfer is unfavorable by over 1.2 eV in the RC, with an even higher barrier. With the protein in the TC, the motions involved in reaction are small, with only P$_\\gamma$ and the catalytic proton moving more than 0.5 \\AA. Examination of the structures reveals that in the RC the active site cleft is not completely closed and there is insufficient space for the phosphorylated serine residue in the product state. Together, these observations imply that the phosphate transfer reaction occurs rapidly and reversibly in a particular conformation of the protein, and that the reaction can be gated by...

  19. Synthesis of supported bimetallic nanoparticles with controlled size and composition distributions for active site elucidation

    Hakim, Sikander H.; Sener, Canan; Alba Rubio, Ana C.; Gostanian, Thomas M.; O' neill, Brandon J; Ribeiro, Fabio H.; Miller, Jeffrey T.; Dumesic, James A

    2015-08-01

    Elucidation of active sites in supported bimetallic catalysts is complicated by the high level of dispersity in the nanoparticle size and composition that is inherent in conventional methods of catalyst preparation. We present a synthesis strategy that leads to highly dispersed, bimetallic nanoparticles with uniform particle size and composition by means of controlled surface reactions. We demonstrate the synthesis of three systems, RhMo, PtMo, and RhRe, consisting of a highly reducible metal with an oxophilic promoter. These catalysts are characterized by FTIR, CO chemisorption, STEM/EDS, TPR, and XAS analysis. The catalytic properties of these bimetallic nanoparticles were probed for the selective CO hydrogenolysis of (hydroxymethyl)tetrahydropyran to produce 1,6 hexanediol. Based on the characterization results and reactivity trends, the active sites in the hydrogenolysis reaction are identified to be small ensembles of the more noble metal (Rh, Pt) adjacent to highly reduced moieties of the more oxophilic metal (Mo, Re).

  20. Use of the Multi-Agency Radiological Laboratory Analytical Protocols Manual (MARLAP) for site cleanup activities

    MARLAP is being developed as a multi-agency guidance manual for project managers and radioanalytical laboratories. The document uses a performance based approach and will provide guidance and a framework to assure that laboratory radioanalytical data meets the specific project or program needs and requirements. MARLAP supports a wide range of data collection activities including site characterization and compliance demonstration activities. Current participants include: US Environmental Protection Agency (EPA), US Department of Energy (DOE), US Nuclear Regulatory Commission (NRC), US Department of Defense (DoD), US National Institutes of Standards and Technology (NIST), US Geologic Survey (USGS), US Food and Drug Administration (FDA), Commonwealth of Kentucky, and the State of California. MARLAP is the radioanalytical laboratory counterpart to the Multi-Agency Radiological Survey and Site Investigation Manual (MARSSIM). MARLAP is currently in a preliminary draft stage. (author)

  1. Computation of Rate Constants for Diffusion of Small Ligands to and from Buried Protein Active Sites.

    Wang, P-H; De Sancho, D; Best, R B; Blumberger, J

    2016-01-01

    The diffusion of ligands to actives sites of proteins is essential to enzyme catalysis and many cellular signaling processes. In this contribution we review our recently developed methodology for calculation of rate constants for diffusion and binding of small molecules to buried protein active sites. The diffusive dynamics of the ligand obtained from molecular dynamics simulation is coarse grained and described by a Markov state model. Diffusion and binding rate constants are then obtained either from the reactive flux formalism or by fitting the time-dependent population of the Markov state model to a phenomenological rate law. The method is illustrated by applications to diffusion of substrate and inhibitors in [NiFe] hydrogenase, CO-dehydrogenase, and myoglobin. We also discuss a recently developed sensitivity analysis that allows one to identify hot spots in proteins, where mutations are expected to have the strongest effects on ligand diffusion rates. PMID:27497172

  2. Conformational changes relevant to channel activity and folding within the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator.

    Hudson, Rhea P; Chong, P Andrew; Protasevich, Irina I; Vernon, Robert; Noy, Efrat; Bihler, Hermann; An, Jian Li; Kalid, Ori; Sela-Culang, Inbal; Mense, Martin; Senderowitz, Hanoch; Brouillette, Christie G; Forman-Kay, Julie D

    2012-08-17

    Deletion of Phe-508 (F508del) in the first nucleotide binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to defects in folding and channel gating. NMR data on human F508del NBD1 indicate that an H620Q mutant, shown to increase channel open probability, and the dual corrector/potentiator CFFT-001 similarly disrupt interactions between β-strands S3, S9, and S10 and the C-terminal helices H8 and H9, shifting a preexisting conformational equilibrium from helix to coil. CFFT-001 appears to interact with β-strands S3/S9/S10, consistent with docking simulations. Decreases in T(m) from differential scanning calorimetry with H620Q or CFFT-001 suggest direct compound binding to a less thermostable state of NBD1. We hypothesize that, in full-length CFTR, shifting the conformational equilibrium to reduce H8/H9 interactions with the uniquely conserved strands S9/S10 facilitates release of the regulatory region from the NBD dimerization interface to promote dimerization and thereby increase channel open probability. These studies enabled by our NMR assignments for F508del NBD1 provide a window into the conformational fluctuations within CFTR that may regulate function and contribute to folding energetics. PMID:22722932

  3. Conformational Changes Relevant to Channel Activity and Folding within the first Nucleotide Binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator*

    Hudson, Rhea P.; Chong, P. Andrew; Protasevich, Irina I.; Vernon, Robert; Noy, Efrat; Bihler, Hermann; An, Jian Li; Kalid, Ori; Sela-Culang, Inbal; Mense, Martin; Senderowitz, Hanoch; Brouillette, Christie G.; Forman-Kay, Julie D.

    2012-01-01

    Deletion of Phe-508 (F508del) in the first nucleotide binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to defects in folding and channel gating. NMR data on human F508del NBD1 indicate that an H620Q mutant, shown to increase channel open probability, and the dual corrector/potentiator CFFT-001 similarly disrupt interactions between β-strands S3, S9, and S10 and the C-terminal helices H8 and H9, shifting a preexisting conformational equilibrium from helix to coil. CFFT-001 appears to interact with β-strands S3/S9/S10, consistent with docking simulations. Decreases in Tm from differential scanning calorimetry with H620Q or CFFT-001 suggest direct compound binding to a less thermostable state of NBD1. We hypothesize that, in full-length CFTR, shifting the conformational equilibrium to reduce H8/H9 interactions with the uniquely conserved strands S9/S10 facilitates release of the regulatory region from the NBD dimerization interface to promote dimerization and thereby increase channel open probability. These studies enabled by our NMR assignments for F508del NBD1 provide a window into the conformational fluctuations within CFTR that may regulate function and contribute to folding energetics. PMID:22722932

  4. Chemistry related to the actives sites of the [Fe]- and [FeFe]-hydrogenases

    Hill, Amanda D.

    2016-01-01

    Hydrogenases are an important group of enzymes found in a range of microorganisms. There are three phylogenetically distinct classes of hydrogenase all of which feature iron-containing complexes. The work contained in this thesis has two main focuses: the synthesis and characterization of novel mimics of the [Fe]-hydrogenase active site, and spectroscopic studies on the interaction of iron-sulfur clusters with CO and CN− relevant to the biosynthesis of the H-cluster of [FeFe]-hydrogenase. ...

  5. Interaction of mining activities and aquatic environment: A review from Greek mine sites.

    Vasileiou, Eleni; Kallioras, Andreas

    2016-04-01

    In Greece a significant amount of mineral and ore deposits have been recorded accompanied by large industrial interest and a long mining history. Today many active and/or abandoned mine sites are scattered within the country; while mining activities take place in different sites for exploiting various deposits (clay, limestone, slate, gypsum, kaolin, mixed sulphide ores (lead, zinc, olivine, pozzolan, quartz lignite, nickel, magnesite, aluminum, bauxite, gold, marbles etc). The most prominent recent ones are: (i) the lignite exploitation that is extended in the area of Ptolemais (Western Macedonia) and Megalopolis (Central Peloponnese); and (ii) the major bauxite deposits located in central Greece within the Parnassos-Ghiona geotectonic zone and on Euboea Island. In the latter area, significant ores of magnesite were exploited and mixed sulphide ores. Centuries of intensive mining exploitation and metallurgical treatment of lead-silver deposits in Greece, have also resulted in significant abandoned sites, such as the one in Lavrion. Mining activities in Lavrio, were initiated in ancient times and continued until the 1980s, resulting in the production of significant waste stockpiles deposited in the area, crucial for the local water resources. Ιn many mining sites, environmental pressures are also recorded after the mine closure to the aquatic environment, as the surface waters flow through waste dump areas and contaminated soils. This paper aims to the geospatial visualization of the mining activities in Greece, in connection to their negative (surface- and/or ground-water pollution; overpumping due to extensive dewatering practices) or positive (enhanced groundwater recharge; pit lakes, improvement of water budget in the catchment scale) impacts on local water resources.

  6. 76 FR 30696 - Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    2011-05-26

    ...This Notice announces revisions to the Department of Energy (DOE) acceptance of claims in FY 2011 from eligible active uranium and thorium processing site licensees for reimbursement under Title X of the Energy Policy Act of 1992. In our Federal Register Notice of November 24, 2010 (75 FR 71677), the Department announced the closing date for the submission of claims in FY 2011 as April 29,......

  7. Identification of the provenience of Majolica from sites in the Caribbean using neutron activation analysis

    Olin, J.S.; Sayre, E.V.

    1975-01-01

    Tin-enamelled earthenware pottery from five early Spanish Colonial sites in the Dominican Republic and Venezuela were sampled and analyzed by neutron activation analysis in an attempt to determine whether these sherds had a common source. The tentative conclusion was that although several sources were indicated for the specimens analyzed the overall similarity in composition indicated that these sources were probably closely related. (JSR)

  8. A Low Affinity Ground State Conformation for the Dynein Microtubule Binding Domain*

    McNaughton, Lynn; Tikhonenko, Irina; Banavali, Nilesh K.; LeMaster, David M.; Koonce, Michael P.

    2010-01-01

    Dynein interacts with microtubules through a dedicated binding domain that is dynamically controlled to achieve high or low affinity, depending on the state of nucleotide bound in a distant catalytic pocket. The active sites for microtubule binding and ATP hydrolysis communicate via conformational changes transduced through a ∼10-nm length antiparallel coiled-coil stalk, which connects the binding domain to the roughly 300-kDa motor core. Recently, an x-ray structure of the murine cytoplasmic...

  9. IgG Conformer's Binding to Amyloidogenic Aggregates.

    Monichan Phay

    Full Text Available Amyloid-reactive IgGs isolated from pooled blood of normal individuals (pAbs have demonstrated clinical utility for amyloid diseases by in vivo targeting and clearing amyloidogenic proteins and peptides. We now report the following three novel findings on pAb conformer's binding to amyloidogenic aggregates: 1 pAb aggregates have greater activity than monomers (HMW species > dimers > monomers, 2 pAbs interactions with amyloidogenic aggregates at least partially involves unconventional (non-CDR interactions of F(ab regions, and 3 pAb's activity can be easily modulated by trace aggregates generated during sample processing. Specifically, we show that HMW aggregates and dimeric pAbs present in commercial preparations of pAbs, intravenous immunoglobulin (IVIg, had up to ~200- and ~7-fold stronger binding to aggregates of Aβ and transthyretin (TTR than the monomeric antibody. Notably, HMW aggregates were primarily responsible for the enhanced anti-amyloid activities of Aβ- and Cibacron blue-isolated IVIg IgGs. Human pAb conformer's binding to amyloidogenic aggregates was retained in normal human sera, and mimicked by murine pAbs isolated from normal pooled plasmas. An unconventional (non-CDR component to pAb's activity was indicated from control human mAbs, generated against non-amyloid targets, binding to aggregated Aβ and TTR. Similar to pAbs, HMW and dimeric mAb conformers bound stronger than their monomeric forms to amyloidogenic aggregates. However, mAbs had lower maximum binding signals, indicating that pAbs were required to saturate a diverse collection of binding sites. Taken together, our findings strongly support further investigations on the physiological function and clinical utility of the inherent anti-amyloid activities of monomeric but not aggregated IgGs.

  10. Measurement of human tissue-type plasminogen activator by a two-site immunoradiometric assay

    A two-site immunoradiometric assay for human extrinsic (tissue-type) plasminogen activator was developed by using rabbit antibodies raised against plasminogen activator purified from human melanoma cell culture fluid. Samples of 100 μl containing 1 to 100 ng/ml plasminogen activator were incubated in the wells of polyvinyl chloride microtiter plates coated with antibody. The amount of bound extrinsic plasminogen activator was quantitated by the subsequent binding of 125I-labeled affinospecific antibody. The mean level of plasma samples taken at rest was 6.6 +/- 2.9 ng/ml (n = 54). This level increased approximately threefold by exhaustive physical exercise, venous occlusion, or infusion of DDAVP. Extrinsic plasminogen activator in plasma is composed of a fibrin-adsorbable and active component (1.9 +/- 1.1 ng/ml, n = 54, in resting conditions) and an inactive component that does not bind to a fibrin clot (probably extrinsic plasminogen activator-proteinase inhibitor complexes). The fibrin-adsorbable fraction increased approximately fivefold to eightfold after physical exercise, venous occlusion, or DDAVP injections. Potential applications of the immunoradiometric assay are illustrated by the measurement of extrinsic plasminogen activator in different tissue extracts, body fluids, and cell culture fluids and in oocyte translation products after injection with mRNA for plasminogen activator

  11. Killing tensors and conformal Killing tensors from conformal Killing vectors

    Koutras has proposed some methods to construct reducible proper conformal Killing tensors and Killing tensors (which are, in general, irreducible) when a pair of orthogonal conformal Killing vectors exist in a given space. We give the completely general result demonstrating that this severe restriction of orthogonality is unnecessary. In addition, we correct and extend some results concerning Killing tensors constructed from a single conformal Killing vector. A number of examples demonstrate that it is possible to construct a much larger class of reducible proper conformal Killing tensors and Killing tensors than permitted by the Koutras algorithms. In particular, by showing that all conformal Killing tensors are reducible in conformally flat spaces, we have a method of constructing all conformal Killing tensors, and hence all the Killing tensors (which will in general be irreducible) of conformally flat spaces using their conformal Killing vectors

  12. Days of dismantling activities of installations and rehabilitation of contaminated sites in France

    The objective of these days, organized by the section environment of the French society of radiation protection, is to present a panorama of the activities of nuclear installations dismantling and contaminated sites rehabilitation in France, by leaning in the same time on practical cases and by stating the French rule and the national and international recommendations on the subject. These days have also for object to approach the stakes associated with the sectors of waste management and the materials generated by these activities and in a more general way, the stakes to come for the different actors of the dismantling and the rehabilitation. (N.C.)

  13. Comparative active-site mutation study of human and Caenorhabditis elegans thymidine kinase 1

    Skovgaard, Tine; Uhlin, Ulla; Munch-Petersen, Birgitte

    2012-01-01

    ligands. To improve our understanding of TK1 substrate specificity, we performed a detailed, mutation-based comparative structure-function study of the active sites of two thymidine kinases: HuTK1 and Caenorhabditis elegans TK1 (CeTK1). Specifically, mutations were introduced into the hydrophobic pocket...... surrounding the substrate base. In CeTK1, some of these mutations led to increased activity with deoxycytidine and deoxyguanosine, two unusual substrates for TK1-like kinases. In HuTK1, mutation of T163 to S resulted in a kinase with a 140-fold lower K(m) for the antiviral nucleoside analogue 3'-azido-3...

  14. Reaction of argininosuccinase with bromomesaconic acid: role of an essential lysine in the active site

    We have undertaken studies on bovine liver argininosuccinase (L-argininosuccinate arginine-lyase with the active site-directed reagent bromo[U-14C]mesaconic acid, an analogue of fumaric acid. Reactivity, measured by enzyme inactivation, followed pseudo-first-order kinetics, and the rate increased with reagent concentration. Argininosuccinate completely protected the enzyme against inactivation, but neither arginine nor fumarate was protective. A plot of the degree of inactivation as a function of alkyl groups incorporated was extrapolated to 4 mol per mol of enzyme, or 1 mol per active site. After large-scale alkylation of the enzyme (and digestion with trypsin), two 14C-labeled tryptic peptides were isolated. These were chemically sequenced by the Edman method. The amino acid sequences proved to be identical with regions of the deduced amino acid sequences or argininosuccinases from human and yeast sources The 14C-labeled tryptic peptide in the active site region had the sequence Gly-Leu-Glu-Xaa-Ala-Gly-Leu-Leu-Thr-Lys; Xaa represents an unknown phenylthiohydantoin derivative detected in cycle 4. The corresponding amino acid was identified as lysine-51 on the basis of sequence similarity with human and yeast amino acid sequences in this region. The reaction of the enzyme with the alkylating agent and the specific protection against inactivation by argininosuccinate suggest that this lysine residue has an essential role in the binding of argininosuccinate to the enzyme and, consequently, is essential for catalysis

  15. Structural analysis of the active site architecture of the VapC toxin from Shigella flexneri.

    Xu, Kehan; Dedic, Emil; Brodersen, Ditlev E

    2016-07-01

    The VapC toxin from the Shigella flexneri 2a virulence plasmid pMYSH6000 belongs to the PIN domain protein family, which is characterized by a conserved fold with low amino acid sequence conservation. The toxin is a bona fide Mg(2+) -dependent ribonuclease and has been shown to target initiator tRNA(fMet) in vivo. Here, we present crystal structures of active site catalytic triad mutants D7A, D7N, and D98N of the VapC toxin in absence of antitoxin. In all structures, as well as in solution, VapC forms a dimer. In the D98N structure, a Hepes molecule occupies both active sites of the dimer and comparison with the structure of RNase H bound to a DNA/RNA hybrid suggests that the Hepes molecule mimics the position of an RNA nucleotide in the VapC active site. Proteins 2016; 84:892-899. © 2016 Wiley Periodicals, Inc. PMID:26833558

  16. Methods for Determining the Activity Concentration Calibration Factor for Ventilation Duct in Cyclotron Sites

    Cyclotrons are commonly used for production of radioactive isotopes for nuclear medicine purposes. The nuclear process requires installation of a ventilation stack. According to the regulations in many countries, the released activity should be monitored and controlled in order to limit air pollution. This requires a stuck radiation detector and determining a converting factor that will translate the radiation detector reading into activity concentration units. Calibrating the conversion factor is done mainly by releasing a known amount of activity. Having a preliminary estimation of the conversion factor, during the site construction stage, is an important option for the duct configuration design, in order to achieve the required detection sensitivity. An algorithm for estimating the stack concentration calibration factor in positron emitting isotopes producing sites was developed. The algorithm which is described in this article is based on three independent methods that consist of MCNP simulations analytical calculations and experimental setup with controlled static calibration releases of gaseous 18F which was produced especially for this purpose by the cyclotron site in Soreq Nuclear Research Center Israel. The specified methods applied on few stack sections with different shapes and sizes

  17. Radiological audit of remedial action activities at the processing site, transfer site, and Cheney disposal site Grand Junction, Colorado: Audit date, August 9--11, 1993. Final report

    1993-08-01

    The Uranium Mill Tailing Remedial Action (UMTRA) Project`s Technical Assistance Contractor (TAC) performed a radiological audit of the Remedial Action Contractor (RAC), MK-Ferguson and CWM Federal Environmental Services, Inc., at the processing site, transfer site, and Cheney disposal site in Grand Junction, Colorado. Jim Hylko and Bill James of the TAC conducted this audit August 9 through 11, 1993. Bob Cornish and Frank Bosiljevec represented the US Department of Energy (DOE). This report presents one programmatic finding, eleven site-specific observations, one good practice, and four programmatic observations.

  18. Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

    Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele; Goedken, Eric R.; Gum, Rebecca J.; Borhani, David W.; Argiriadi, Maria; Groebe, Duncan R.; Jia, Yong; Clampit, Jill E.; Haasch, Deanna L.; Smith, Harriet T.; Wang, Sanyi; Song, Danying; Coen, Michael L.; Cloutier, Timothy E.; Tang, Hua; Cheng, Xueheng; Quinn, Christopher; Liu, Bo; Xin, Zhili; Liu, Gang; Fry, Elizabeth H.; Stoll, Vincent; Ng, Teresa I.; Banach, David; Marcotte, Doug; Burns, David J.; Calderwood, David J.; Hajduk, Philip J. (Abbott)

    2012-03-02

    Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38{alpha} (involved in the formation of TNF{alpha} and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional {sup 1}H/{sup 13}C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38{alpha} both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in

  19. Charged conformal Killing spinors

    We study the twistor equation on pseudo-Riemannian Spinc-manifolds whose solutions we call charged conformal Killing spinors (CCKSs). We derive several integrability conditions for the existence of CCKS and study their relations to spinor bilinears. A construction principle for Lorentzian manifolds admitting CCKS with nontrivial charge starting from CR-geometry is presented. We obtain a partial classification result in the Lorentzian case under the additional assumption that the associated Dirac current is normal conformal and complete the classification of manifolds admitting CCKS in all dimensions and signatures ≤5 which has recently been initiated in the study of supersymmetric field theories on curved space

  20. A kinetic description for sodium and potassium effects on (Na+ plus K+)-adenosine triphosphatase: a model for a two-nonequivalent site potassium activation and an analysis of multiequivalent site models for sodium activation.

    Lindenmayer, G E; Schwartz, A; Thompson, H K

    1974-01-01

    1. Dissociation constants for sodium and potassium of a site that modulates the rate of ouabain-(Na(+)+K(+))-ATPase interaction were applied to models for potassium activation of (Na(+)+K(+))-ATPase. The constants for potassium (0.213 mM) and for sodium (13.7 mM) were defined, respectively, as activation constant, K(a) and inhibitory constant, K(i).2. Tests of the one- and the two-equivalent site models, that describe sodium and potassium competition, revealed that neither model adequately predicts the activation effects of potassium in the presence of 100 or 200 mM sodium.3. The potassium-activation data, obtained at low potassium and high sodium, were explained by a two-nonequivalent site model where the dissociation constants of the first site are 0.213 mM for potassium and 13.7 mM for sodium. The second site was characterized by dissociation constants of 0.091 mM for potassium and 74.1 mM for sodium.4. The two-nonequivalent site model adequately predicted the responses to concentrations of potassium between 0.25 and 5 mM in the presence of 100-500 mM sodium. At lower sodium concentrations the predicted responses formed an upper limit for the function of observed activities. This limit was reached at lower concentrations of potassium and higher concentrations of sodium, which inferred saturation of the sodium-activation sites with sodium.5. Sodium-activation data were corrected for sodium interaction with potassium-activation sites by use of the two-nonequivalent site model for potassium activation. Tests of equivalent site models suggested that the corrected data for sodium activation may be most consistent with a model that has three-equivalent sites. Other multiequivalent site models (n = 2, 4, 5 or 6), however, cannot be statistically eliminated as possibilities. The three-equivalent site activation model was characterized by dissociation constants of 1.39 mM for sodium and 11.7 mM for potassium. The system theoretically would be half-maximally activated by

  1. Study of Molecular Conformation and Activity-Related Properties of Lipase Immobilized onto Core-Shell Structured Polyacrylic Acid-Coated Magnetic Silica Nanocomposite Particles.

    Esmaeilnejad-Ahranjani, Parvaneh; Kazemeini, Mohammad; Singh, Gurvinder; Arpanaei, Ayyoob

    2016-04-01

    A facile approach for the preparation of core-shell structured poly(acrylic acid) (PAA)-coated Fe3O4 cluster@SiO2 nanocomposite particles as the support materials for the lipase immobilization is reported. Low- or high-molecular-weight (1800 and 100 000, respectively) PAA molecules were covalently attached onto the surface of amine-functionalized magnetic silica nanoacomposite particles. The successful preparation of particles were verified by scanning transmission electron microscopy (STEM), X-ray diffraction (XRD), vibrating sample magnetometer (VSM), thermogravimetric analysis (TGA), zeta potential measurement, and Fourier-transform infrared (FTIR) techniques. Once lipase is covalently immobilized onto the particles with an average diameter of 210 ± 50 nm, resulting from high binding sites concentrations on the low- and high-molecular-weight PAA-coated particles, high lipase immobilization efficiencies (86.2% and 89.9%, respectively), and loading capacities (786 and 816 mg g(-1), respectively) are obtained. Results from circular dichroism (CD) analysis and catalytic activity tests reveal an increase in the β-sheet content of lipase molecules upon immobilization, along with an enhancement in their activities and stabilities. The lipases immobilized onto the low- and high-molecular-weight PAA-coated particles show maximum activities at 55 and 50 °C, respectively, which are ∼28% and ∼15% higher than that of the free lipase at its own optimum temperature (40 °C), respectively. The immobilized lipases exhibit excellent performance at broader temperature and pH ranges and high thermal and storage stabilities, as well as superior reusability. These prepared magnetic nanocomposite particles can be offered as suitable support materials for efficient immobilization of enzymes and improvement of the immobilized enzymes properties. PMID:26986897

  2. Three-dimensional representations of salt-dome margins at four active strategic petroleum reserve sites.

    Rautman, Christopher Arthur; Stein, Joshua S.

    2003-01-01

    Existing paper-based site characterization models of salt domes at the four active U.S. Strategic Petroleum Reserve sites have been converted to digital format and visualized using modern computer software. The four sites are the Bayou Choctaw dome in Iberville Parish, Louisiana; the Big Hill dome in Jefferson County, Texas; the Bryan Mound dome in Brazoria County, Texas; and the West Hackberry dome in Cameron Parish, Louisiana. A new modeling algorithm has been developed to overcome limitations of many standard geological modeling software packages in order to deal with structurally overhanging salt margins that are typical of many salt domes. This algorithm, and the implementing computer program, make use of the existing interpretive modeling conducted manually using professional geological judgement and presented in two dimensions in the original site characterization reports as structure contour maps on the top of salt. The algorithm makes use of concepts of finite-element meshes of general engineering usage. Although the specific implementation of the algorithm described in this report and the resulting output files are tailored to the modeling and visualization software used to construct the figures contained herein, the algorithm itself is generic and other implementations and output formats are possible. The graphical visualizations of the salt domes at the four Strategic Petroleum Reserve sites are believed to be major improvements over the previously available two-dimensional representations of the domes via conventional geologic drawings (cross sections and contour maps). Additionally, the numerical mesh files produced by this modeling activity are available for import into and display by other software routines. The mesh data are not explicitly tabulated in this report; however an electronic version in simple ASCII format is included on a PC-based compact disk.

  3. Functional properties of the two redox-active sites in yeast protein disulphide isomerase in vitro and in vivo

    Westphal, V; Darby, N J; Winther, Jakob R.

    1999-01-01

    Protein folding catalysed by protein disulphide isomerase (PDI) has been studied both in vivo and in vitro using different assays. PDI contains a CGHC active site in each of its two catalytic domains (a and a'). The relative importance of each active site in PDI from Saccharomyces cerevisiae (y...

  4. Targeting mechanisms at sites of complement activation for imaging and therapy.

    Holers, V Michael

    2016-06-01

    The complement system plays a key role in many acute injury states as well as chronic autoimmune and inflammatory diseases. Localized complement activation and alternative pathway-mediated amplification on diverse target surfaces promote local recruitment of pro-inflammatory cells and elaboration of other mediators. Despite a general understanding of the architecture of the system, though, many of the mechanisms that underlie site-specific complement activation and amplification in vivo are incompletely understood. In addition, there is no capability yet to measure the level of local tissue site-specific complement activation in patients without performing biopsies to detect products using immunohistochemical techniques. Herein is reviewed emerging evidence obtained through clinical research studies of human rheumatoid arthritis along with translational studies of its disease models which demonstrate that several parallel mechanisms are involved in site-specific amplification of activation of the complement system in vivo. Among these processes are de-regulation of the alternative pathway, effector pathway-catalyzed amplification of proximal complement activation, recognition of injury-associated ligands by components of the lectin pathway, and engagement of pathogenic natural antibodies that recognize a limited set of injury-associated neoepitopes. Studies suggest that each of these inter-related processes can play key roles in amplification of complement-dependent injury on self-tissues in vivo. These findings, in addition to development of an imaging strategy described herein designed to quantitatively measure local complement C3 fixation, have relevance to therapeutic and diagnostic strategies targeting the complement system. PMID:25979851

  5. Modeling emissions and dispersion of contaminants from cleanup activities at a mixed waste site to estimate air impacts and risks

    The transport and dispersion of contaminants via the air pathway is a major concern during cleanup of contaminated sites. Impacts to air quality and human health during cleanup were evaluated for the Weldon Spring site by using site-specific information for source areas, activities, and receptor locations. In order to ensure protection of human health and the environment, results are being used to focus on those cleanup activities for which release controls should be emphasized

  6. Comparative analysis of the impact of geological activity on astronomical sites of the Canary Islands, Hawaii and Chile

    Eff-Darwich, A.; Garcia-Lorenzo, B.; Rodriguez-Losada, J. A.; de la Nuez, J.; Hernandez-Gutierrez, L. E.; Romero-Ruiz, M. C.

    2009-01-01

    An analysis of the impact of seismic and volcanic activity was carried out at selected astronomical sites, namely the observatories of El Teide (Tenerife, Canary Islands), Roque de los Muchachos (La Palma, Canary Islands), Mauna Kea (Hawaii) and Paranal (Chile) and the candidate site of Cerro Ventarrones (Chile). Hazard associated to volcanic activity is low or negligible at all sites, whereas seismic hazard is very high in Chile and Hawaii. The lowest geological hazard in both seismic and vo...

  7. Site selection and licensing issues: Southwest Compact low-level radioactive waste disposal site

    Grant, J.L.

    1989-11-01

    The low-level radioactive waste disposal site in California is being selected through a three-phase program. Phase 1 is a systematic statewide, regional, and local screening study. This program was conducted during 1986 and 1987, and culminated in the selection of three candidate sites fur further study. The candidate sites are identified as the Panamint, Silurian, and Ward Valley sites. Phase 2 comprises site characterization and environmental and socio-economic impact study activities at the three candidate sites. Based upon the site characterization studies, the candidate sites are ranked according to the desirability and conformance with regulatory requirements. Phase 3 comprises preparation of a license application for the selected candidate site. The license application will include a detailed characterization of the site, detailed design and operations plans for the proposed facility, and assessments of potential impacts of the site upon the environment and the local communities. Five types of siting criteria were developed to govern the site selection process. These types are: technical suitability exclusionary criteria, high-avoidance criteria beyond technical suitability requirements, discretionary criteria, public acceptance, and schedule requirements of the LLWR Policy Act Amendments. This paper discusses the application of the hydrological and geotechnical criteria during the siting and licensing studies in California. These criteria address site location and performance, and the degree to which present and future site behavior can be predicted. Primary regulatory requirements governing the suitability of a site are that the site must be hydrologically and geologically simple enough for the confident prediction of future behavior, and that the site must be stable enough that frequent or intensive maintenance of the closed site will not be required. This paper addresses the methods to measure site suitability at each stage of the process, methods to

  8. The conforming brain and deontological resolve.

    Melanie Pincus

    Full Text Available Our personal values are subject to forces of social influence. Deontological resolve captures how strongly one relies on absolute rules of right and wrong in the representation of one's personal values and may predict willingness to modify one's values in the presence of social influence. Using fMRI, we found that a neurobiological metric for deontological resolve based on relative activity in the ventrolateral prefrontal cortex (VLPFC during the passive processing of sacred values predicted individual differences in conformity. Individuals with stronger deontological resolve, as measured by greater VLPFC activity, displayed lower levels of conformity. We also tested whether responsiveness to social reward, as measured by ventral striatal activity during social feedback, predicted variability in conformist behavior across individuals but found no significant relationship. From these results we conclude that unwillingness to conform to others' values is associated with a strong neurobiological representation of social rules.

  9. Conformational States of HIV-1 Reverse Transcriptase for Nucleotide Incorporation vs Pyrophosphorolysis-Binding of Foscarnet.

    Das, Kalyan; Balzarini, Jan; Miller, Matthew T; Maguire, Anita R; DeStefano, Jeffrey J; Arnold, Eddy

    2016-08-19

    HIV-1 reverse transcriptase (RT) catalytically incorporates individual nucleotides into a viral DNA strand complementing an RNA or DNA template strand; the polymerase active site of RT adopts multiple conformational and structural states while performing this task. The states associated are dNTP binding at the N site, catalytic incorporation of a nucleotide, release of a pyrophosphate, and translocation of the primer 3'-end to the P site. Structural characterization of each of these states may help in understanding the molecular mechanisms of drug activity and resistance and in developing new RT inhibitors. Using a 38-mer DNA template-primer aptamer as the substrate mimic, we crystallized an RT/dsDNA complex that is catalytically active, yet translocation-incompetent in crystals. The ability of RT to perform dNTP binding and incorporation in crystals permitted obtaining a series of structures: (I) RT/DNA (P-site), (II) RT/DNA/AZTTP ternary, (III) RT/AZT-terminated DNA (N-site), and (IV) RT/AZT-terminated DNA (N-site)/foscarnet complexes. The stable N-site complex permitted the binding of foscarnet as a pyrophosphate mimic. The Mg(2+) ions dissociated after catalytic addition of AZTMP in the pretranslocated structure III, whereas ions A and B had re-entered the active site to bind foscarnet in structure IV. The binding of foscarnet involves chelation with the Mg(2+) (B) ion and interactions with K65 and R72. The analysis of interactions of foscarnet and the recently discovered nucleotide-competing RT inhibitor (NcRTI) α-T-CNP in two different conformational states of the enzyme provides insights for developing new classes of polymerase active site RT inhibitors. PMID:27192549

  10. Extende conformal field theories

    Taormina, A. (Chicago Univ., IL (USA). Enrico Fermi Inst.)

    1990-08-01

    Some extended conformal field theories are briefly reviewed. They illustrate how non minimal models of the Virasoro algebra (c{ge}1) can become minimal with respect to a larger algebra. The accent is put on N-extended superconformal algebras, which are relevant in superstring compactification. (orig.).

  11. Extended conformal field theories

    Taormina, Anne

    1990-08-01

    Some extended conformal field theories are briefly reviewed. They illustrate how non minimal models of the Virasoro algebra (c≥1) can become minimal with respect to a larger algebra. The accent is put on N-extended superconformal algebras, which are relevant in superstring compactification.

  12. Conformal special relativity

    It is shown that the information loss/recovery theorem based on the ADS/CFT correspondence is not consistent with the stability of the Schwarzschild or Reissner-Nordstrom black holes. Nonetheless, the conformal invariance of Yang-Mills theory points to new relativity principle compatible with quantum unitarity near those black holes

  13. Digital library conformance checklist

    Ross, Seamus; CASTELLI, Donatella; Ioannidis, Yannis; Vullo, Giuseppina; Innocenti, Perla; Candela, Leonardo; Nika, Ana; El Raheb, Katerina; Katifori, Akrivi

    2011-01-01

    This booklet is abstracted and abridged from "The Digital Library Reference Model"- D3.2 DL.org Project Deliverable. The check list is designed to verify whether or not a digital library conforms to the Digital Library Reference Model.

  14. Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity

    Hartmann, Jens; Linke, Dennis; Bönniger, Christine; Tholey, Andreas; Sauter, Margret

    2015-01-01

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser696 and Ser698 in the JM (juxtamembrane) region and probably Ser886 and/or Ser893 in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser717 in the JM, and at Ser733, Thr752, Ser783, Ser864, Ser911, Ser958 and Thr9...

  15. Cernavoda Unit 2: - BOP 3D model proposal for a possible organization of site activities

    The scope of this activity is to define characteristics and advantages of the 3D model of Cernavoda BOP to this set up at site for engineering and construction activities. This model will provide a modern and proven tool able to strongly support the site activities with particular regard to the following: 1. engineering activities, - plant arrangement 'double check' for resolution of clashing; - easy management of future design changes; - real time plant configuration updating as soon as any design modification is approved and integrated in the model; - preparation of high quality documentation for procurement, construction and commissioning; - prompt availability of the as built configuration of the plant as soon as the last modification is frozen; 2. material procurement activities, - definition of the priorities in the construction material procurement according to the construction planning by area; - inventory list of equipment, pipes, fittings, valves, cable trays and ventilation ducts to be installed in each construction area; 3. construction activities, - definition of construction sequences, with particular reference in the congested areas, for piping cable trays (electrical and C-and-I) and ventilations ducts; - definition of piping spools by construction contractors; - follow-up of the activities in each area (i.e. construction, painting, insulation, flushing, pressure testing, etc); 4. turn-over and commissioning, - check of the progress. The success of this approach is based on the following: i) proper management of the remote workstations providing easy and reliable access to the model; ii) subdivision of the Integrated Building in construction areas, whose detail design may be allotted to Romanian organizations with multidisciplinary tasks; iii) integration in the model of the remote developed engineering in order to validate the details of the design. (authors)

  16. O2 activation by binuclear Cu sites: Noncoupled versus exchange coupled reaction mechanisms

    Chen, Peng; Solomon, Edward I.

    2004-09-01

    Binuclear Cu proteins play vital roles in O2 binding and activation in biology and can be classified into coupled and noncoupled binuclear sites based on the magnetic interaction between the two Cu centers. Coupled binuclear Cu proteins include hemocyanin, tyrosinase, and catechol oxidase. These proteins have two Cu centers strongly magnetically coupled through direct bridging ligands that provide a mechanism for the 2-electron reduction of O2 to a µ-2:2 side-on peroxide bridged species. This side-on bridged peroxo-CuII2 species is activated for electrophilic attack on the phenolic ring of substrates. Noncoupled binuclear Cu proteins include peptidylglycine -hydroxylating monooxygenase and dopamine -monooxygenase. These proteins have binuclear Cu active sites that are distant, that exhibit no exchange interaction, and that activate O2 at a single Cu center to generate a reactive CuII/O2 species for H-atom abstraction from the C-H bond of substrates. O2 intermediates in the coupled binuclear Cu enzymes can be trapped and studied spectroscopically. Possible intermediates in noncoupled binuclear Cu proteins can be defined through correlation to mononuclear CuII/O2 model complexes. The different intermediates in these two classes of binuclear Cu proteins exhibit different reactivities that correlate with their different electronic structures and exchange coupling interactions between the binuclear Cu centers. These studies provide insight into the role of exchange coupling between the Cu centers in their reaction mechanisms.

  17. Tricyclic covalent inhibitors selectively target Jak3 through an active site thiol.

    Goedken, Eric R; Argiriadi, Maria A; Banach, David L; Fiamengo, Bryan A; Foley, Sage E; Frank, Kristine E; George, Jonathan S; Harris, Christopher M; Hobson, Adrian D; Ihle, David C; Marcotte, Douglas; Merta, Philip J; Michalak, Mark E; Murdock, Sara E; Tomlinson, Medha J; Voss, Jeffrey W

    2015-02-20

    The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. We have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). We found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC50 < 100 nm) inhibit Jak3 activity in cell-based assays. These results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases. PMID:25552479

  18. Exploring the active site structure of photoreceptor proteins by Raman optical activity

    Unno, Masashi

    2015-03-01

    Understanding protein function at the atomic level is a major challenge in a field of biophysics and requires the combined efforts of structural and functional methods. We use photoreceptor proteins as a model system to understand in atomic detail how a chromophore and a protein interact to sense light and send a biological signal. A potential technique for investigating molecular structures is Raman optical activity (ROA), which is a spectroscopic method with a high sensitivity to the structural details of chiral molecules. However, its application to photoreceptor proteins has not been reported. Thus we have constructed ROA spectrometer using near-infrared (NIR) laser excitation at 785 nm. The NIR excitation enables us to measure ROA spectra for a variety of biological samples, including photoreceptor proteins, without fluorescence from the samples. In the present study, we have applied the NIR-ROA to bacteriorhodopsin (BR) and photoactive yellow protein (PYP). BR is a light-driven proton pump and contains a protonated Schiff base of retinal as a chromophore. PYP is a blue light receptor, and this protein has the 4-hydroxycinnamyl chromophore, which is covalently linked to Cys69 through a thiolester bond. We have successfully obtained the ROA spectra of the chromophore within a protein environment. Furthermore, calculations of the ROA spectra utilizing density functional theory provide detailed structural information, such as data on out-of-plane distortions of the chromophore. The structural information obtained from the ROA spectra includes the positions of hydrogen atoms, which are usually not detected in the crystal structures of biological samples.

  19. Integral communication activities in support of the repository site selection in Slovenia

    The siting and licensing of a radioactive waste repository requires a complete public consensus that is very difficult to obtain. The main reasons for the public reluctance to accept the radioactive waste repository are the feeling of being ignored in the decision making process and inadequate understanding of radioactivity. Therefore communication and information activities, as early as possible in the siting process, are very important for reducing the potential conflicts of interests between the local community and the investor of the radioactive waste repository on the potential repository sites. ARAO communication activities are based on research on public opinion and public knowledge about radioactive waste management. Communication strategies that provide two-way communication channels, such as interactive web pages, workshops, study circles, visitors' centre, are preferred. Different educational materials (leaflets, CD-ROMs, articles in the local newspapers, yearly magazine, and posters) are also being produced. Collaboration with non-governmental environmental organizations has also proved to be helpful in confidence building, as well as in informing the public. It is also very important for establishing competent public participation in the decision making process. (author)

  20. Stress-induced enhancement of leukocyte trafficking into sites of surgery or immune activation

    Viswanathan, Kavitha; Dhabhar, Firdaus S.

    2005-04-01

    Effective immunoprotection requires rapid recruitment of leukocytes into sites of surgery, wounding, infection, or vaccination. In contrast to immunosuppressive chronic stressors, short-term acute stressors have immunoenhancing effects. Here, we quantify leukocyte infiltration within a surgical sponge to elucidate the kinetics, magnitude, subpopulation, and chemoattractant specificity of an acute stress-induced increase in leukocyte trafficking to a site of immune activation. Mice acutely stressed before sponge implantation showed 200-300% higher neutrophil, macrophage, natural killer cell, and T cell infiltration than did nonstressed animals. We also quantified the effects of acute stress on lymphotactin- (LTN; a predominantly lymphocyte-specific chemokine), and TNF-- (a proinflammatory cytokine) stimulated leukocyte infiltration. An additional stress-induced increase in infiltration was observed for neutrophils, in response to TNF-, macrophages, in response to TNF- and LTN, and natural killer cells and T cells in response to LTN. These results show that acute stress initially increases trafficking of all major leukocyte subpopulations to a site of immune activation. Tissue damage-, antigen-, or pathogen-driven chemoattractants subsequently determine which subpopulations are recruited more vigorously. Such stress-induced increases in leukocyte trafficking may enhance immunoprotection during surgery, vaccination, or infection, but may also exacerbate immunopathology during inflammatory (cardiovascular disease or gingivitis) or autoimmune (psoriasis, arthritis, or multiple sclerosis) diseases. chemokine | psychophysiological stress | surgical sponge | wound healing | lymphotactin