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Sample records for active site molecular

  1. Molecular Imprint of Enzyme Active Site by Camel Nanobodies

    PubMed Central

    Li, Jiang-Wei; Xia, Lijie; Su, Youhong; Liu, Hongchun; Xia, Xueqing; Lu, Qinxia; Yang, Chunjin; Reheman, Kalbinur

    2012-01-01

    Screening of inhibitory Ab1 antibodies is a critical step for producing catalytic antibodies in the anti-idiotypic approach. However, the incompatible surface of the active site of the enzyme and the antigen-binding site of heterotetrameric conventional antibodies become the limiting step. Because camelid-derived nanobodies possess the potential to preferentially bind to the active site of enzymes due to their small size and long CDR3, we have developed a novel approach to produce antibodies with alliinase activities by exploiting the molecular mimicry of camel nanobodies. By screening the camelid-derived variable region of the heavy chain cDNA phage display library with alliinase, we obtained an inhibitory nanobody VHHA4 that recognizes the active site. Further screening with VHHA4 from the same variable domain of the heavy chain of a heavy-chain antibody library led to a higher incidence of anti-idiotypic Ab2 abzymes with alliinase activities. One of the abzymes, VHHC10, showed the highest activity that can be inhibited by Ab1 VHHA4 and alliinase competitive inhibitor penicillamine and significantly suppressed the B16 tumor cell growth in the presence of alliin in vitro. The results highlight the feasibility of producing abzymes via anti-idiotypic nanobody approach. PMID:22374998

  2. Molecular Active Sites in Heterogeneous Ir-La/C-Catalyzed Carbonylation of Methanol to Acetates.

    PubMed

    Kwak, Ja Hun; Dagle, Robert; Tustin, Gerald C; Zoeller, Joseph R; Allard, Lawrence F; Wang, Yong

    2014-02-01

    We report that when Ir and La halides are deposited on carbon, exposure to CO spontaneously generates a discrete molecular heterobimetallic structure, containing an Ir-La covalent bond that acts as a highly active, selective, and stable heterogeneous catalyst for the carbonylation of methanol to produce acetic acid. This catalyst exhibits a very high productivity of ∼1.5 mol acetyl/mol Ir·s with >99% selectivity to acetyl (acetic acid and methyl acetate) without detectable loss in activity or selectivity for more than 1 month of continuous operation. The enhanced activity can be mechanistically rationalized by the presence of La within the ligand sphere of the discrete molecular Ir-La heterobimetallic structure, which acts as a Lewis acid to accelerate the normally rate-limiting CO insertion in Ir-catalyzed carbonylation. Similar approaches may provide opportunities for attaining molecular (single site) behavior similar to homogeneous catalysis on heterogeneous surfaces for other industrial applications. PMID:26276610

  3. Molecular dioxygen enters the active site of 12/15-lipoxygenase via dynamic oxygen access channels.

    PubMed

    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

  4. Evidence from molecular dynamics simulations of conformational preorganization in the ribonuclease H active site

    PubMed Central

    Stafford, Kate A.; Palmer III, Arthur G.

    2014-01-01

    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. PMID:25075292

  5. A Frontier Molecular Orbital determination of the active sites on dispersed metal catalysts

    SciTech Connect

    Augustine, R.L.; Lahanas, K.M.

    1992-01-01

    An angular overlap calculation has been used to determine the s, p and d orbital energy levels of the different types of surface sites present on a dispersed metal catalysts. The basis for these calculations is the reported finding that a large number of catalyzed reactions take place on single atom active sites on the metal surface. Thus, these sites can be considered as surface complexes made up of the central active atom surrounded by near-neighbor metal atom ligands'' with localized surface orbitals perturbed only by these ligands''. These complexes'' are based on a twelve coordinate species with the ligands'' attached to the t{sub 2g} orbitals and the coordinate axes coincident with the direction of the e{sub g} orbitals on the central atom. These data can permit a Frontier Molecular Orbital treatment of specific site activities as long as the surface orbital availability for overlap with adsorbed substrates is considered along with its energy value and symmetry.

  6. A Frontier Molecular Orbital determination of the active sites on dispersed metal catalysts

    SciTech Connect

    Augustine, R.L.; Lahanas, K.M.

    1992-11-01

    An angular overlap calculation has been used to determine the s, p and d orbital energy levels of the different types of surface sites present on a dispersed metal catalysts. The basis for these calculations is the reported finding that a large number of catalyzed reactions take place on single atom active sites on the metal surface. Thus, these sites can be considered as surface complexes made up of the central active atom surrounded by near-neighbor metal atom ``ligands`` with localized surface orbitals perturbed only by these ``ligands``. These ``complexes`` are based on a twelve coordinate species with the ``ligands`` attached to the t{sub 2g} orbitals and the coordinate axes coincident with the direction of the e{sub g} orbitals on the central atom. These data can permit a Frontier Molecular Orbital treatment of specific site activities as long as the surface orbital availability for overlap with adsorbed substrates is considered along with its energy value and symmetry.

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

    SciTech Connect

    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.

  8. Analysis of structural changes in active site of luciferase adsorbed on nanofabricated hydrophilic Si surface by molecular-dynamics simulations

    SciTech Connect

    Nishiyama, Katsuhiko; Hoshino, Tadatsugu

    2007-05-21

    Interactions between luciferase and a nanofabricated hydrophilic Si surface were explored by molecular-dynamics simulations. The structural changes in the active-site residues, the residues affecting the luciferin binding, and the residues affecting the bioluminescence color were smaller on the nanofabricated hydrophilic Si surface than on both a hydrophobic Si surface and a hydrophilic Si surface. The nanofabrication and wet-treatment techniques are expected to prevent the decrease in activity of luciferase on the Si surface.

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

    PubMed

    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

  10. Improving the activity of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation.

    PubMed

    Weng, Meizhi; Deng, Xiongwei; Bao, Wei; Zhu, Li; Wu, Jieyuan; Cai, Yongjun; Jia, Yan; Zheng, Zhongliang; Zou, Guolin

    2015-09-25

    Nattokinase (NK), a bacterial serine protease from Bacillus subtilis var. natto, is a potential cardiovascular drug exhibiting strong fibrinolytic activity. To broaden its commercial and medical applications, we constructed a single-mutant (I31L) and two double-mutants (M222A/I31L and T220S/I31L) by site-directed mutagenesis. Active enzymes were expressed in Escherichia coli with periplasmic secretion and were purified to homogeneity. The kinetic parameters of enzymes were examined by spectroscopy assay and isothermal titration calorimetry (ITC), and their fibrinolytic activities were determined by fibrin plate method. The substitution of Leu(31) for Ile(31) resulted in about 2-fold enhancement of catalytic efficiency (Kcat/KM) compared with wild-type NK. The specific activities of both double-mutants (M222A/I31L and T220S/I31L) were significantly increased when compared with the single-mutants (M222A and T220S) and the oxidative stability of M222A/I31L mutant was enhanced with respect to wild-type NK. This study demonstrates the feasibility of improving activity of NK by site-directed mutagenesis and shows successful protein engineering cases to improve the activity of NK as a potent therapeutic agent. PMID:26291268

  11. Molecular mimicry of substrate oxygen atoms by water molecules in the beta-amylase active site.

    PubMed

    Pujadas, G; Palau, J

    2001-08-01

    Soybean beta-amylase (EC 3.2.1.2) has been crystallized both free and complexed with a variety of ligands. Four water molecules in the free-enzyme catalytic cleft form a multihydrogen-bond network with eight strategic residues involved in enzyme-ligand hydrogen bonds. We show here that the positions of these four water molecules are coincident with the positions of four potential oxygen atoms of the ligands within the complex. Some of these waters are displaced from the active site when the ligands bind to the enzyme. How many are displaced depends on the shape of the ligand. This means that when one of the four positions is not occupied by a ligand oxygen atom, the corresponding water remains. We studied the functional/structural role of these four waters and conclude that their presence means that the conformation of the eight side chains is fixed in all situations (free or complexed enzyme) and preserved from unwanted or forbidden conformational changes that could hamper the catalytic mechanism. The water structure at the active pocket of beta-amylase is therefore essential for providing the ligand recognition process with plasticity. It does not affect the protein active-site geometry and preserves the overall hydrogen-bonding network, irrespective of which ligand is bound to the enzyme. We also investigated whether other enzymes showed a similar role for water. Finally, we discuss the potential use of these results for predicting whether water molecules can mimic ligand atoms in the active center. PMID:11468361

  12. Computational Investigation of Locked Nucleic Acid (LNA) Nucleotides in the Active Sites of DNA Polymerases by Molecular Docking Simulations

    PubMed Central

    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

  13. Molecular protein adaptor with genetically encoded interaction sites guiding the hierarchical assembly of plasmonically active nanoparticle architectures

    NASA Astrophysics Data System (ADS)

    Schreiber, Andreas; Huber, Matthias C.; Cölfen, Helmut; Schiller, Stefan M.

    2015-03-01

    The control over the defined assembly of nano-objects with nm-precision is important to create systems and materials with enhanced properties, for example, metamaterials. In nature, the precise assembly of inorganic nano-objects with unique features, for example, magnetosomes, is accomplished by efficient and reliable recognition schemes involving protein effectors. Here we present a molecular approach using protein-based ‘adaptors/connectors’ with genetically encoded interaction sites to guide the assembly and functionality of different plasmonically active gold nanoparticle architectures (AuNP). The interaction of the defined geometricaly shaped protein adaptors with the AuNP induces the self-assembly of nanoarchitectures ranging from AuNP encapsulation to one-dimensional chain-like structures, complex networks and stars. Synthetic biology and bionanotechnology are applied to co-translationally encode unnatural amino acids as additional site-specific modification sites to generate functionalized biohybrid nanoarchitectures. This protein adaptor-based nano-object assembly approach might be expanded to other inorganic nano-objects creating biohybrid materials with unique electronic, photonic, plasmonic and magnetic properties.

  14. Molecular protein adaptor with genetically encoded interaction sites guiding the hierarchical assembly of plasmonically active nanoparticle architectures.

    PubMed

    Schreiber, Andreas; Huber, Matthias C; Cölfen, Helmut; Schiller, Stefan M

    2015-01-01

    The control over the defined assembly of nano-objects with nm-precision is important to create systems and materials with enhanced properties, for example, metamaterials. In nature, the precise assembly of inorganic nano-objects with unique features, for example, magnetosomes, is accomplished by efficient and reliable recognition schemes involving protein effectors. Here we present a molecular approach using protein-based 'adaptors/connectors' with genetically encoded interaction sites to guide the assembly and functionality of different plasmonically active gold nanoparticle architectures (AuNP). The interaction of the defined geometricaly shaped protein adaptors with the AuNP induces the self-assembly of nanoarchitectures ranging from AuNP encapsulation to one-dimensional chain-like structures, complex networks and stars. Synthetic biology and bionanotechnology are applied to co-translationally encode unnatural amino acids as additional site-specific modification sites to generate functionalized biohybrid nanoarchitectures. This protein adaptor-based nano-object assembly approach might be expanded to other inorganic nano-objects creating biohybrid materials with unique electronic, photonic, plasmonic and magnetic properties. PMID:25813537

  15. Evidence for the Role of Active Site Residues in the Hairpin Ribozyme from Molecular Simulations along the Reaction Path

    PubMed Central

    2015-01-01

    The hairpin ribozyme accelerates a phosphoryl transfer reaction without catalytic participation of divalent metal ions. Residues A38 and G8 have been implicated as playing roles in general acid and base catalysis, respectively. Here we explore the structure and dynamics of key active site residues using more than 1 μs of molecular dynamics simulations of the hairpin ribozyme at different stages along the catalytic pathway. Analysis of results indicates hydrogen bond interactions between the nucleophile and proR nonbridging oxygen are correlated with active inline attack conformations. Further, the simulation results suggest a possible alternative role for G8 to promote inline fitness and facilitate activation of the nucleophile by hydrogen bonding, although this does not necessarily exclude an additional role as a general base. Finally, we suggest that substitution of G8 with N7- or N3-deazaguanosine which have elevated pKa values, both with and without thio modifications at the 5′ leaving group position, would provide valuable insight into the specific role of G8 in catalysis. PMID:24842535

  16. New Insights into Active Site Conformation Dynamics of E. coli PNP Revealed by Combined H/D Exchange Approach and Molecular Dynamics Simulations

    NASA Astrophysics Data System (ADS)

    Kazazić, Saša; Bertoša, Branimir; Luić, Marija; Mikleušević, Goran; Tarnowski, Krzysztof; Dadlez, Michal; Narczyk, Marta; Bzowska, Agnieszka

    2016-01-01

    The biologically active form of purine nucleoside phosphorylase (PNP) from Escherichia coli (EC 2.4.2.1) is a homohexamer unit, assembled as a trimer of dimers. Upon binding of phosphate, neighboring monomers adopt different active site conformations, described as open and closed. To get insight into the functions of the two distinctive active site conformations, virtually inactive Arg24Ala mutant is complexed with phosphate; all active sites are found to be in the open conformation. To understand how the sites of neighboring monomers communicate with each other, we have combined H/D exchange (H/DX) experiments with molecular dynamics (MD) simulations. Both methods point to the mobility of the enzyme, associated with a few flexible regions situated at the surface and within the dimer interface. Although H/DX provides an average extent of deuterium uptake for all six hexamer active sites, it was able to indicate the dynamic mechanism of cross-talk between monomers, allostery. Using this technique, it was found that phosphate binding to the wild type (WT) causes arrest of the molecular motion in backbone fragments that are flexible in a ligand-free state. This was not the case for the Arg24Ala mutant. Upon nucleoside substrate/inhibitor binding, some release of the phosphate-induced arrest is observed for the WT, whereas the opposite effects occur for the Arg24Ala mutant. MD simulations confirmed that phosphate is bound tightly in the closed active sites of the WT; conversely, in the open conformation of the active site of the WT phosphate is bound loosely moving towards the exit of the active site. In Arg24Ala mutant binary complex Pi is bound loosely, too.

  17. A site-renormalized molecular fluid theory

    NASA Astrophysics Data System (ADS)

    Dyer, Kippi M.; Perkyns, John S.; Pettitt, B. Montgomery

    2007-11-01

    The orientation-dependent pair distribution function for molecular fluids on site-site potentials is expanded in a topological analog of the diagrammatically proper site-site theory of liquids [D. Chandler et al., Mol. Phys. 46, 1335 (1982)]. The resulting functions are then used to diagrammatically renormalize the molecular fluid theory. A result is that the diagrammatically proper interaction site model theory is shown to be a linearized, minimal angular basis set approximation to this site-renormalized molecular theory. This framework is used to propose a new, exact, and proper closure to the diagrammatically proper interaction site model theory. The resulting equation system contains a bridge function expansion in the proper site-site theory. In addition, the construction of the theory is such that the molecular pair distribution function, in full dimensionality, is intrinsic to the theory. Furthermore, the theory is equivalent to the molecular Ornstein-Zernike treatment of site-site molecules in the basis set expansion of Blum and Torruella [J. Chem. Phys. 56, 303 (1971)]. A significant formal result of the theory is the demonstration that certain classes of diagrams which would otherwise be considered improper in the interaction site model formalism are included in the angular expansion of molecular interactions. Numerical results for several apolar homonuclear models and an apolar heteronuclear model are shown to quantitatively improve upon those of reference interaction site model and our recent proper variant with respect to simulation. Significant numerical results are that the various thermodynamic quantities obey the exact symmetries and sum rules within numerical error for the different sites in the heteronuclear case, even for the low order approximation used in this work, and the theory is independent of the so-called auxiliary site problem common to previous site-site theories.

  18. Molecular recognition at the active site of subtilisin BPN': crystallographic studies using genetically engineered proteinaceous inhibitor SSI (Streptomyces subtilisin inhibitor).

    PubMed

    Takeuchi, Y; Noguchi, S; Satow, Y; Kojima, S; Kumagai, I; Miura, K; Nakamura, K T; Mitsui, Y

    1991-06-01

    Unlike trypsin-like serine proteases having only one conspicuous binding pocket in the active site, subtilisin BPN' has two such pockets, the S1 and S4 pockets, which accommodate the P1 and P4 residues of ligands (after Schechter and Berger notation) respectively. Using computer graphics, the geometrical nature of the two pockets was carefully examined and strategies for site-directed mutagenesis studies were set up against a protein SSI (Streptomyces subtilisin inhibitor), which is a strong proteinaceous inhibitor (or a substrate analogue) of subtilisin BPN'. It was decided to convert the P1 residue, methionine 73, into lysine (M73K) with or without additional conversion of the P4 residue, methionine 70, into glycine (M70G). The crystal structures of the two complexes of subtilisin BPN', one with the single mutant SSI (M73K) and the other with the double mutant SSI (M73K, M70G) were solved showing that (i) small 'electrostatic induced-fit movement' occurs in the S1 pocket upon introducing the terminal plus charge of the lysine side chain, and (ii) large 'mechanical induced-fit movement' occurs in the S4 pocket upon reducing the size of the P4 side chain from methionine to glycine. In both (i) and (ii), the induced-fit movement occurred in a concerted fashion involving both the enzyme and 'substrate' amino acid residues. The term 'substrate-assisted stabilization' was coined to stress the cooperative nature of the induced-fit movements. PMID:1891457

  19. Probing the importance of hydrogen bonds in the active site of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation.

    PubMed

    Zheng, Zhong-liang; Ye, Mao-qing; Zuo, Zhen-yu; Liu, Zhi-gang; Tai, Keng-chang; Zou, Guo-lin

    2006-05-01

    Hydrogen bonds occurring in the catalytic triad (Asp32, His64 and Ser221) and the oxyanion hole (Asn155) are very important to the catalysis of peptide bond hydrolysis by serine proteases. For the subtilisin NK (nattokinase), a bacterial serine protease, construction and analysis of a three-dimensional structural model suggested that several hydrogen bonds formed by four residues function to stabilize the transition state of the hydrolysis reaction. These four residues are Ser33, Asp60, Ser62 and Thr220. In order to remove the effect of these hydrogen bonds, four mutants (Ser33-->Ala33, Asp60-->Ala60, Ser62-->Ala62, and Thr220-->Ala220) were constructed by site-directed mutagenesis. The results of enzyme kinetics indicated that removal of these hydrogen bonds increases the free-energy of the transition state (DeltaDeltaG(T)). We concluded that these hydrogen bonds are more important for catalysis than for binding the substrate, because removal of these bonds mainly affects the kcat but not the K(m) values. A substrate, SUB1 (succinyl-Ala-Ala-Pro-Phe-p-nitroanilide), was used during enzyme kinetics experiments. In the present study we have also shown the results of FEP (free-energy perturbation) calculations with regard to the binding and catalysis reactions for these mutant subtilisins. The calculated difference in FEP also suggested that these four residues are more important for catalysis than binding of the substrate, and the simulated values compared well with the experimental values from enzyme kinetics. The results of MD (molecular dynamics) simulations further demonstrated that removal of these hydrogen bonds partially releases Asp32, His64 and Asn155 so that the stability of the transition state decreases. Another substrate, SUB2 (H-D-Val-Leu-Lys-p-nitroanilide), was used for FEP calculations and MD simulations. PMID:16411898

  20. Probing the importance of hydrogen bonds in the active site of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation

    PubMed Central

    Zheng, Zhong-liang; Ye, Mao-qing; Zuo, Zhen-yu; Liu, Zhi-gang; Tai, Keng-chang; Zou, Guo-lin

    2006-01-01

    Hydrogen bonds occurring in the catalytic triad (Asp32, His64 and Ser221) and the oxyanion hole (Asn155) are very important to the catalysis of peptide bond hydrolysis by serine proteases. For the subtilisin NK (nattokinase), a bacterial serine protease, construction and analysis of a three-dimensional structural model suggested that several hydrogen bonds formed by four residues function to stabilize the transition state of the hydrolysis reaction. These four residues are Ser33, Asp60, Ser62 and Thr220. In order to remove the effect of these hydrogen bonds, four mutants (Ser33→Ala33, Asp60→Ala60, Ser62→Ala62, and Thr220→Ala220) were constructed by site-directed mutagenesis. The results of enzyme kinetics indicated that removal of these hydrogen bonds increases the free-energy of the transition state (ΔΔGT). We concluded that these hydrogen bonds are more important for catalysis than for binding the substrate, because removal of these bonds mainly affects the kcat but not the Km values. A substrate, SUB1 (succinyl-Ala-Ala-Pro-Phe-p-nitroanilide), was used during enzyme kinetics experiments. In the present study we have also shown the results of FEP (free-energy perturbation) calculations with regard to the binding and catalysis reactions for these mutant subtilisins. The calculated difference in FEP also suggested that these four residues are more important for catalysis than binding of the substrate, and the simulated values compared well with the experimental values from enzyme kinetics. The results of MD (molecular dynamics) simulations further demonstrated that removal of these hydrogen bonds partially releases Asp32, His64 and Asn155 so that the stability of the transition state decreases. Another substrate, SUB2 (H-D-Val-Leu-Lys-p-nitroanilide), was used for FEP calculations and MD simulations. PMID:16411898

  1. Protonation states of the key active site residues and structural dynamics of glmS riboswitch as reveled by molecular dynamics

    PubMed Central

    Banáš, Pavel; Walter, Nils G.

    2010-01-01

    The glmS catalytic riboswitch is part of the 5'-untranslated region of mRNAs encoding glucosamine-6-phosphate (GlcN6P) synthetase (glmS) in numerous Gram-positive bacteria. Binding of the cofactor GlcN6P induces site-specific self-cleavage of the RNA. However, detailed reaction mechanism as well as protonation state of glmS reactive form remains still elusive. To probe the dominant protonation states of key active site residues, we carried out explicit solvent molecular dynamic simulations involving various protonation states of three crucial active site moieties observed in the available crystal structures: (i) guanine G40 (following the T. tengcongensis numbering), (ii) the GlcN6P amino/ammonium group, and (iii) the GlcN6P phosphate moiety. We found that a deprotonated G40− seems incompatible with the observed glmS active site architecture. Our data suggest that the canonical form of G40 plays a structural role by stabilizing an in-line attack conformation of the cleavage site A-1(2'-OH) nucleophile, rather than a more direct chemical role. In addition, we observe weakened cofactor binding upon protonation of the GlcN6P phosphate moiety, which explains the experimentally observed increase of Km with decreasing pH. Finally, we discuss a possible role of cofactor binding and its interaction with the G65 and G1 purines in structural stabilization of the A-1(2'-OH) in-line attack conformation. Based on the identified dominant protonation state of the reaction precursor, we propose a hypothesis of self-cleavage mechanism, in which A-1(2'-OH) is activated as nucleophile by the G1(pro-Rp) non-bridging oxygen of the scissile phosphate, whereas the ammonium group of GlcN6P acts as the general acid protonating the G1(O5') leaving group. PMID:20536206

  2. ER–endosome contact sites: molecular compositions and functions

    PubMed Central

    Raiborg, Camilla; Wenzel, Eva M; Stenmark, Harald

    2015-01-01

    Recent studies have revealed the existence of numerous contact sites between the endoplasmic reticulum (ER) and endosomes in mammalian cells. Such contacts increase during endosome maturation and play key roles in cholesterol transfer, endosome positioning, receptor dephosphorylation, and endosome fission. At least 7 distinct contact sites between the ER and endosomes have been identified to date, which have diverse molecular compositions. Common to these contact sites is that they impose a close apposition between the ER and endosome membranes, which excludes membrane fusion while allowing the flow of molecular signals between the two membranes, in the form of enzymatic modifications, or ion, lipid, or protein transfer. Thus, ER–endosome contact sites ensure coordination of molecular activities between the two compartments while keeping their general compositions intact. Here, we review the molecular architectures and cellular functions of known ER–endosome contact sites and discuss their implications for human health. PMID:26041457

  3. Physical nature of intermolecular interactions inside Sir2 homolog active site: molecular dynamics and ab initio study.

    PubMed

    Czeleń, Przemysław; Czyżnikowska, Żaneta

    2016-06-01

    In the present study, we analyze the interactions of NAD+-dependent deacetylase (Sir2 homolog yeast Hst2) with carba-nicotinamide-adenine-dinucleotide (ADP-HPD). For the Sir2 homolog, a yeast Hst2 docking procedure was applied. The structure of the protein-ADP-HPD complex obtained during the docking procedure was used as a starting point for molecular dynamics simulation. The intermolecular interaction energy partitioning was performed for protein-ADP-HPD complex resulting from molecular dynamics simulation. The analysis was performed for ADP-HPD and 15 amino acids forming a deacetylase binding pocket. Although the results indicate that the first-order electrostatic interaction energy is substantial, the presence of multiple hydrogen bonds in investigated complexes can lead to significant value of induction component. PMID:27154340

  4. An Insight into the Environmental Effects of the Pocket of the Active Site of the Enzyme. Ab initio ONIOM-Molecular Dynamics (MD) Study on Cytosine Deaminase

    SciTech Connect

    Matsubara, Toshiaki; Dupuis, Michel; Aida, Misako

    2008-02-01

    We applied the ONIOM-molecular dynamics (MD) method to cytosine deaminase to examine the environmental effects of the amino acid residues in the pocket of the active site on the substrate taking account of their thermal motion. The ab initio ONIOM-MD simulations show that the substrate uracil is strongly perturbed by the amino acid residue Ile33, which sandwiches the uracil with His62, through the steric contact due to the thermal motion. As a result, the magnitude of the thermal oscillation of the potential energy and structure of the substrate uracil significantly increases. TM and MA were partly supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan.MD was supported by the Division of Chemical Sciences, Office of Basic Energy Sciences, and by the Office of Biological and Environmental Research of the U.S. Department of Energy DOE. Battelle operates Pacific Northwest National Laboratory for DOE.

  5. Molecular Dynamics Study of Twister Ribozyme: Role of Mg(2+) Ions and the Hydrogen-Bonding Network in the Active Site.

    PubMed

    Ucisik, Melek N; Bevilacqua, Philip C; Hammes-Schiffer, Sharon

    2016-07-12

    The recently discovered twister ribozyme is thought to utilize general acid-base catalysis in its self-cleavage mechanism, but the roles of nucleobases and metal ions in the mechanism are unclear. Herein, molecular dynamics simulations of the env22 twister ribozyme are performed to elucidate the structural and equilibrium dynamical properties, as well as to examine the role of Mg(2+) ions and possible candidates for the general base and acid in the self-cleavage mechanism. The active site region and the ends of the pseudoknots were found to be less mobile than other regions of the ribozyme, most likely providing structural stability and possibly facilitating catalysis. A purported catalytic Mg(2+) ion and the closest neighboring Mg(2+) ion remained chelated and relatively immobile throughout the microsecond trajectories, although removal of these Mg(2+) ions did not lead to any significant changes in the structure or equilibrium motions of the ribozyme on the microsecond time scale. In addition, a third metal ion, a Na(+) ion remained close to A1(O5'), the leaving group atom, during the majority of the microsecond trajectories, suggesting that it might stabilize the negative charge on A1(O5') during self-cleavage. The locations of these cations and their interactions with key nucleotides in the active site suggest that they may be catalytically relevant. The P1 stem is partially melted at its top and bottom in the crystal structure and further unwinds in the trajectories. The simulations also revealed an interconnected network comprised of hydrogen-bonding and π-stacking interactions that create a relatively rigid network around the self-cleavage site. The nucleotides involved in this network are among the highly conserved nucleotides in twister ribozymes, suggesting that this interaction network may be important to structure and function. PMID:27295275

  6. Molecular modeling benzo[a]pyrene N2-dG adducts in the two overlapping active sites of the Y-family DNA polymerase Dpo4.

    PubMed

    Chandani, Sushil; Loechler, Edward L

    2007-01-01

    The potent, ubiquitous environmental mutagen/carcinogen benzo[a]pyrene (B[a]P) induces a single major adduct [+ta]-B[a]P-N2-dG, whose bypass in most cases results in either no mutation (dCTP insertion) or a G-->T mutation (dATP insertion). Translesion synthesis (TLS) of [+ta]-B[a]P-N2-dG generally requires DNA polymerases (DNAPs) in the Y-family, which exist in cells to bypass DNA damage caused by chemicals and radiation. A molecular dynamics (MD) study is described with dCTP opposite [+ta]-B[a]P-N2-dG in Dpo4, which is the best studied Y-family DNAP from a structural point of view. Two orientations of B[a]P-N2-dG (BPmi5 and BPmi3) are considered, along with two orientations of the dCTP (AS1 and AS2), as outlined next. Based on NMR studies, the pyrene moiety of B[a]P-N2-dG is in the minor groove, when paired with dC, and can point toward either the base on the 5'-side (BPmi5) or the 3'-side (BPmi3). Based on published X-ray structures, Dpo4 appears to have two partially overlapping active sites. The architecture of active site 1 (AS1) is similar to all other families of DNAPs (e.g., the shape of the dNTP). Active site 2 (AS2), however, is non-canonical (e.g., the beta- and gamma-phosphates in AS2 are approximately where the alpha- and beta-phosphates are in AS1). In the Dpo4 models generated herein, using the BPmi3 orientation the pyrene moiety of [+ta]-B[a]P-N2-dG points toward the duplex region of the DNA, and is accommodated without distortions in AS1, but with distortions in AS2. Considering the BPmi5 orientation, the pyrene moiety points toward the ss-region of DNA in Dpo4, and sits in a hole defined by the fingers and little fingers domain ("chimney"); BPmi5 is accommodated in AS2 without significant distortions, but poorly in AS1. In summary, when dCTP is paired with [+ta]-B[a]P-N2-dG in the two overlapping active sites in Dpo4, the pyrene in the BPmi3 orientation is accommodated better in active site 1 (AS1), while the pyrene in the BPmi5 orientation is

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

    SciTech Connect

    Matak, Mehdi Youssefi; Moghaddam, Majid Erfani

    2009-12-11

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

  8. Molecular dynamics simulations of apo, holo, and inactivator bound GABA-at reveal the role of active site residues in PLP dependent enzymes.

    PubMed

    Gökcan, Hatice; Monard, Gerald; Sungur Konuklar, F Aylin

    2016-07-01

    The pyridoxal 5-phosphate (PLP) cofactor is a significant organic molecule in medicinal chemistry. It is often found covalently bound to lysine residues in proteins to form PLP dependent enzymes. An example of this family of PLP dependent enzymes is γ-aminobutyric acid aminotransferase (GABA-AT) which is responsible for the degradation of the neurotransmitter GABA. Its inhibition or inactivation can be used to prevent the reduction of GABA concentration in brain which is the source of several neurological disorders. As a test case for PLP dependent enzymes, we have performed molecular dynamics simulations of GABA-AT to reveal the roles of the protein residues and its cofactor. Three different states have been considered: the apoenzyme, the holoenzyme, and the inactive state obtained after the suicide inhibition by vigabatrin. Different protonation states have also been considered for PLP and two key active site residues: Asp298 and His190. Together, 24 independent molecular dynamics trajectories have been simulated for a cumulative total of 2.88 µs. Our results indicate that, unlike in aqueous solution, the PLP pyridine moiety is protonated in GABA-AT. This is a consequence of a pKa shift triggered by a strong charge-charge interaction with an ionic "diad" formed by Asp298 and His190 that would help the activation of the first half-reaction of the catalytic mechanism in GABA-AT: the conversion of PLP to free pyridoxamine phosphate (PMP). In addition, our MD simulations exhibit additional strong hydrogen bond networks between the protein and PLP: the phosphate group is held in place by the donation of at least three hydrogen bonds while the carbonyl oxygen of the pyridine ring interacts with Gln301; Phe181 forms a π-π stacking interaction with the pyridine ring and works as a gate keeper with the assistance of Val300. All these interactions are hypothesized to help maintain free PMP in place inside the protein active site to facilitate the second half

  9. Probing the Effect of the Non-Active-Site Mutation Y229W in New Delhi Metallo-β-lactamase-1 by Site-Directed Mutagenesis, Kinetic Studies, and Molecular Dynamics Simulations

    PubMed Central

    Shi, Yun; Hu, Feng; Lao, Xingzhen; Gao, Xiangdong; Zheng, Heng; Yao, Wenbing

    2013-01-01

    New Delhi metallo-β-lactmase-1 (NDM-1) has attracted extensive attention for its high catalytic activities of hydrolyzing almost all β-lactam antibiotics. NDM-1 shows relatively higher similarity to subclass B1 metallo-β-lactmases (MβLs), but its residue at position 229 is identical to that of B2/B3 MβLs, which is a Tyr instead of a B1-MβL-conserved Trp. To elucidate the possible role of Y229 in the bioactivity of NDM-1, we performed mutagenesis study and molecular dynamics (MD) simulations. Although residue Y229 is spatially distant from the active site and not contacting directly with the substrate or zinc ions, the Y229W mutant was found to have higher kcat and Km values than those of wild-type NDM-1, resulting in 1∼7 fold increases in kcat/Km values against tested antibiotics. In addition, our MD simulations illustrated the enhanced flexibility of Loop 2 upon Y229W mutation, which could increase the kinetics of both substrate entrance (kon) and product egress (koff). The enhanced flexibility of Loop 2 might allow the enzyme to adjust the geometry of its active site to accommodate substrates with different structures, broadening its substrate spectrum. This study indicated the possible role of the residue at position 229 in the evolution of NDM-1. PMID:24339993

  10. Molecular design of substrate binding sites

    SciTech Connect

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

    1991-12-31

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

  11. Molecular design of substrate binding sites

    SciTech Connect

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

    1991-01-01

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

  12. Identification of protein functions from a molecular surface database, eF-site.

    PubMed

    Kinoshita, Kengo; Furui, Jun'ichi; Nakamura, Haruki

    2002-01-01

    A bioinformatics method was developed to identify the protein surface around the functional site and to estimate the biochemical function, using a newly constructed molecular surface database named the eF-site (electrostatic surface of Functional site. Molecular surfaces of protein molecules were computed based on the atom coordinates, and the eF-site database was prepared by adding the physical properties on the constructed molecular surfaces. The electrostatic potential on each molecular surface was individually calculated solving the Poisson-Boltzmann equation numerically for the precise continuum model, and the hydrophobicity information of each residue was also included. The eF-site database is accessed by the internet (http://pi.protein.osaka-u.ac.jp/eF-site/). We have prepared four different databases, eF-site/antibody, eF-site/prosite, eF-site/P-site, and eF-site/ActiveSite, corresponding to the antigen binding sites of antibodies with the same orientations, the molecular surfaces for the individual motifs in PROSITE database, the phosphate binding sites, and the active site surfaces for the representatives of the individual protein family, respectively. An algorithm using the clique detection method as an applied graph theory was developed to search of the eF-site database, so as to recognize and discriminate the characteristic molecular surfaces of the proteins. The method identifies the active site having the similar function to those of the known proteins. PMID:12836670

  13. Reversible Switching of Redox-Active Molecular Orbitals and Electron Transfer Pathways in Cu(A) Sites of Cytochrome c Oxidase.

    PubMed

    Zitare, Ulises; Alvarez-Paggi, Damián; Morgada, Marcos N; Abriata, Luciano A; Vila, Alejandro J; Murgida, Daniel H

    2015-08-10

    The Cu(A) site of cytochrome c oxidase is a redox hub that participates in rapid electron transfer at low driving forces with two redox cofactors in nearly perpendicular orientations. Spectroscopic and electrochemical characterizations performed on first and second-sphere mutants have allowed us to experimentally detect the reversible switching between two alternative electronic states that confer different directionalities to the redox reaction. Specifically, the M160H variant of a native Cu(A) shows a reversible pH transition that allows to functionally probe both states in the same protein species. Alternation between states exerts a dramatic impact on the kinetic redox parameters, thereby suggesting this effect as the mechanism underlying the efficiency and directionality of Cu(A) electron transfer in vivo. These findings may also prove useful for the development of molecular electronics. PMID:26118421

  14. Identification of essential residues for binding and activation in the human 5-HT7(a) serotonin receptor by molecular modeling and site-directed mutagenesis.

    PubMed

    Impellizzeri, Agata Antonina Rita; Pappalardo, Matteo; Basile, Livia; Manfra, Ornella; Andressen, Kjetil Wessel; Krobert, Kurt Allen; Messina, Angela; Levy, Finn Olav; Guccione, Salvatore

    2015-01-01

    The human 5-HT7 receptor is expressed in both the central nervous system and peripheral tissues and is a potential drug target in behavioral and psychiatric disorders. We examined molecular determinants of ligand binding and G protein activation by the human 5-HT7(a) receptor. The role of several key residues in the 7th transmembrane domain (TMD) and helix 8 were elucidated combining in silico and experimental mutagenesis. Several single and two double point mutations of the 5-HT7(a) wild type receptor were made (W7.33V, E7.35T, E7.35R, E7.35D, E7.35A, R7.36V, Y7.43A, Y7.43F, Y7.43T, R8.52D, D8.53K; E7.35T-R7.36V, R8.52D-D8.53K), and their effects upon ligand binding were assessed by radioligand binding using a potent agonist (5-CT) and a potent antagonist (SB269970). In addition, the ability of the mutated 5-HT7(a) receptors to activate G protein after 5-HT-stimulation was determined through activation of adenylyl cyclase. In silico investigation on mutated receptors substantiated the predicted importance of TM7 and showed critical roles of residues E7.35, W7.33, R7.36 and Y7.43 in agonist and antagonist binding and conformational changes of receptor structure affecting adenylyl cyclase activation. Experimental data showed that mutants E7.35T and E7.35R were incapable of ligand binding and adenylyl cyclase activation, consistent with a requirement for a negatively charged residue at this position. The mutant R8.52D was unable to activate adenylyl cyclase, despite unaffected ligand binding, consistent with the R8.52 residue playing an important role in the receptor-G protein interface. The mutants Y7.43A and Y7.43T displayed reduced agonist binding and AC agonist potency, not seen in Y7.43F, consistent with a requirement for an aromatic residue at this position. Knowledge of the molecular interactions important in h5-HT7 receptor ligand binding and G protein activation will aid the design of selective h5-HT7 receptor ligands for potential pharmacological use. PMID

  15. Identification of essential residues for binding and activation in the human 5-HT7(a) serotonin receptor by molecular modeling and site-directed mutagenesis

    PubMed Central

    Impellizzeri, Agata Antonina Rita; Pappalardo, Matteo; Basile, Livia; Manfra, Ornella; Andressen, Kjetil Wessel; Krobert, Kurt Allen; Messina, Angela; Levy, Finn Olav; Guccione, Salvatore

    2015-01-01

    The human 5-HT7 receptor is expressed in both the central nervous system and peripheral tissues and is a potential drug target in behavioral and psychiatric disorders. We examined molecular determinants of ligand binding and G protein activation by the human 5-HT7(a) receptor. The role of several key residues in the 7th transmembrane domain (TMD) and helix 8 were elucidated combining in silico and experimental mutagenesis. Several single and two double point mutations of the 5-HT7(a) wild type receptor were made (W7.33V, E7.35T, E7.35R, E7.35D, E7.35A, R7.36V, Y7.43A, Y7.43F, Y7.43T, R8.52D, D8.53K; E7.35T-R7.36V, R8.52D-D8.53K), and their effects upon ligand binding were assessed by radioligand binding using a potent agonist (5-CT) and a potent antagonist (SB269970). In addition, the ability of the mutated 5-HT7(a) receptors to activate G protein after 5-HT-stimulation was determined through activation of adenylyl cyclase. In silico investigation on mutated receptors substantiated the predicted importance of TM7 and showed critical roles of residues E7.35, W7.33, R7.36 and Y7.43 in agonist and antagonist binding and conformational changes of receptor structure affecting adenylyl cyclase activation. Experimental data showed that mutants E7.35T and E7.35R were incapable of ligand binding and adenylyl cyclase activation, consistent with a requirement for a negatively charged residue at this position. The mutant R8.52D was unable to activate adenylyl cyclase, despite unaffected ligand binding, consistent with the R8.52 residue playing an important role in the receptor-G protein interface. The mutants Y7.43A and Y7.43T displayed reduced agonist binding and AC agonist potency, not seen in Y7.43F, consistent with a requirement for an aromatic residue at this position. Knowledge of the molecular interactions important in h5-HT7 receptor ligand binding and G protein activation will aid the design of selective h5-HT7 receptor ligands for potential pharmacological use. PMID

  16. Salt site performance assessment activities

    SciTech Connect

    Kircher, J.F.; Gupta, S.K.

    1983-01-01

    During this year the first selection of the tools (codes) for performance assessments of potential salt sites have been tentatively selected and documented; the emphasis has shifted from code development to applications. During this period prior to detailed characterization of a salt site, the focus is on bounding calculations, sensitivity and with the data available. The development and application of improved methods for sensitivity and uncertainty analysis is a focus for the coming years activities and the subject of a following paper in these proceedings. Although the assessments to date are preliminary and based on admittedly scant data, the results indicate that suitable salt sites can be identified and repository subsystems designed which will meet the established criteria for protecting the health and safety of the public. 36 references, 5 figures, 2 tables.

  17. Molecular mechanisms of microglial activation.

    PubMed

    Zielasek, J; Hartung, H P

    1996-01-01

    Microglial cells are brain macrophages which serve specific functions in the defense of the central nervous system (CNS) against microorganisms, the removal of tissue debris in neurodegenerative diseases or during normal development, and in autoimmune inflammatory disorders of the brain. In cultured microglial cells, several soluble inflammatory mediators such as cytokines and bacterial products like lipopolysaccharide (LPS) were demonstrated to induce a wide range of microglial activities, e.g. increased phagocytosis, chemotaxis, secretion of cytokines, activation of the respiratory burst and induction of nitric oxide synthase. Since heightened microglial activation was shown to play a role in the pathogenesis of experimental inflammatory CNS disorders, understanding the molecular mechanisms of microglial activation may lead to new treatment strategies for neurodegenerative disorders, multiple sclerosis and bacterial or viral infections of the nervous system. PMID:8876774

  18. Molecular characteristics versus biological activity

    USGS Publications Warehouse

    Applegate, Vernon C.; Smith, Manning A.; Willeford, Bennett R.

    1967-01-01

    The molecular characteristics of mononitrophenols containing halogens not only play a key role in their biological activity but provide a novel example of selective toxicity among vertebrate animals. It has been reported that efforts to control the parasitic sea lamprey in the Great Lakes are directed at present to the applications of a selective toxicant to streams inhabited by lamprey larvae. Since 1961, the larvicide that has been used almost exclusively in the control program has been 3-trifluoromethyl-4-nitrophenol (TFM). However, this is only one of about 15 closely related compounds, all halogen-containing mononitrophenols, that display a selectively toxic action upon lampreys. Although not all of the halogenated mononitrophenols are selectively toxic to lampreys (in fact, fewer than half of those tested), no other group of related compounds has displayed any useful larvicidal activity except for the substituted nitrosalicylanilides.

  19. High-Molecular-Weight Protein (pUL48) of Human Cytomegalovirus Is a Competent Deubiquitinating Protease: Mutant Viruses Altered in Its Active-Site Cysteine or Histidine Are Viable†

    PubMed Central

    Wang, Jianlei; Loveland, Amy N.; Kattenhorn, Lisa M.; Ploegh, Hidde L.; Gibson, Wade

    2006-01-01

    We show here that the high-molecular-weight protein (HMWP or pUL48; 253 kDa) of human cytomegalovirus (HCMV) is a functionally competent deubiquitinating protease (DUB). By using a suicide substrate probe specific for ubiquitin-binding cysteine proteases (DUB probe) to screen lysates of HCMV-infected cells, we found just one infected-cell-specific DUB. Characteristics of this protein, including its large size, expression at late times of infection, presence in extracellular virus particles, and reactivity with an antiserum to the HMWP, identified it as the HMWP. This was confirmed by constructing mutant viruses with substitutions in two of the putative active-site residues, Cys24Ile and His162Ala. HMWP with these mutations either failed to bind the DUB probe (C24I) or had significantly reduced reactivity with it (H162A). More compellingly, the deubiquitinating activity detected in wild-type virus particles was completely abolished in both the C24I and H162A mutants, thereby directly linking HMWP with deubiquitinating enzyme activity. Mutations in these active-site residues were not lethal to virus replication but slowed production of infectious virus relative to wild type and mutations of other conserved residues. Initial studies, by electron microscopy, of cells infected with the mutants revealed no obvious differences at late times of replication in the general appearance of the cells or in the distribution, relative numbers, or appearance of virus particles in the cytoplasm or nucleus. PMID:16731939

  20. Site-directed deep electronic tunneling through a molecular network

    SciTech Connect

    Caspary, Maytal; Peskin, Uri

    2005-10-15

    Electronic tunneling in a complex molecular network of N(>2) donor/acceptor sites, connected by molecular bridges, is analyzed. The 'deep' tunneling dynamics is formulated using a recursive perturbation expansion, yielding a McConnell-type reduced N-level model Hamiltonian. Applications to models of molecular junctions demonstrate that the donor-bridge contact parameters can be tuned in order to control the tunneling dynamics and particularly to direct the tunneling pathway to either one of the various acceptors.

  1. Molecular anatomy of the antibody binding site.

    PubMed

    Novotný, J; Bruccoleri, R; Newell, J; Murphy, D; Haber, E; Karplus, M

    1983-12-10

    The binding region of immunoglobulins, which includes the portion of the molecule having the most variability in its amino acid sequence, is shown to have a surprisingly constant structure that can be characterized in terms of a simple, well-defined model. The binding region is composed of the antigen combining site plus its immediate vicinity and arises by noncovalent association of the light and heavy chain variable domains (VL and VH, respectively). The antigen combining site itself consists of six polypeptide chain segments ("hypervariable loops") which comprise some 80 amino acid residues and are attached to a framework of VL and VH beta-sheet bilayers. Having analyzed refined x-ray crystallographic coordinates for three antigen-binding fragments (Fab KOL (Marquart, M., Deisenhofer, J., and Huber, R. (1980) J. Mol. Biol. 141, 369-391), MCPC 603 (Segal, D., Padlan, E. A., Cohen, G. H., Rudikoff, S., Potter, M., and Davies, D. R. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 4298-4302), and NEW (Saul, F. A., Amzel, L. M., and Poljak, R. J. (1978) J. Biol. Chem. 253, 585-597] we use the results to introduce a general model for the VL-VH interface forming the binding region. The region consists of two closely packed beta-sheets, and its geometry corresponds to a 9-stranded, cylindrical barrel of average radius 0.84 nm with an average angle of -53 degrees between its two constituent beta-sheets. The barrel forms the bottom and sides of the antigen combining site. The model demonstrates that the structural variability of the binding region is considerably less than was thought previously. Amino acid residues which are part of the domain-domain interface and appear not to be accessible to solvent or antigen contribute to antibody specificity. PMID:6643494

  2. Amino acid sequence and molecular structure of an alkaline amylopullulanase from Bacillus that hydrolyzes alpha-1,4 and alpha-1,6 linkages in polysaccharides at different active sites.

    PubMed

    Hatada, Y; Igarashi, K; Ozaki, K; Ara, K; Hitomi, J; Kobayashi, T; Kawai, S; Watabe, T; Ito, S

    1996-09-27

    An amylopullulanase from alkalophilic Bacillus sp. KSM-1378 hydrolyzes both alpha-1,6 linkages in pullulan and alpha-1,4 linkages in other polysaccharides, with maximum activity in each case at an alkaline pH, to generate oligosaccharides (Ara, K., Saeki, K., Igarashi, K., Takaiwa, M., Uemura, T., Hagihara, H., Kawai, S., and Ito, S. (1995) Biochim. Biophys. Acta 1243, 315-324). Here, we report the molecular cloning and sequencing of the gene for and the structure of this enzyme and show that its dual hydrolytic activities are associated with two independent active sites. The structural gene contained a single, long open reading frame of 5,814 base pairs, corresponding to 1,938 amino acids that included a signal peptide of 32 amino acids. The molecular mass of the extracellular mature enzyme (Glu33 through Leu1938) was calculated to be 211,450 Da, a value close to the 210 kDa determined for the amylopullulanase produced by Bacillus sp. KSM-1378. The amylase and the pullulanase domains were located in the amino-terminal half and in the carboxyl-terminal half of the enzyme, respectively, being separated by a tandem repeat of a sequence of 35 amino acids. Four regions, designated I, II, III, and IV, were highly conserved in each catalytic domain, and they included a putative catalytic triad Asp550-Glu579-Asp645 for the amylase activity and Asp1464-Glu1493-Asp1581 for the pullulanase activity. The purified enzyme was rotary shadowed at a low angle and observed by transmission electron microscopy; it appeared to be a "castanet-like" or "bent dumbbell-like" molecule with a diameter of approximately 25 nm. PMID:8798645

  3. Energy transfer at the active sites of heme proteins

    SciTech Connect

    Dlott, D.D.; Hill, J.R.

    1995-12-31

    Experiments using a picosecond pump-probe apparatus at the Picosecond Free-electron Laser Center at Stanford University, were performed to investigate the relaxation of carbon monoxide bound to the active sites of heme proteins. The significance of these experiments is two-fold: (1) they provide detailed information about molecular dynamics occurring at the active sites of proteins; and (2) they provide insight into the nature of vibrational relaxation processes in condensed matter. Molecular engineering is used to construct various molecular systems which are studied with the FEL. We have studied native proteins, mainly myoglobin obtained from different species, mutant proteins produced by genetic engineering using recombinant DNA techniques, and a variety of model systems which mimic the structures of the active sites of native proteins, which are produced using molecular synthesis. Use of these different systems permits us to investigate how specific molecular structural changes affect dynamical processes occurring at the active sites. This research provides insight into the problems of how different species needs are fulfilled by heme proteins which have greatly different functionality, which is induced by rather small structural changes.

  4. Role of the Active Site Guanine in the glmS Ribozyme Self-Cleavage Mechanism: Quantum Mechanical/Molecular Mechanical Free Energy Simulations

    PubMed Central

    2015-01-01

    The glmS ribozyme catalyzes a self-cleavage reaction at the phosphodiester bond between residues A-1 and G1. This reaction is thought to occur by an acid–base mechanism involving the glucosamine-6-phosphate cofactor and G40 residue. Herein quantum mechanical/molecular mechanical free energy simulations and pKa calculations, as well as experimental measurements of the rate constant for self-cleavage, are utilized to elucidate the mechanism, particularly the role of G40. Our calculations suggest that an external base deprotonates either G40(N1) or possibly A-1(O2′), which would be followed by proton transfer from G40(N1) to A-1(O2′). After this initial deprotonation, A-1(O2′) starts attacking the phosphate as a hydroxyl group, which is hydrogen-bonded to deprotonated G40, concurrent with G40(N1) moving closer to the hydroxyl group and directing the in-line attack. Proton transfer from A-1(O2′) to G40 is concomitant with attack of the scissile phosphate, followed by the remainder of the cleavage reaction. A mechanism in which an external base does not participate, but rather the proton transfers from A-1(O2′) to a nonbridging oxygen during nucleophilic attack, was also considered but deemed to be less likely due to its higher effective free energy barrier. The calculated rate constant for the favored mechanism is in agreement with the experimental rate constant measured at biological Mg2+ ion concentration. According to these calculations, catalysis is optimal when G40 has an elevated pKa rather than a pKa shifted toward neutrality, although a balance among the pKa’s of A-1, G40, and the nonbridging oxygen is essential. These results have general implications, as the hammerhead, hairpin, and twister ribozymes have guanines at a similar position as G40. PMID:25526516

  5. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

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

    1990-10-01

    DOE Order 5820.2A requires that low-level waste (LLW) disposal sites active on or after September 1988 and all transuranic (TRU) waste storage sites be monitored periodically to assure that radioactive contamination does not escape from the waste sites and pose a threat to the public or to the environment. This plan describes such a monitoring program for the active LLW disposal sites in SWSA 6 and the TRU waste storage sites in SWSA 5 North. 14 refs., 8 figs.

  6. MOLECULAR OPTICAL SPECTROSCOPIC TECHNIQUES FOR HAZARDOUS WASTE SITE SCREENING

    EPA Science Inventory

    The U.S. Environmental Protection Agency is interested in field screening hazardous waste sites for contaminants in the soil and surface and ground water. his study is an initial technical overview of the principal molecular spectroscopic techniques and instrumentation currently ...

  7. Paramagnetic 1H and 13C NMR studies on cobalt-substituted human carbonic anhydrase I carboxymethylated at active site histidine-200: molecular basis for the changes in catalytic properties induced by the modification.

    PubMed

    Khalifah, R G; Rogers, J I; Harmon, P; Morely, P J; Carroll, S B

    1984-07-01

    Using bromo[1-13C]acetate to modify N tau of His-200 of human carbonic anhydrase isozyme I leads to the introduction of a useful 13C NMR probe into the active site. To complement our previous diamagnetic NMR studies with this probe, we have now succeeded in directly observing the paramagnetically perturbed resonance of the carboxylate in the cobalt-substituted modified enzyme above pH 8. In the pH range 8-10, the resonance undergoes a pH-dependent slow-exchange process, with the more alkaline form having a much smaller pseudocontact shift and a narrower line width. Below pH 8, the resonance apparently undergoes a very large paramagnetic downfield shift that was estimated by extrapolation. An ionization of approximate pK of 6 appears to control this process. Paramagnetic spin-relaxation studies on the resonance under conditions where it was directly observed yielded distance measurements between the carboxylate carbon and the active site cobalt ion. In inhibitor complexes, this distance was in the range of 5-7 A. In the absence of inhibitors, the distance was approximately 3.0-3.2 A at pH 7.9, consistent with the coordination of the carboxylate to the metal. However, at pH 10, the distance was increased to 4.8 A. These distance determinations were aided by relaxation measurements of a paramagnetically shifted proton resonance at 60-65 ppm downfield assigned by others to a proton of a ligand histidine of metal and confirmed by us to be 5.2 +/- 0.1 A from the metal. Our findings provide a molecular basis for the observed changes in catalytic properties that accompany the carboxymethylation. PMID:6432037

  8. Detection of Binding Site Molecular Interaction Field Similarities.

    PubMed

    Chartier, Matthieu; Najmanovich, Rafael

    2015-08-24

    Protein binding-site similarity detection methods can be used to predict protein function and understand molecular recognition, as a tool in drug design for drug repurposing and polypharmacology, and for the prediction of the molecular determinants of drug toxicity. Here, we present IsoMIF, a method able to identify binding site molecular interaction field similarities across protein families. IsoMIF utilizes six chemical probes and the detection of subgraph isomorphisms to identify geometrically and chemically equivalent sections of protein cavity pairs. The method is validated using six distinct data sets, four of those previously used in the validation of other methods. The mean area under the receiver operator curve (AUC) obtained across data sets for IsoMIF is higher than those of other methods. Furthermore, while IsoMIF obtains consistently high AUC values across data sets, other methods perform more erratically across data sets. IsoMIF can be used to predict function from structure, to detect potential cross-reactivity or polypharmacology targets, and to help suggest bioisosteric replacements to known binding molecules. Given that IsoMIF detects spatial patterns of molecular interaction field similarities, its predictions are directly related to pharmacophores and may be readily translated into modeling decisions in structure-based drug design. IsoMIF may in principle detect similar binding sites with distinct amino acid arrangements that lead to equivalent interactions within the cavity. The source code to calculate and visualize MIFs and MIF similarities are freely available. PMID:26158641

  9. Educational Activity Sites for High School Students

    ERIC Educational Resources Information Center

    Troutner, Joanne

    2005-01-01

    Finding quality Internet resources for high school students is a continuing challenge. Several high-quality web sites are presented for educators and students. These sites offer activities to learn how an art conservator looks at paintings, create a newspaper, research and develop an end product, build geometry and physics skills, explore science…

  10. Molecular description of flexibility in an antibody combining site.

    PubMed

    Zimmermann, Jörg; Romesberg, Floyd E; Brooks, Charles L; Thorpe, Ian F

    2010-06-01

    Mature antibodies (Abs) that are exquisitely specific for virtually any foreign molecule may be produced by affinity maturation of naïve (or germline) Abs. However, the finite number of germline Abs available suggests that, in contrast to mature Abs, germline Abs must be broadly polyspecific so that they are able to recognize a wide range of ligands. Thus, affinity maturation must play a role in mediating Ab specificity. One biophysical property that distinguishes polyspecificity from specificity is protein flexibility; a flexible combining site is able to adopt different conformations that recognize different foreign molecules (or antigens), while a rigid combining site is locked into a conformation that is specific for a given antigen. Recent studies (Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 8821-8826) have examined, at the atomic level, the structural properties that mediate changes in flexibility at four stages of affinity maturation in the 4-4-20 Ab. These studies employed molecular dynamics simulations to reveal a network of residue interactions that mediate the flexibility changes accompanying maturation. The flexibility of the Ab combining sites in these molecular systems was originally measured using three-pulse photon echo spectroscopy (3PEPS). The present investigation extends this work by providing a concrete link between structural properties of the Ab molecules and features of the spectroscopic measurements used to characterize their flexibility. Results obtained from the simulations are in good qualitative agreement with the experimental measurements and indicate that the spectroscopic signal is sensitive to protein dynamics distributed throughout the entire combining site. Thus, the simulations provide a molecular-level interpretation of the changes induced by affinity maturation of the Ab. The results suggest that 3PEPS spectroscopy in combination with molecular dynamics simulations can provide a detailed description of protein dynamics and, in

  11. Water in the Active Site of Ketosteroid Isomerase

    PubMed Central

    Hanoian, Philip; Hammes-Schiffer, Sharon

    2011-01-01

    Classical molecular dynamics simulations were utilized to investigate the structural and dynamical properties of water in the active site of ketosteroid isomerase (KSI) to provide insight into the role of these water molecules in the enzyme-catalyzed reaction. This reaction is thought to proceed via a dienolate intermediate that is stabilized by hydrogen bonding with residues Tyr16 and Asp103. A comparative study was performed for the wild-type (WT) KSI and the Y16F, Y16S, and Y16F/Y32F/Y57F (FFF) mutants. These systems were studied with three different bound ligands: equilenin, which is an intermediate analog, and the intermediate states of two steroid substrates. Several distinct water occupation sites were identified in the active site of KSI for the WT and mutant systems. Three additional sites were identified in the Y16S mutant that were not occupied in WT KSI or the other mutants studied. The number of water molecules directly hydrogen bonded to the ligand oxygen was approximately two waters in the Y16S mutant, one water in the Y16F and FFF mutants, and intermittent hydrogen bonding of one water molecule in WT KSI. The molecular dynamics trajectories of the Y16F and FFF mutants reproduced the small conformational changes of residue 16 observed in the crystal structures of these two mutants. Quantum mechanical/molecular mechanical calculations of 1H NMR chemical shifts of the protons in the active site hydrogen-bonding network suggest that the presence of water in the active site does not prevent the formation of short hydrogen bonds with far-downfield chemical shifts. The molecular dynamics simulations indicate that the active site water molecules exchange much more frequently for WT KSI and the FFF mutant than for the Y16F and Y16S mutants. This difference is most likely due to the hydrogen-bonding interaction between Tyr57 and an active site water molecule that is persistent in the Y16F and Y16S mutants but absent in the FFF mutant and significantly less

  12. Molecular regulation of osteoclast activity.

    PubMed

    Bruzzaniti, Angela; Baron, Roland

    2006-06-01

    Osteoclasts are multinucleated cells derived from hematopoietic precursors that are primarily responsible for the degradation of mineralized bone during bone development, homeostasis and repair. In various skeletal disorders such as osteoporosis, hypercalcemia of malignancy, tumor metastases and Paget's disease, bone resorption by osteoclasts exceeds bone formation by osteoblasts leading to decreased bone mass, skeletal fragility and bone fracture. The overall rate of osteoclastic bone resorption is regulated either at the level of differentiation of osteoclasts from their monocytic/macrophage precursor pool or through the regulation of key functional proteins whose specific activities in the mature osteoclast control its attachment, migration and resorption. Thus, reducing osteoclast numbers and/or decreasing the bone resorbing activity of osteoclasts are two common therapeutic approaches for the treatment of hyper-resorptive skeletal diseases. In this review, several of the key functional players involved in the regulation of osteoclast activity will be discussed. PMID:16951988

  13. The molecular architecture of protein-protein binding sites.

    PubMed

    Reichmann, Dana; Rahat, Ofer; Cohen, Mati; Neuvirth, Hani; Schreiber, Gideon

    2007-02-01

    The formation of specific protein interactions plays a crucial role in most, if not all, biological processes, including signal transduction, cell regulation, the immune response and others. Recent advances in our understanding of the molecular architecture of protein-protein binding sites, which facilitates such diversity in binding affinity and specificity, are enabling us to address key questions. What is the amino acid composition of binding sites? What are interface hotspots? How are binding sites organized? What are the differences between tight and weak interacting complexes? How does water contribute to binding? Can the knowledge gained be translated into protein design? And does a universal code for binding exist, or is it the architecture and chemistry of the interface that enable diverse but specific binding solutions? PMID:17239579

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

    PubMed

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed Central

    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

  17. A molecular site-site integral equation that yields the dielectric constant

    NASA Astrophysics Data System (ADS)

    Dyer, Kippi M.; Perkyns, John S.; Stell, George; Pettitt, B. Montgomery

    2008-09-01

    Our recent derivation [K. M. Dyer et al., J. Chem. Phys. 127, 194506 (2007)] of a diagrammatically proper, site-site, integral equation theory using molecular angular expansions is extended to polar fluids. With the addition of atomic site charges we take advantage of the formal long-ranged potential field cancellations before renormalization to generate a set of numerically stable equations. Results for calculations in a minimal (spherical) angular basis set are presented for the radial distribution function, the first dipolar (110) projection, and the dielectric constant for two model diatomic systems. All results, when compared to experiment and simulation, are a significant quantitative and qualitative improvement over previous site-site theories. More importantly, the dielectric constant is not trivial and close to simulation and experiment.

  18. Site-site direct correlation functions for three popular molecular models of liquid water

    NASA Astrophysics Data System (ADS)

    Zhao, Shuangliang; Liu, Yu; Liu, Honglai; Wu, Jianzhong

    2013-08-01

    Direct correlation functions (DCFs) play a pivotal role in the applications of classical density functional theory (DFT) to addressing the thermodynamic properties of inhomogeneous systems beyond the local-density or mean-field approximations. Whereas numerous studies have been dedicated to the radial distribution functions of liquid water - the most important solvent on earth, relatively little attention has been given to the site-site DCFs. The water DCFs are long-ranged and difficult to calculate directly by simulation, and the predictions from conventional liquid-state theories have been rarely calibrated. Here we report a computational procedure for accurate evaluation of the site-site DCFs of liquid water based on three popular molecular models (viz., SPC, SPC/E, and TIP3P). The numerical results provide a benchmark for calibration of conventional liquid-state theories and fresh insights into development of new DFT methods. We show that: (1) the long-range behavior of the site-site DCFs depends on both the molecular model and the thermodynamic condition; (2) the asymptotic limit of DCFs at large distance does not follow the mean-spherical approximation (MSA); (3) individual site-site DCFs are long ranged (˜40 nm) but a summation of all DCF pairs exhibits only short-range behavior (˜1 nm or a few water diameters); (4) the site-site bridge correlation functions behave as the DCFs, i.e., they are also long-ranged while the summation of all bridge correlation functions is short ranged. Our analytical and numerical analyses of the DCFs provide some simple strategies for possible improvement of the numerical performance of conventional liquid-state theories.

  19. Low dielectric response in enzyme active site

    PubMed Central

    Mertz, Edward L.; Krishtalik, Lev I.

    2000-01-01

    The kinetics of charge transfer depend crucially on the dielectric reorganization of the medium. In enzymatic reactions that involve charge transfer, atomic dielectric response of the active site and of its surroundings determines the efficiency of the protein as a catalyst. We report direct spectroscopic measurements of the reorganization energy associated with the dielectric response in the active site of α-chymotrypsin. A chromophoric inhibitor of the enzyme is used as a spectroscopic probe. We find that water strongly affects the dielectric reorganization in the active site of the enzyme in solution. The reorganization energy of the protein matrix in the vicinity of the active site is similar to that of low-polarity solvents. Surprisingly, water exhibits an anomalously high dielectric response that cannot be described in terms of the dielectric continuum theory. As a result, sequestering the active site from the aqueous environment inside low-dielectric enzyme body dramatically reduces the dielectric reorganization. This reduction is particularly important for controlling the rate of enzymatic reactions. PMID:10681440

  20. Solution 1H NMR investigation of the active site molecular and electronic structures of substrate-bound, cyanide-inhibited HmuO, a bacterial heme oxygenase from Corynebacterium diphtheriae.

    PubMed

    Li, Yiming; Syvitski, Ray T; Chu, Grace C; Ikeda-Saito, Masao; Mar, Gerd N La

    2003-02-28

    The molecular structure and dynamic properties of the active site environment of HmuO, a heme oxygenase (HO) from the pathogenic bacterium Corynebacterium diphtheriae, have been investigated by (1)H NMR spectroscopy using the human HO (hHO) complex as a homology model. It is demonstrated that not only the spatial contacts among residues and between residues and heme, but the magnetic axes that can be related to the direction and magnitude of the steric tilt of the FeCN unit are strongly conserved in the two HO complexes. The results indicate that very similar contributions of steric blockage of several meso positions and steric tilt of the attacking ligand are operative. A distal H-bond network that involves numerous very strong H-bonds and immobilized water molecules is identified in HmuO that is analogous to that previously identified in hHO (Li, Y., Syvitski, R. T., Auclair, K., Wilks, A., Ortiz de Montellano, P. R., and La Mar, G. N. (2002) J. Biol. Chem. 277, 33018-33031). The NMR results are completely consistent with the very recent crystal structure of the HmuO.substrate complex. The H-bond network/ordered water molecules are proposed to orient the distal water molecule near the catalytically key Asp(136) (Asp(140) in hHO) that stabilizes the hydroperoxy intermediate. The dynamic stability of this H-bond network in HmuO is significantly greater than in hHO and may account for the slower catalytic rate in bacterial HO compared with mammalian HO. PMID:12480929

  1. Morphological and molecular heterogeneity in release sites of single neurons.

    PubMed

    Morgenthaler, Florence D; Knott, Graham W; Floyd Sarria, J-C; Wang, Xin; Staple, Julie K; Catsicas, Stefan; Hirling, Harald

    2003-04-01

    We have previously shown that labelling intensities for synaptic proteins vary strongly among synaptic boutons. Here we addressed the questions as to whether there are heterogeneous levels of integral membrane synaptic vesicle proteins at distinct active release sites of single neurons and if these sites possess the ultrastructural features of synapses. By double-immunostaining with specific antibodies against synaptophysin, synaptotagmin I, VAMP1 and VAMP2, we identified different relative levels of these integral membrane proteins of synaptic vesicles in comparison to boutons of the same rat cortical neuron. This heterogeneity could also be observed between the two isoforms VAMP1 and VAMP2. By studying pairs of these proteins implicated in neurotransmitter release, including both VAMP isoforms, we also show that the sites that contained predominantly one protein were nevertheless functional, as they internalized and released FM1-43 upon potassium stimulation. Using electron microscopy, we show that these active sites could have either synaptic specializations, or the features of vesicle-containing varicosities without a postsynaptic target. Different varicosities of the same neuron showed different intensities for synaptic vesicle proteins; some varicosities were capable of internalizing and releasing FM1-43, while others were silent. These results show that integral membrane synaptic vesicle proteins are differentially distributed among functional release sites of the same neuron. PMID:12713639

  2. Tunable Molecular MoS2 Edge-Site Mimics for Catalytic Hydrogen Production.

    PubMed

    Garrett, Benjamin R; Polen, Shane M; Click, Kevin A; He, Mingfu; Huang, Zhongjie; Hadad, Christopher M; Wu, Yiying

    2016-04-18

    Molybdenum sulfides represent state-of-the-art, non-platinum electrocatalysts for the hydrogen evolution reaction (HER). According to the Sabatier principle, the hydrogen binding strength to the edge active sites should be neither too strong nor too weak. Therefore, it is of interest to develop a molecular motif that mimics the catalytic sites structurally and possesses tunable electronic properties that influence the hydrogen binding strength. Furthermore, molecular mimics will be important for providing mechanistic insight toward the HER with molybdenum sulfide catalysts. In this work, a modular method to tune the catalytic properties of the S-S bond in MoO(S2)2L2 complexes is described. We studied the homogeneous electrocatalytic hydrogen production performance metrics of three catalysts with different bipyridine substitutions. By varying the electron-donating abilities, we present the first demonstration of using the ligand to tune the catalytic properties of the S-S bond in molecular MoS2 edge-site mimics. This work can shed light on the relationship between the structure and electrocatalytic activity of molecular MoS2 catalysts and thus is of broad importance from catalytic hydrogen production to biological enzyme functions. PMID:27022836

  3. Lysostaphin: molecular changes that preserve staphylolytic activity.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lysostaphin is a potent staphylolytic enzyme with known resistance mechanisms. To avoid resistance development, this lab is developing lysostaphin-LysK fusion proteins harboring three synergistic lytic activities in one protein. Some of the molecular changes to lysostaphin and their effects on sta...

  4. Rat intestinal trehalase. Studies of the active site.

    PubMed

    Chen, C C; Guo, W J; Isselbacher, K J

    1987-11-01

    Rat intestinal trehalase was solubilized, purified and reconstituted into proteoliposomes. With octyl glucoside as the solubilizing detergent, the purified protein appeared as a single band on SDS/polyacrylamide-gel electrophoresis with an apparent molecular mass of 67 kDa. Kinetic studies indicated that the active site of this enzyme can be functionally divided into two adjacent regions, namely a binding site (with pKa 4.8) and a catalytic site (with pKa 7.2). Other findings suggested that the catalytic site contains a functional thiol group, which is sensitive to inhibition by N-ethylmaleimide, Hg2+ and iodoacetate. Substrate protection and iodoacetate labelling of the thiol group demonstrated that only a protein of 67 kDa was labelled. Furthermore, sucrose and phlorizin protected the thiol group, but Tris-like inhibitors did not. Structure-inhibition analysis of Tris-like inhibitors, the pH effect of Tris inhibition and Tris protection of 1-(3-dimethylaminopropyl)-3-ethylcarbodi-imide inactivation permitted characterization and location of a separate site containing a carboxy group for Tris binding, which may also be the binding region. On the basis of these findings, a possible structure for the active site of trehalase is proposed. PMID:3426558

  5. Active site specificity of plasmepsin II.

    PubMed Central

    Westling, J.; Cipullo, P.; Hung, S. H.; Saft, H.; Dame, J. B.; Dunn, B. M.

    1999-01-01

    Members of the aspartic proteinase family of enzymes have very similar three-dimensional structures and catalytic mechanisms. Each, however, has unique substrate specificity. These distinctions arise from variations in amino acid residues that line the active site subsites and interact with the side chains of the amino acids of the peptides that bind to the active site. To understand the unique binding preferences of plasmepsin II, an enzyme of the aspartic proteinase class from the malaria parasite, Plasmodium falciparum, chromogenic octapeptides having systematic substitutions at various positions in the sequence were analyzed. This enabled the design of new, improved substrates for this enzyme (Lys-Pro-Ile-Leu-Phe*Nph-Ala/Glu-Leu-Lys, where * indicates the cleavage point). Additionally, the crystal structure of plasmepsin II was analyzed to explain the binding characteristics. Specific amino acids (Met13, Ser77, and Ile287) that were suspected of contributing to active site binding and specificity were chosen for site-directed mutagenesis experiments. The Met13Glu and Ile287Glu single mutants and the Met13Glu/Ile287Glu double mutant gain the ability to cleave substrates containing Lys residues. PMID:10548045

  6. Visible-Light-Activated Molecular Switches.

    PubMed

    Bléger, David; Hecht, Stefan

    2015-09-21

    The ability to influence key properties of molecular systems by using light holds much promise for the fields of materials science and life sciences. The cornerstone of such systems is molecules that are able to reversibly photoisomerize between two states, commonly referred to as photoswitches. One serious restriction to the development of functional photodynamic systems is the necessity to trigger switching in at least one direction by UV light, which is often damaging and penetrates only partially through most media. This review provides a summary of the different conceptual strategies for addressing molecular switches in the visible and near-infrared regions of the optical spectrum. Such visible-light-activated molecular switches tremendously extend the scope of photoswitchable systems for future applications and technologies. PMID:26096635

  7. Corrosion Research And Web Site Activities

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2001-01-01

    This report covers corrosion-related activities at the NASA Kennedy Space Center during the summer of 2000. The NASA Kennedy Space Center's corrosion web site, corrosion.ksc.nasa.gov, was updated with new information based on feedback over the past two years. The methodology for a two-year atmospheric exposure testing program to study the effectiveness of commercial chemicals sold for rinsing aircraft and other equipment was developed and some preliminary laboratory chemical analyses are presented.

  8. Corrosion Research and Web Site Activities

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2002-01-01

    This report covers corrosion-related activities at the NASA Kennedy Space Center during the summer of 2000. The NASA Kennedy Space Center's corrosion web site, corrosion.ksc.nasa.gov, was updated with new information based on feedback over the past two years. The methodology for a two-year atmospheric exposure testing program to study the effectiveness of commercial chemicals sold for rinsing aircraft and other equipment was developed and some preliminary laboratory chemical analyses are presented.

  9. Identification of inhibitor binding site in human sirtuin 2 using molecular docking and dynamics simulations.

    PubMed

    Sakkiah, Sugunadevi; Arooj, Mahreen; Kumar, Manian Rajesh; Eom, Soo Hyun; Lee, Keun Woo

    2013-01-01

    The ability to identify the site of a protein that can bind with high affinity to small, drug-like compounds has been an important goal in drug design. Sirtuin 2 (SIRT2), histone deacetylase protein family, plays a central role in the regulation of various pathways. Hence, identification of drug for SIRT2 has attracted great interest in the drug discovery community. To elucidate the molecular basis of the small molecules interactions to inhibit the SIRT2 function we employed the molecular docking, molecular dynamics simulations, and the molecular mechanism Poisson-Boltzmann/surface area (MM-PBSA) calculations. Five well know inhibitors such as suramin, mol-6, sirtinol, 67, and nf675 were selected to establish the nature of the binding mode of the inhibitors in the SIRT2 active site. The molecular docking and dynamics simulations results revealed that the hydrogen bonds between Arg97 and Gln167 are crucial to inhibit the function of SIRT2. In addition, the MM-PBSA calculations revealed that binding of inhibitors to SIRT2 is mainly driven by van der Waals/non-polar interactions. Although the five inhibitors are very different in structure, shape, and electrostatic potential, they are able to fit in the same binding pocket. These findings from this study provide insights to elucidate the binding pattern of SIRT2 inhibitors and help in the rational structure-based design of novel SIRT2 inhibitors with improved potency and better resistance profile. PMID:23382805

  10. Molecular Imaging with SERS-Active Nanoparticles

    PubMed Central

    Zhang, Yin; Hong, Hao; Myklejord, Duane V.; Cai, Weibo

    2011-01-01

    Lead-in Raman spectroscopy has been explored for various biomedical applications (e.g. cancer diagnosis) because it can provide detailed information on the chemical composition of cells and tissues. For imaging applications, several variations of Raman spectroscopy have been developed to enhance its sensitivity. To date, a wide variety of molecular targets and biological events have been investigated using surface-enhanced Raman scattering (SERS)-active nanoparticles. The superb multiplexing capability of SERS-based Raman imaging, already successfully demonstrated in live animals, can be extremely powerful in future research where different agents can be attached to different Raman tags to enable the simultaneous interrogation of multiple biological events. Over the last several years, molecular imaging with SERS-active nanoparticles has advanced significantly and many pivotal proof-of-principle experiments have been successfully carried out. It is expected that SERS-based imaging will continue to be a dynamic research field over the next decade. PMID:21932216

  11. Molecular simulations of Taxawallin I inside classical taxol binding site of β-tubulin.

    PubMed

    Khan, Inamullah; Nisar, Muhammad; Ahmad, Manzoor; Shah, Hamidullah; Iqbal, Zafar; Saeed, Muhammad; Halimi, Syed Muhammad Ashhad; Kaleem, Waqar Ahmad; Qayum, Mughal; Aman, Akhter; Abdullah, Syed Muhammad

    2011-03-01

    A new taxoid Taxawallin I (1) along with two known taxoids (2-3) were isolated from methanolic bark extract of Taxus wallichiana Zucc. Structural characterization was confirmed by mass and NMR spectral techniques. Taxawallin I exhibited significant in-vitro anticancer activity against HepG2, A498, NCI-H226 and MDR 2780AD cancer lines. Tubulin binding assay was performed to assess its tubulin binding activity. Molecular docking analysis was performed to study the potential binding mode inside the taxol binding site of β-tubulin. PMID:20969934

  12. [Molecular mechanisms of niclosamide antitumor activity].

    PubMed

    Moskaleva, E Yu; Perevozchikova, V G; Zhirnik, A S; Severin, S E

    2015-01-01

    In this review the recent data regarding the antitumor activity of niclosamide and the molecular mechanisms of its antitumor activity are presented. Niclosamide has been used in the clinic for the treatment of intestinal parasite infections. In recent years in several screening investigations of various drugs and chemical compounds niclosamide was identified as a potential anticancer agent. Niclosamide not only inhibits the Wnt/β-catenin, mTORC1, STAT3, NF-κB and Notch signaling pathways, but also targets mitochondria in cancer cells to induce growth inhibition and apoptosis. A number of studies have established the anticancer activity of niclosamide in both in vitro and in vivo in xenotransplantation models using human tumors and immunodeficient mice. It is important that niclosamide is active not only against tumor cells but also cancer stem cells. Normal cells are resistant to niclosamide. The accumulated experimental data suggest niclosamide is a promising drug for the treatment of various types of cancer. PMID:26716739

  13. The calculation of surface orbital energies for specific types of active sites on dispersed metal catalysts

    SciTech Connect

    Augustine, R.L.; Lahanas, K.M.; Cole, F.

    1992-11-01

    An angular overlap calculation has been used to determine the s, p, and d orbital energy levels of the different types of surface sites present on dispersed metal catalysts. These data can permit a Frontier Molecular Orbital treatment of specific site activities as long as the surface orbital availability for overlap with adsorbed substrates is considered along with its energy value and symmetry.

  14. The calculation of surface orbital energies for specific types of active sites on dispersed metal catalysts

    SciTech Connect

    Augustine, R.L.; Lahanas, K.M.; Cole, F.

    1992-01-01

    An angular overlap calculation has been used to determine the s, p, and d orbital energy levels of the different types of surface sites present on dispersed metal catalysts. These data can permit a Frontier Molecular Orbital treatment of specific site activities as long as the surface orbital availability for overlap with adsorbed substrates is considered along with its energy value and symmetry.

  15. Molecular docking studies in factor XIa binding site

    NASA Astrophysics Data System (ADS)

    Balaji, Govardhan A.; Balaji, Vitukudi N.; Rao, Shashidhar N.

    2016-03-01

    Factor XIa inhibitors have been identified to have potential as anticoagulants with robust efficacy and low bleeding risks. In light of their significance and the availability of their 3-D X-ray data in the PDB, we present molecular docking studies carried out with a view to obtain docking protocols that will successfully reproduce the experimentally observed protein-ligand interactions in the case of various X-ray ligands. In this context, we have specifically investigated the efficacy of various cross-docking protocols in reproducing experimental data. Our studies demonstrate that an ensemble of the three apo proteins is capable of accurately docking a majority of the X-ray ligands accurately without invoking any additional conformational flexibility than that present in their experimental structures. Further, we demonstrate that such an ensemble is successfully able to enrich a collection of known active factor XIa inhibitors embedded in a decoy database of drug-like molecules.

  16. Allosteric site-mediated active site inhibition of PBP2a using Quercetin 3-O-rutinoside and its combination.

    PubMed

    Rani, Nidhi; Vijayakumar, Saravanan; P T V, Lakshmi; Arunachalam, Annamalai

    2016-08-01

    Recent crystallographic study revealed the involvement of allosteric site in active site inhibition of penicillin binding protein (PBP2a), where one molecule of Ceftaroline (Cef) binds to the allosteric site of PBP2a and paved way for the other molecule (Cef) to bind at the active site. Though Cef has the potency to inhibit the PBP2a, its adverse side effects are of major concern. Previous studies have reported the antibacterial property of Quercetin derivatives, a group of natural compounds. Hence, the present study aims to evaluate the effect of Quercetin 3-o-rutinoside (Rut) in allosteric site-mediated active site inhibition of PBP2a. The molecular docking studies between allosteric site and ligands (Rut, Que, and Cef) revealed a better binding efficiency (G-score) of Rut (-7.790318) and Cef (-6.194946) with respect to Que (-5.079284). Molecular dynamic (MD) simulation studies showed significant changes at the active site in the presence of ligands (Rut and Cef) at allosteric site. Four different combinations of Rut and Cef were docked and their G-scores ranged between -6.320 and -8.623. MD studies revealed the stability of the key residue (Ser403) with Rut being at both sites, compared to other complexes. Morphological analysis through electron microscopy confirmed that combination of Rut and Cefixime was able to disturb the bacterial cell membrane in a similar fashion to that of Rut and Cefixime alone. The results of this study indicate that the affinity of Rut at both sites were equally good, with further validations Rut could be considered as an alternative for inhibiting MRSA growth. PMID:26360629

  17. Identification of Ice Nucleation Active Sites on Silicate Dust Particles

    NASA Astrophysics Data System (ADS)

    Zolles, Tobias; Burkart, Julia; Häusler, Thomas; Pummer, Bernhard; Hitzenberger, Regina; Grothe, Hinrich

    2015-04-01

    Mineral dusts originating from Earth's crust are known to be important atmospheric ice nuclei. In agreement with earlier studies, feldspar was found as the most active of the tested natural mineral dusts [1-3]. Nevertheless, among those structures K-feldspar showed by far the highest ice nucleation activity. In this study, the reasons for its activity and the difference in the activity of the different feldspars were investigated in closer details. Conclusions are drawn from scanning electron microscopy, X-ray powder diffraction, infrared spectroscopy, and oil-immersion freezing experiments. We give a potential explanation of the increased ice nucleation activity of K-feldspar. The ice nucleating sites are very much dependent on the alkali ion present by altering the water structure and the feldspar surface. The higher activity of K-feldspar can be attributed to the presence of potassium ions on the surface and surface bilayer. The alkali-ions have different hydration shells and thus an influence on the ice nucleation activity of feldspar. Chaotropic behavior of Calcium and Sodium ions are lowering the ice nucleation potential of the other feldspars, while kosmotropic Potassium has a neutral or even positive effect. Furthermore we investigated the influence of milling onto the ice nucleation of quartz particles. The ice nucleation activity can be increased by mechanical milling, by introducing more molecular, nucleation active defects to the particle surface. This effect is larger than expected by plane surface increase. [1] Atkinson et al. The Importance of Feldspar for Ice Nucleation by Mineral Dust in Mixed-Phase Clouds. Nature 2013, 498, 355-358. [2] Yakobi-Hancock et al.. Feldspar Minerals as Efficient Deposition Ice Nuclei. Atmos. Chem. Phys. 2013, 13, 11175-11185. [3] Zolles et al. Identification of Ice Nucleation Active Sites on Feldspar Dust Particles. J. Phys. Chem. A 2015 accepted.

  18. Active site of ribulosebisphosphate carboxylase/oxygenase

    SciTech Connect

    Hartman, F.C.; Stringer, C.D.; Milanez, S.; Lee, E.H.

    1985-01-01

    Previous affinity labeling studies and comparative sequence analyses have identified two different lysines at the active site of ribulosebisphosphate carboxylase/oxygenase and have suggested their essentiality to function. The essential lysines occupy positions 166 and 329 in the Rhodospirillum rubrum enzyme and positions 175 and 334 in the spinach enzyme. Based on the pH-dependencies of inactivations of the two enzymes by trinitrobenzene sulfonate, Lys-166 (R. rubrum enzyme) exhibits a pK/sub a/ of 7.9 and Lys-334 (spinach enzyme) exhibits a pK/sub a/ of 9.0. These low pK/sub a/ values as well as the enhanced nucleophilicities of the lysyl residues argue that both are important to catalysis rather than to substrate binding. Lys-166 may correspond to the essential base that initiates catalysis and that displays a pK/sub a/ of 7.5 in the pH-curve for V/sub max//K/sub m/. Cross-linking experiments with 4,4'-diisothiocyano-2,2'-disulfonate stilbene demonstrate that the two active-site lysines are within 12 A. 50 refs., 7 figs., 1 tab.

  19. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

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

    1992-02-01

    The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of transuranic (TRU) waste and active low-level waste (LLW) facilities at Oak Ridge National Laboratory (ORNL) in accordance with US Department of Energy (DOE) Order 5820.2A. Active LLW facilities in Solid Waste Storage Area (SWSA) 6 include Tumulus I and Tumulus II, the Interim Waste Management Facility (IWMF), LLW silos, high-range wells, asbestos silos, and fissile wells. The tumulus pads and IWMF are aboveground, high-strength concrete pads on which concrete vaults containing metal boxes of LLW are placed; the void space between the boxes and vaults is filled with grout. Eventually, these pads and vaults will be covered by an engineered multilayered cap. All other LLW facilities in SWSA 6 are below ground. In addition, this plan includes monitoring of the Hillcut Disposal Test Facility (HDTF) in SWSA 6, even though this facility was completed prior to the data of the DOE order. In SWSA 5 North, the TRU facilities include below-grade engineered caves, high-range wells, and unlined trenches. All samples from SWSA 6 are screened for alpha and beta activity, counted for gamma-emitting isotopes, and analyzed for tritium. In addition to these analytes, samples from SWSA 5 North are analyzed for specific transuranic elements.

  20. Molecular hydrogen suppresses activated Wnt/β-catenin signaling.

    PubMed

    Lin, Yingni; Ohkawara, Bisei; Ito, Mikako; Misawa, Nobuaki; Miyamoto, Kentaro; Takegami, Yasuhiko; Masuda, Akio; Toyokuni, Shinya; Ohno, Kinji

    2016-01-01

    Molecular hydrogen (H2) is effective for many diseases. However, molecular bases of H2 have not been fully elucidated. Cumulative evidence indicates that H2 acts as a gaseous signal modulator. We found that H2 suppresses activated Wnt/β-catenin signaling by promoting phosphorylation and degradation οf β-catenin. Either complete inhibition of GSK3 or mutations at CK1- and GSK3-phosphorylation sites of β-catenin abolished the suppressive effect of H2. H2 did not increase GSK3-mediated phosphorylation of glycogen synthase, indicating that H2 has no direct effect on GSK3 itself. Knock-down of adenomatous polyposis coli (APC) or Axin1, which form the β-catenin degradation complex, minimized the suppressive effect of H2 on β-catenin accumulation. Accordingly, the effect of H2 requires CK1/GSK3-phosphorylation sites of β-catenin, as well as the β-catenin degradation complex comprised of CK1, GSK3, APC, and Axin1. We additionally found that H2 reduces the activation of Wnt/β-catenin signaling in human osteoarthritis chondrocytes. Oral intake of H2 water tended to ameliorate cartilage degradation in a surgery-induced rat osteoarthritis model through attenuating β-catenin accumulation. We first demonstrate that H2 suppresses abnormally activated Wnt/β-catenin signaling, which accounts for the protective roles of H2 in a fraction of diseases. PMID:27558955

  1. Molecular hydrogen suppresses activated Wnt/β-catenin signaling

    PubMed Central

    Lin, Yingni; Ohkawara, Bisei; Ito, Mikako; Misawa, Nobuaki; Miyamoto, Kentaro; Takegami, Yasuhiko; Masuda, Akio; Toyokuni, Shinya; Ohno, Kinji

    2016-01-01

    Molecular hydrogen (H2) is effective for many diseases. However, molecular bases of H2 have not been fully elucidated. Cumulative evidence indicates that H2 acts as a gaseous signal modulator. We found that H2 suppresses activated Wnt/β-catenin signaling by promoting phosphorylation and degradation οf β-catenin. Either complete inhibition of GSK3 or mutations at CK1- and GSK3-phosphorylation sites of β-catenin abolished the suppressive effect of H2. H2 did not increase GSK3-mediated phosphorylation of glycogen synthase, indicating that H2 has no direct effect on GSK3 itself. Knock-down of adenomatous polyposis coli (APC) or Axin1, which form the β-catenin degradation complex, minimized the suppressive effect of H2 on β-catenin accumulation. Accordingly, the effect of H2 requires CK1/GSK3-phosphorylation sites of β-catenin, as well as the β-catenin degradation complex comprised of CK1, GSK3, APC, and Axin1. We additionally found that H2 reduces the activation of Wnt/β-catenin signaling in human osteoarthritis chondrocytes. Oral intake of H2 water tended to ameliorate cartilage degradation in a surgery-induced rat osteoarthritis model through attenuating β-catenin accumulation. We first demonstrate that H2 suppresses abnormally activated Wnt/β-catenin signaling, which accounts for the protective roles of H2 in a fraction of diseases. PMID:27558955

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

    SciTech Connect

    Miao, Yinglong; Baudry, Jerome Y

    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.

  3. Molecular Imaging of the ATM Kinase Activity

    SciTech Connect

    Williams, Terence M.; Nyati, Shyam; Ross, Brian D.; Rehemtulla, Alnawaz

    2013-08-01

    Purpose: Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including from DNA double-strand breaks. ATM activation results in the initiation of a complex cascade of events including DNA damage repair, cell cycle checkpoint control, and survival. We sought to create a bioluminescent reporter that dynamically and noninvasively measures ATM kinase activity in living cells and subjects. Methods and Materials: Using the split luciferase technology, we constructed a hybrid cDNA, ATM-reporter (ATMR), coding for a protein that quantitatively reports on changes in ATM kinase activity through changes in bioluminescence. Results: Treatment of ATMR-expressing cells with ATM inhibitors resulted in a dose-dependent increase in bioluminescence activity. In contrast, induction of ATM kinase activity upon irradiation resulted in a decrease in reporter activity that correlated with ATM and Chk2 activation by immunoblotting in a time-dependent fashion. Nuclear targeting improved ATMR sensitivity to both ATM inhibitors and radiation, whereas a mutant ATMR (lacking the target phosphorylation site) displayed a muted response. Treatment with ATM inhibitors and small interfering (si)RNA-targeted knockdown of ATM confirm the specificity of the reporter. Using reporter expressing xenografted tumors demonstrated the ability of ATMR to report in ATM activity in mouse models that correlated in a time-dependent fashion with changes in Chk2 activity. Conclusions: We describe the development and validation of a novel, specific, noninvasive bioluminescent reporter that enables monitoring of ATM activity in real time, in vitro and in vivo. Potential applications of this reporter include the identification and development of novel ATM inhibitors or ATM-interacting partners through high-throughput screens and in vivo pharmacokinetic/pharmacodynamic studies of ATM inhibitors in preclinical models.

  4. Computational approaches to the determination of active site structures and reaction mechanisms in heterogeneous catalysts.

    PubMed

    Catlow, C R A; French, S A; Sokol, A A; Thomas, J M

    2005-04-15

    We apply quantum chemical methods to the study of active site structures and reaction mechanisms in mesoporous silica and metal oxide catalysts. Our approach is based on the use of both molecular cluster and embedded cluster (QM/MM) techniques, where the active site and molecular complex are described using density functional theory (DFT) and the embedding matrix simulated by shell model potentials. We consider three case studies: alkene epoxidation over the microporous TS-1 catalyst; methanol synthesis on ZnO and Cu/ZnO and C-H bond activation over Li-doped MgO. PMID:15901543

  5. Site-controlled Ag nanocrystals grown by molecular beam epitaxy-Towards plasmonic integration technology

    SciTech Connect

    Urbanczyk, Adam; Noetzel, Richard

    2012-12-15

    We demonstrate site-controlled growth of epitaxial Ag nanocrystals on patterned GaAs substrates by molecular beam epitaxy with high degree of long-range uniformity. The alignment is based on lithographically defined holes in which position controlled InAs quantum dots are grown. The Ag nanocrystals self-align preferentially on top of the InAs quantum dots. No such ordering is observed in the absence of InAs quantum dots, proving that the ordering is strain-driven. The presented technique facilitates the placement of active plasmonic nanostructures at arbitrarily defined positions enabling their integration into complex devices and plasmonic circuits.

  6. Site-renormalised molecular fluid theory: on the utility of a two-site model of water

    PubMed Central

    Dyer, Kippi M.; Perkyns, John S.; Stell, George; Pettitt, B. Montgomery

    2009-01-01

    We propose a simple, two-site model of water, using the familiar three-site Simple Point Charge (SPC) model as a guide. We briefly examine the resulting dielectric and solvation properties of the bulk fluid, both pure and in a three component mixture of apolar or ionic simple fluid solutes, using integral equation methods. The results confirm a practical utility of this simplified model, and the essential predictive properties of the site-renormalised molecular fluid theory. PMID:19920881

  7. The bacterial and mitochondrial ribosomal A-site molecular switches possess different conformational substates

    PubMed Central

    Kondo, Jiro; Westhof, Eric

    2008-01-01

    The A site of the small ribosomal subunit participates in the fidelity of decoding by switching between two states, a resting ‘off’ state and an active decoding ‘on’ state. Eight crystal structures of RNA duplexes containing two minimal decoding A sites of the Homo sapiens mitochondrial wild-type, the A1555G mutant or bacteria have been solved. The resting ‘off’ state of the mitochondrial wild-type A site is surprisingly different from that of the bacterial A site. The mitochondrial A1555G mutant has two types of the ‘off’ states; one is similar to the mitochondrial wild-type ‘off’ state and the other is similar to the bacterial ‘off’ state. Our present results indicate that the dynamics of the A site in bacteria and mitochondria are different, a property probably related to the small number of tRNAs used for decoding in mitochondria. Based on these structures, we propose a hypothesis for the molecular mechanism of non-syndromic hearing loss due to the mitochondrial A1555G mutation. PMID:18346970

  8. Molecular Mechanisms of Large-Conductance Ca2+-Activated Potassium Channel Activation by Ginseng Gintonin

    PubMed Central

    Choi, S. H.; Lee, B. H.; Hwang, S. H.; Kim, H. J.; Lee, S. M.; Kim, H. C.; Rhim, H. W.; Nah, S. Y.

    2013-01-01

    Gintonin is a unique lysophosphatidic acid (LPA) receptor ligand found in Panax ginseng. Gintonin induces transient [Ca2+]i through G protein-coupled LPA receptors. Large-conductance Ca2+-activated K+ (BKCa) channels are expressed in blood vessels and neurons and play important roles in blood vessel relaxation and attenuation of neuronal excitability. BKCa channels are activated by transient [Ca2+]i and are regulated by various Ca2+-dependent kinases. We investigated the molecular mechanisms of BKCa channel activation by gintonin. BKCa channels are heterologously expressed in Xenopus oocytes. Gintonin treatment induced BKCa channel activation in oocytes expressing the BKCa channel α subunit in a concentration-dependent manner (EC50 = 0.71 ± 0.08 µg/mL). Gintonin-mediated BKCa channel activation was blocked by a PKC inhibitor, calphostin, and by the calmodulin inhibitor, calmidazolium. Site-directed mutations in BKCa channels targeting CaM kinase II or PKC phosphorylation sites but not PKA phosphorylation sites attenuated gintonin action. Mutations in the Ca2+ bowl and the regulator of K+ conductance (RCK) site also blocked gintonin action. These results indicate that gintonin-mediated BKCa channel activations are achieved through LPA1 receptor-phospholipase C-IP3-Ca2+-PKC-calmodulin-CaM kinase II pathways and calcium binding to the Ca2+ bowl and RCK domain. Gintonin could be a novel contributor against blood vessel constriction and over-excitation of neurons. PMID:23662129

  9. Different cleavage site for high molecular weight kininogen in vivo following intravenous injection of dextran sulfate in the rabbit

    SciTech Connect

    Wiggins, R.C.

    1986-04-01

    Purified radiolabeled rabbit Hageman factor, prekallikrein, and high molecular weight kininogen were used to examine Hageman factor system molecular dynamics after the intravenous injection of heparin-like dextran sulfate polymer in the rabbit. Hageman factor system proteins rapidly disappeared from the circulation following dextran sulfate injection, as measured by radial immunodiffusion, by kaolin-releasable kinin formation, and by measuring circulating levels of radiolabeled Hageman factor, prekallikrein, and high molecular weight kininogen. /sup 125/I-Hageman factor was distributed mainly to lung, liver, and spleen following dextran sulfate injection. Proteolysis of circulating /sup 125/I-Hageman factor occurred at a site within a disulfide loop into fragments of 50,000 and 30,000 molecular weight. Proteolysis of /sup 125/I-prekallikrein also occurred with visualization of a 50,000 molecular weight fragment. Although extensive proteolysis of /sup 131/I-high molecular weight kininogen was observed, the cleavage fragments were not the same as those generated during contact activation in vitro. The major fragment of high molecular weight kininogen observed in vivo was at 80,000 molecular weight, in contrast to the 65,000 molecular weight fragment generated by kallikrein in vitro. These results indicate that high molecular weight kininogen can undergo proteolysis in vivo into fragments not known to be associated with kinin release.

  10. X-ray Analyses of the Ribosomal A-Site Molecular Switches

    NASA Astrophysics Data System (ADS)

    Kondo, Jiro

    The aminoacyl-tRNA decoding site (A-site) on the small ribosomal subunit is an RNA molecular switch guaranteeing high translation fidelity. Due to the similarity of the secondary structure of the A-site, it has long been believed that the functional characteristics and tertiary structure of the A-site molecular switch are basically conserved in three main cell types, bacteria, mitochondria and eukaryotic cytoplasm. However, these three cell types are noticeably different in their biological properties such as life cycle, genome size, structural component of ribosome and number of tRNA species. In our structural studies, we have shown how a small difference of nucleotide sequences affects the dynamics of the A-site molecular switches underlying the decoding mechanism adapted to their biological properties and environments. The observed structural insights into the decoding process allowed us to understand molecular mechanisms of non-syndromic hearing loss and toxicity mechanism of aminoglycoside antibiotics.

  11. Molecular Ice Nucleation Activity of Birch Pollen

    NASA Astrophysics Data System (ADS)

    Felgitsch, Laura; Bichler, Magdalena; Häusler, Thomas; Weiss, Victor U.; Marchetti-Deschmann, Martina; Allmaier, Günter; Grothe, Hinrich

    2015-04-01

    Heterogeneous ice nucleation plays a major part in ecosystem and climate. Due to the triggering of ice cloud formation it influences the radiation balance of the earth, but also on the ground it can be found to be important in many processes of nature. So far the process of heterogeneous ice nucleation is not fully understood and many questions remain to be answered. Biological ice nucleation is hereby from great interest, because it shows the highest freezing temperatures. Several bacteria and fungi act as ice nuclei. A famous example is Pseudomonas syringae, a bacterium in commercial use (Snomax®), which increases the freezing from homogeneous freezing temperatures of approx. -40° C (for small volumes as in cloud droplets) to temperatures up to -2° C. In 2001 it was found that birch pollen can trigger ice nucleation (Diehl et al. 2001; Diehl et al. 2002). For a long time it was believed that this is due to macroscopic features of the pollen surface. Recent findings of Bernhard Pummer (2012) show a different picture. The ice nuclei are not attached on the pollen surface directly, but on surface material which can be easily washed off. This shows that not only the surface morphology, but also specific molecules or molecular structures are responsible for the ice nucleation activity of birch pollen. With various analytic methods we work on elucidating the structure of these molecules as well as the mechanism with which they trigger ice nucleation. To solve this we use various instrumental analytic techniques like Nuclear Magnetic Resonance spectroscopy (NMR), Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS), and Gas-phase Electrophoretic Mobility Molecular Analysis (GEMMA). Also standard techniques like various chromatographic separation techniques and solvent extraction are in use. We state here that this feature might be due to the aggregation of small molecules, with agglomerates showing a specific surface structure. Our results

  12. Quantifying the density and utilization of active sites in non-precious metal oxygen electroreduction catalysts.

    PubMed

    Sahraie, Nastaran Ranjbar; Kramm, Ulrike I; Steinberg, Julian; Zhang, Yuanjian; Thomas, Arne; Reier, Tobias; Paraknowitsch, Jens-Peter; Strasser, Peter

    2015-01-01

    Carbon materials doped with transition metal and nitrogen are highly active, non-precious metal catalysts for the electrochemical conversion of molecular oxygen in fuel cells, metal air batteries, and electrolytic processes. However, accurate measurement of their intrinsic turn-over frequency and active-site density based on metal centres in bulk and surface has remained difficult to date, which has hampered a more rational catalyst design. Here we report a successful quantification of bulk and surface-based active-site density and associated turn-over frequency values of mono- and bimetallic Fe/N-doped carbons using a combination of chemisorption, desorption and (57)Fe Mössbauer spectroscopy techniques. Our general approach yields an experimental descriptor for the intrinsic activity and the active-site utilization, aiding in the catalyst development process and enabling a previously unachieved level of understanding of reactivity trends owing to a deconvolution of site density and intrinsic activity. PMID:26486465

  13. Quantifying the density and utilization of active sites in non-precious metal oxygen electroreduction catalysts

    NASA Astrophysics Data System (ADS)

    Sahraie, Nastaran Ranjbar; Kramm, Ulrike I.; Steinberg, Julian; Zhang, Yuanjian; Thomas, Arne; Reier, Tobias; Paraknowitsch, Jens-Peter; Strasser, Peter

    2015-10-01

    Carbon materials doped with transition metal and nitrogen are highly active, non-precious metal catalysts for the electrochemical conversion of molecular oxygen in fuel cells, metal air batteries, and electrolytic processes. However, accurate measurement of their intrinsic turn-over frequency and active-site density based on metal centres in bulk and surface has remained difficult to date, which has hampered a more rational catalyst design. Here we report a successful quantification of bulk and surface-based active-site density and associated turn-over frequency values of mono- and bimetallic Fe/N-doped carbons using a combination of chemisorption, desorption and 57Fe Mössbauer spectroscopy techniques. Our general approach yields an experimental descriptor for the intrinsic activity and the active-site utilization, aiding in the catalyst development process and enabling a previously unachieved level of understanding of reactivity trends owing to a deconvolution of site density and intrinsic activity.

  14. Quantifying the density and utilization of active sites in non-precious metal oxygen electroreduction catalysts

    PubMed Central

    Sahraie, Nastaran Ranjbar; Kramm, Ulrike I.; Steinberg, Julian; Zhang, Yuanjian; Thomas, Arne; Reier, Tobias; Paraknowitsch, Jens-Peter; Strasser, Peter

    2015-01-01

    Carbon materials doped with transition metal and nitrogen are highly active, non-precious metal catalysts for the electrochemical conversion of molecular oxygen in fuel cells, metal air batteries, and electrolytic processes. However, accurate measurement of their intrinsic turn-over frequency and active-site density based on metal centres in bulk and surface has remained difficult to date, which has hampered a more rational catalyst design. Here we report a successful quantification of bulk and surface-based active-site density and associated turn-over frequency values of mono- and bimetallic Fe/N-doped carbons using a combination of chemisorption, desorption and 57Fe Mössbauer spectroscopy techniques. Our general approach yields an experimental descriptor for the intrinsic activity and the active-site utilization, aiding in the catalyst development process and enabling a previously unachieved level of understanding of reactivity trends owing to a deconvolution of site density and intrinsic activity. PMID:26486465

  15. Molecular Mapping of General Anesthetic Sites in a Voltage-Gated Ion Channel

    PubMed Central

    Barber, Annika F.; Liang, Qiansheng; Amaral, Cristiano; Treptow, Werner; Covarrubias, Manuel

    2011-01-01

    Several voltage-gated ion channels are modulated by clinically relevant doses of general anesthetics. However, the structural basis of this modulation is not well understood. Previous work suggested that n-alcohols and inhaled anesthetics stabilize the closed state of the Shaw2 voltage-gated (Kv) channel (K-Shaw2) by directly interacting with a discrete channel site. We hypothesize that the inhibition of K-Shaw2 channels by general anesthetics is governed by interactions between binding and effector sites involving components of the channel's activation gate. To investigate this hypothesis, we applied Ala/Val scanning mutagenesis to the S4-S5 linker and the post-PVP S6 segment, and conducted electrophysiological analysis to evaluate the energetic impact of the mutations on the inhibition of the K-Shaw2 channel by 1-butanol and halothane. These analyses identified residues that determine an apparent binding cooperativity and residue pairs that act in concert to modulate gating upon anesthetic binding. In some instances, due to their critical location, key residues also influence channel gating. Complementing these results, molecular dynamics simulations and in silico docking experiments helped us visualize possible anesthetic sites and interactions. We conclude that the inhibition of K-Shaw2 by general anesthetics results from allosteric interactions between distinct but contiguous binding and effector sites involving inter- and intrasubunit interfaces. PMID:21961587

  16. Virtual Screening of Receptor Sites for Molecularly Imprinted Polymers.

    PubMed

    Bates, Ferdia; Cela-Pérez, María Concepción; Karim, Kal; Piletsky, Sergey; López-Vilariño, José Manuel

    2016-08-01

    Molecularly Imprinted Polymers (MIPs) are highly advantageous in the field of analytical chemistry. However, interference from secondary molecules can also impede capture of a target by a MIP receptor. This greatly complicates the design process and often requires extensive laboratory screening which is time consuming, costly, and creates substantial waste products. Herein, is presented a new technique for screening of "virtually imprinted receptors" for rebinding of the molecular template as well as secondary structures, correlating the virtual predictions with experimentally acquired data in three case studies. This novel technique is particularly applicable to the evaluation and prediction of MIP receptor specificity and efficiency in complex aqueous systems. PMID:27076379

  17. Lipid interaction sites on channels, transporters and receptors: Recent insights from molecular dynamics simulations.

    PubMed

    Hedger, George; Sansom, Mark S P

    2016-10-01

    Lipid molecules are able to selectively interact with specific sites on integral membrane proteins, and modulate their structure and function. Identification and characterization of these sites are of importance for our understanding of the molecular basis of membrane protein function and stability, and may facilitate the design of lipid-like drug molecules. Molecular dynamics simulations provide a powerful tool for the identification of these sites, complementing advances in membrane protein structural biology and biophysics. We describe recent notable biomolecular simulation studies which have identified lipid interaction sites on a range of different membrane proteins. The sites identified in these simulation studies agree well with those identified by complementary experimental techniques. This demonstrates the power of the molecular dynamics approach in the prediction and characterization of lipid interaction sites on integral membrane proteins. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg. PMID:26946244

  18. Molecular mechanism of APC/C activation by mitotic phosphorylation.

    PubMed

    Zhang, Suyang; Chang, Leifu; Alfieri, Claudio; Zhang, Ziguo; Yang, Jing; Maslen, Sarah; Skehel, Mark; Barford, David

    2016-05-12

    In eukaryotes, the anaphase-promoting complex (APC/C, also known as the cyclosome) regulates the ubiquitin-dependent proteolysis of specific cell-cycle proteins to coordinate chromosome segregation in mitosis and entry into the G1 phase. The catalytic activity of the APC/C and its ability to specify the destruction of particular proteins at different phases of the cell cycle are controlled by its interaction with two structurally related coactivator subunits, Cdc20 and Cdh1. Coactivators recognize substrate degrons, and enhance the affinity of the APC/C for its cognate E2 (refs 4-6). During mitosis, cyclin-dependent kinase (Cdk) and polo-like kinase (Plk) control Cdc20- and Cdh1-mediated activation of the APC/C. Hyperphosphorylation of APC/C subunits, notably Apc1 and Apc3, is required for Cdc20 to activate the APC/C, whereas phosphorylation of Cdh1 prevents its association with the APC/C. Since both coactivators associate with the APC/C through their common C-box and Ile-Arg tail motifs, the mechanism underlying this differential regulation is unclear, as is the role of specific APC/C phosphorylation sites. Here, using cryo-electron microscopy and biochemical analysis, we define the molecular basis of how phosphorylation of human APC/C allows for its control by Cdc20. An auto-inhibitory segment of Apc1 acts as a molecular switch that in apo unphosphorylated APC/C interacts with the C-box binding site and obstructs engagement of Cdc20. Phosphorylation of the auto-inhibitory segment displaces it from the C-box-binding site. Efficient phosphorylation of the auto-inhibitory segment, and thus relief of auto-inhibition, requires the recruitment of Cdk-cyclin in complex with a Cdk regulatory subunit (Cks) to a hyperphosphorylated loop of Apc3. We also find that the small-molecule inhibitor, tosyl-l-arginine methyl ester, preferentially suppresses APC/C(Cdc20) rather than APC/C(Cdh1), and interacts with the binding sites of both the C-box and Ile-Arg tail motifs. Our

  19. The Use of Molecular Techniques at Hazardous Waste Sites

    EPA Science Inventory

    It is clear that typical protocols used for soil analysis would certainly fail to adequately interrogate ground-water treatment systems unless they were substantially modified. The modifications found necessary to compensate for the low biomass include molecular tools and techniq...

  20. Automated Site-Directed Drug Design: The Formation of Molecular Templates in Primary Structure Generation

    NASA Astrophysics Data System (ADS)

    Lewis, R. A.; Dean, P. M.

    1989-03-01

    In this paper the spacer skeleton concept is used to produce molecular graphs of putative ligands for binding sites. The skeletons are transformed into molecular templates within the constraints of the accessible surface of the ligand-binding site. A distance-matrix method is used to compare ligand points with vertices of the spacer skeleton through a permutation of all possible correspondences. A tolerance parameter is used to screen for poor matches. As a result. a small number of matched vertices and ligand points are produced. These are fitted into the site by a constrained optimization routine using an analytical function. Ligand points fall within the site and are optimally positioned adjacent to the corresponding site points, other vertices of the spacer skeleton lying beneath the accessible surface of the site are clipped off. A molecular template is thereby formed with its vertices linked to the ligand points. The final step is to verify that the bonding integrity of the skeleton remains. The computational methods outlined in this paper have been tested at two binding sites the pteridine binding site in dihydrofolate reductase and the amidinophenylpyruvate site of trypsin. Molecular graphs for both sites were generated automatically, they showed strong similarity to those of the natural ligands.

  1. Dissecting the active site of a photoreceptor protein

    NASA Astrophysics Data System (ADS)

    Hoff, Wouter; Hara, Miwa; Ren, Jie; Moghadam, Farzaneh; Xie, Aihua; Kumauchi, Masato

    While enzymes are quite large molecules, functionally important chemical events are often limited to a small region of the protein: the active site. The physical and chemical properties of residues at such active sites are often strongly altered compared to the same groups dissolved in water. Understanding such effects is important for unraveling the mechanisms underlying protein function and for protein engineering, but has proven challenging. Here we report on our ongoing efforts on using photoactive yellow protein (PYP), a bacterial photoreceptor, as a model system for such effects. We will report on the following questions: How many residues affect active site properties? Are these residues in direct physical contact with the active site? Can functionally important residues be recognized in the crystal structure of a protein? What structural resolution is needed to understand active sites? What spectroscopic techniques are most informative? Which weak interactions dominate active site properties?

  2. Active site densities, oxygen activation and adsorbed reactive oxygen in alcohol activation on npAu catalysts.

    PubMed

    Wang, Lu-Cun; Friend, C M; Fushimi, Rebecca; Madix, Robert J

    2016-07-01

    The activation of molecular O2 as well as the reactivity of adsorbed oxygen species is of central importance in aerobic selective oxidation chemistry on Au-based catalysts. Herein, we address the issue of O2 activation on unsupported nanoporous gold (npAu) catalysts by applying a transient pressure technique, a temporal analysis of products (TAP) reactor, to measure the saturation coverage of atomic oxygen, its collisional dissociation probability, the activation barrier for O2 dissociation, and the facility with which adsorbed O species activate methanol, the initial step in the catalytic cycle of esterification. The results from these experiments indicate that molecular O2 dissociation is associated with surface silver, that the density of reactive sites is quite low, that adsorbed oxygen atoms do not spill over from the sites of activation onto the surrounding surface, and that methanol reacts quite facilely with the adsorbed oxygen atoms. In addition, the O species from O2 dissociation exhibits reactivity for the selective oxidation of methanol but not for CO. The TAP experiments also revealed that the surface of the npAu catalyst is saturated with adsorbed O under steady state reaction conditions, at least for the pulse reaction. PMID:27376884

  3. Mars Surveyor Project Landing Site Activities

    NASA Technical Reports Server (NTRS)

    Gulick, Virginia C.; Briggs, Geoffrey; Saunders, R. Stephen; Gilmore, Martha; Soderblom, Larry

    1999-01-01

    The Mars Surveyor Program --now a cooperative program led by NASA and CNES along with other international partners -- is underway. It has the primary science objective of furthering our understanding of the biological potential and possible biological history of Mars and has the complementary objective of improving our understanding of martian climate evolution and planetary history The missions will develop technology and acquire data necessary for eventual human Exploration. Launches of orbiters, landers and rovers will take place in 2001 and in 2003; in 2005 a complete system will be launched capable of returning samples to Earth by 2008. A key aspect of the program is the selection of landing sites. This abstract 1) reports on the status of the landing site selection process that begins with the 2001 lander mission and 2) outlines be opportunities for the Mars community to provide input into the landing site selection process.

  4. Mars Surveyor Project Landing Site Activities

    NASA Technical Reports Server (NTRS)

    Gulick, V. C.; Briggs, Geoffrey; Saunders, R. Stephen; Gilmore, Martha; Soderblom, Larry

    1999-01-01

    The Mars Surveyor Program -- now a cooperative program led by NASA and CNES along with other international partners -- is underway. It has the primary science objective of furthering our understanding of the biological potential and possible biological history of Mars and has the complementary objective of improving our understanding of martian climate evolution and planetary history. The missions will develop technology and acquire data necessary for eventual human exploration. Launches of orbiters, landers and rovers will take place in 2001 and in 2003; in 2005 a complete system will be launched capable of returning samples to Earth by 2008. A key aspect of the program is the selection of landing sites. This abstract 1) reports on the status of the landing site selection process that begins with the 2001 lander mission and 2) outlines the opportunities for the Mars community to provide input into the landing site selection process.

  5. The Molecular Mechanism of P2Y1 Receptor Activation.

    PubMed

    Yuan, Shuguang; Chan, H C Stephen; Vogel, Horst; Filipek, Slawomir; Stevens, Raymond C; Palczewski, Krzysztof

    2016-08-22

    Human purinergic G protein-coupled receptor P2Y1 (P2Y1 R) is activated by adenosine 5'-diphosphate (ADP) to induce platelet activation and thereby serves as an important antithrombotic drug target. Crystal structures of P2Y1 R revealed that one ligand (MRS2500) binds to the extracellular vestibule of this GPCR, whereas another (BPTU) occupies the surface between transmembrane (TM) helices TM2 and TM3. We introduced a total of 20 μs all-atom long-timescale molecular dynamic (MD) simulations to inquire why two molecules in completely different locations both serve as antagonists while ADP activates the receptor. Our results indicate that BPTU acts as an antagonist by stabilizing extracellular helix bundles leading to an increase of the lipid order, whereas MRS2500 blocks signaling by occupying the ligand binding site. Both antagonists stabilize an ionic lock within the receptor. However, binding of ADP breaks this ionic lock, forming a continuous water channel that leads to P2Y1 R activation. PMID:27460867

  6. Interfacial activation-based molecular bioimprinting of lipolytic enzymes.

    PubMed Central

    Mingarro, I; Abad, C; Braco, L

    1995-01-01

    Interfacial activation-based molecular (bio)-imprinting (IAMI) has been developed to rationally improve the performance of lipolytic enzymes in nonaqueous environments. The strategy combinedly exploits (i) the known dramatic enhancement of the protein conformational rigidity in a water-restricted milieu and (ii) the reported conformational changes associated with the activation of these enzymes at lipid-water interfaces, which basically involves an increased substrate accessibility to the active site and/or an induction of a more competent catalytic machinery. Six model enzymes have been assayed in several model reactions in nonaqueous media. The results, rationalized in light of the present biochemical and structural knowledge, show that the IAMI approach represents a straightforward, versatile method to generate manageable, activated (kinetically trapped) forms of lipolytic enzymes, providing under optimal conditions nonaqueous rate enhancements of up to two orders of magnitude. It is also shown that imprintability of lipolytic enzymes depends not only on the nature of the enzyme but also on the "quality" of the interface used as the template. PMID:7724558

  7. The Molecular Mechanism of P2Y1 Receptor Activation

    PubMed Central

    Chan, H. C. Stephen; Vogel, Horst; Filipek, Slawomir

    2016-01-01

    Human purinergic G protein-coupled receptor P2Y1 (P2Y1R) is activated by adenosine 5’-diphosphate (ADP) to induce platelet activation and thereby serves as an important antithrombotic drug target. Crystal structures of P2Y1R revealed that one ligand (MRS2500) binds to the extracellular vestibule of this GPCR, whereas another (BPTU) occupies the surface between transmembrane (TM) helices TM2 and TM3. We introduced a total of 20 µs all-atom long-timescale molecular dynamic (MD) simulations to inquire why two molecules in completely different locations both serve as antagonists while ADP activates the receptor. Our results indicate that BPTU acts as an antagonist by stabilizing extracellular helix bundles leading to an increase of the lipid order, whereas MRS2500 blocks signaling by occupying the ligand binding site. Both antagonists stabilize an ionic lock within the receptor. However, binding of ADP breaks this ionic lock, forming a continuous water channel that leads to P2Y1R activation. PMID:27460867

  8. The bifunctional active site of s-adenosylmethionine synthetase. Roles of the active site aspartates.

    PubMed

    Taylor, J C; Markham, G D

    1999-11-12

    S-Adenosylmethionine (AdoMet) synthetase catalyzes the biosynthesis of AdoMet in a unique enzymatic reaction. Initially the sulfur of methionine displaces the intact tripolyphosphate chain (PPP(i)) from ATP, and subsequently PPP(i) is hydrolyzed to PP(i) and P(i) before product release. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site contains four aspartate residues. Aspartate residues Asp-16* and Asp-271 individually provide the sole protein ligand to one of the two required Mg(2+) ions (* denotes a residue from a second subunit); aspartates Asp-118 and Asp-238* are proposed to interact with methionine. Each aspartate has been changed to an uncharged asparagine, and the metal binding residues were also changed to alanine, to assess the roles of charge and ligation ability on catalytic efficiency. The resultant enzyme variants all structurally resemble the wild type enzyme as indicated by circular dichroism spectra and are tetramers. However, all have k(cat) reductions of approximately 10(3)-fold in AdoMet synthesis, whereas the MgATP and methionine K(m) values change by less than 3- and 8-fold, respectively. In the partial reaction of PPP(i) hydrolysis, mutants of the Mg(2+) binding residues have >700-fold reduced catalytic efficiency (k(cat)/K(m)), whereas the D118N and D238*N mutants are impaired less than 35-fold. The catalytic efficiency for PPP(i) hydrolysis by Mg(2+) site mutants is improved by AdoMet, like the wild type enzyme. In contrast AdoMet reduces the catalytic efficiency for PPP(i) hydrolysis by the D118N and D238*N mutants, indicating that the events involved in AdoMet activation are hindered in these methionyl binding site mutants. Ca(2+) uniquely activates the D271A mutant enzyme to 15% of the level of Mg(2+), in contrast to the approximately 1% Ca(2+) activation of the wild type enzyme. This indicates that the Asp-271 side chain size is a discriminator between the activating ability of Ca(2+) and the

  9. Formation of target-specific binding sites in enzymes: solid-phase molecular imprinting of HRP

    NASA Astrophysics Data System (ADS)

    Czulak, J.; Guerreiro, A.; Metran, K.; Canfarotta, F.; Goddard, A.; Cowan, R. H.; Trochimczuk, A. W.; Piletsky, S.

    2016-05-01

    Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates.Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike

  10. The active site of ribulose-bisphosphate carboxylase/oxygenase

    SciTech Connect

    Hartman, F.C.

    1991-01-01

    The active site of ribulose-bisphosphate carboxylase/oxygenase requires interacting domains of adjacent, identical subunits. Most active-site residues are located within the loop regions of an eight-stranded {beta}/{alpha}-barrel which constitutes the larger C-terminal domain; additional key residues are located within a segment of the smaller N-terminal domain which partially covers the mouth of the barrel. Site-directed mutagenesis of the gene encoding the enzyme from Rhodospirillum rubrum has been used to delineate functions of active-site residues. 6 refs., 2 figs.

  11. A study on the flexibility of enzyme active sites

    PubMed Central

    2011-01-01

    Background A common assumption about enzyme active sites is that their structures are highly conserved to specifically distinguish between closely similar compounds. However, with the discovery of distinct enzymes with similar reaction chemistries, more and more studies discussing the structural flexibility of the active site have been conducted. Results Most of the existing works on the flexibility of active sites focuses on a set of pre-selected active sites that were already known to be flexible. This study, on the other hand, proposes an analysis framework composed of a new data collecting strategy, a local structure alignment tool and several physicochemical measures derived from the alignments. The method proposed to identify flexible active sites is highly automated and robust so that more extensive studies will be feasible in the future. The experimental results show the proposed method is (a) consistent with previous works based on manually identified flexible active sites and (b) capable of identifying potentially new flexible active sites. Conclusions This proposed analysis framework and the former analyses on flexibility have their own advantages and disadvantage, depending on the cause of the flexibility. In this regard, this study proposes an alternative that complements previous studies and helps to construct a more comprehensive view of the flexibility of enzyme active sites. PMID:21342563

  12. Safety Oversight of Decommissioning Activities at DOE Nuclear Sites

    SciTech Connect

    Zull, Lawrence M.; Yeniscavich, William

    2008-01-15

    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.

  13. Changes in active site histidine hydrogen bonding trigger cryptochrome activation.

    PubMed

    Ganguly, Abir; Manahan, Craig C; Top, Deniz; Yee, Estella F; Lin, Changfan; Young, Michael W; Thiel, Walter; Crane, Brian R

    2016-09-01

    Cryptochrome (CRY) is the principal light sensor of the insect circadian clock. Photoreduction of the Drosophila CRY (dCRY) flavin cofactor to the anionic semiquinone (ASQ) restructures a C-terminal tail helix (CTT) that otherwise inhibits interactions with targets that include the clock protein Timeless (TIM). All-atom molecular dynamics (MD) simulations indicate that flavin reduction destabilizes the CTT, which undergoes large-scale conformational changes (the CTT release) on short (25 ns) timescales. The CTT release correlates with the conformation and protonation state of conserved His378, which resides between the CTT and the flavin cofactor. Poisson-Boltzmann calculations indicate that flavin reduction substantially increases the His378 pKa Consistent with coupling between ASQ formation and His378 protonation, dCRY displays reduced photoreduction rates with increasing pH; however, His378Asn/Arg variants show no such pH dependence. Replica-exchange MD simulations also support CTT release mediated by changes in His378 hydrogen bonding and verify other responsive regions of the protein previously identified by proteolytic sensitivity assays. His378 dCRY variants show varying abilities to light-activate TIM and undergo self-degradation in cellular assays. Surprisingly, His378Arg/Lys variants do not degrade in light despite maintaining reactivity toward TIM, thereby implicating different conformational responses in these two functions. Thus, the dCRY photosensory mechanism involves flavin photoreduction coupled to protonation of His378, whose perturbed hydrogen-bonding pattern alters the CTT and surrounding regions. PMID:27551082

  14. DOE site performance assessment activities. Radioactive Waste Technical Support Program

    SciTech Connect

    Not Available

    1990-07-01

    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.

  15. Savannah River Site prioritization of transition activities

    SciTech Connect

    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.

  16. Activation of molecular catalysts using semiconductor quantum dots

    DOEpatents

    Meyer, Thomas J.; Sykora, Milan; Klimov, Victor I.

    2011-10-04

    Photocatalytic materials based on coupling of semiconductor nanocrystalline quantum dots (NQD) and molecular catalysts. These materials have capability to drive or catalyze non-spontaneous chemical reactions in the presence of visible radiation, ultraviolet radiation, or both. The NQD functions in these materials as a light absorber and charge generator. Following light absorption, the NQD activates a molecular catalyst adsorbed on the surface of the NQD via transfer of one or more charges (either electrons or electron-holes) from the NQD to the molecular catalyst. The activated molecular catalyst can then drive a chemical reaction. A photoelectrolytic device that includes such photocatalytic materials is also described.

  17. Dynamical Observations of Local Bio-molecular Sites Using Nanocrystals

    SciTech Connect

    Sasaki, Yuji C.; Okumura, Yasuaki; Ohishi, Noboru

    2004-05-12

    Recently, we succeeded time-resolved x-ray observations of picometer-scale slow Brownian motions of individual protein molecules in aqueous solutions. In this work, we investigated the relationship between individual protein molecules and the labeled nanocrystals. In order to control the number of the bonding sites on the labeled gold nanocrystals, we utilized both the periodical structure in Actin filaments and using the mercury compound. As a result, we observed free Brownian Motions of the C-terminal in F-actin, when single gold nanocrystal is linked to single G-actin.

  18. Multiple-site replacement analogs of glucagon. A molecular basis for antagonist design.

    PubMed

    Unson, C G; Wu, C R; Fitzpatrick, K J; Merrifield, R B

    1994-04-29

    Extensive structure activity analysis has allowed us to identify specific residues in the glucagon sequence that are responsible for either receptor recognition or signal transduction. For instance, we have demonstrated that aspartic acid 9 and histidine 1 are essential for activation, and that an ionic interaction between the negative carboxylate and the protonated imidazole may contribute to the activation reaction at the molecular level. In the absence of the carboxylic group at position 9, aspartic 21 or aspartic 15 might furnish distal electrostatic effects to maintain partial agonism. Further investigation established that each of the 4 serine residues in the hormone play distinct roles. Serine 8 provides an important determinant of binding. Whereas neither serines 2, 11, nor 16 are required for receptor recognition. We have shown that serine 16 is essential for signal transduction and thus have identified it to be the third residue in glucagon to participate in a putative catalytic triad together with aspartic 9 and histidine 1, in the transduction of the glucagon response. In this work, we utilized insights into the functional significance of particular residues in the peptide appropriated from our structure-function assignments, as the basis of a molecular approach for the design of active-site directed antagonists of glucagon. The importance as well as the accuracy of our findings are confirmed by the synthesis of a series of improved glucagon antagonists based on replacements at positions 1, 9, 11, 16, and 21. The inhibition index, (I/A)50, of our best antagonist des-His1-[Nle9-Ala11-Ala16]glucagon amide, has been improved 10-fold over the previous best glucagon inhibitor. PMID:8175663

  19. Ionizable Side Chains at Catalytic Active Sites of Enzymes

    PubMed Central

    Jimenez-Morales, David; Liang, Jie

    2012-01-01

    Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1072 Å3. The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes. PMID:22484856

  20. Powerful Molecular Outflows in Nearby Active Galaxies

    NASA Astrophysics Data System (ADS)

    Veilleux, Sylvain; Meléndez, Marcio

    2014-07-01

    We report the results from a systematic search for molecular (OH 119 μm) outflows with Herschel-PACS† in a sample of 43 nearby (z < 0.3) galaxy mergers, mostly ultraluminous infrared galaxies (ULIRGs) and QSOs. We find that the character of the OH feature (strength of the absorption relative to the emission) correlates with that of the 9.7-μm silicate feature, a measure of obscuration in ULIRGs. Unambiguous evidence for molecular outflows, based on the detection of OH absorption profiles with median velocities more blueshifted than -50 km s-1, is seen in 26 (70%) of the 37 OH-detected targets, suggesting a wide-angle (~ 145°) outflow geometry. Conversely, unambiguous evidence for molecular inflows, based on the detection of OH absorption profiles with median velocities more redshifted than +50 km s-1, is seen in only 4 objects, suggesting a planar or filamentary geometry for the inflowing gas. Terminal outflow velocities of ~ -1000 km s-1 are measured in several objects, but median outflow velocities are typically ~ -200 km s-1. While the outflow velocities show no statistically significant dependence on the star formation rate, they are distinctly more blueshifted among systems with large AGN fractions and luminosities [log (L AGN/L ⊙) >= 11.8 +/- 0.3]. The quasars in these systems play a dominant role in driving the molecular outflows. In contrast, the most AGN dominated systems, where OH is seen purely in emission, show relatively modest OH line widths, despite their large AGN luminosities, perhaps indicating that molecular outflows subside once the quasar has cleared a path through the obscuring material.

  1. Correlated structural kinetics and retarded solvent dynamics at the metalloprotease active site

    PubMed Central

    Grossman, Moran; Born, Benjamin; Heyden, Matthias; Tworowski, Dmitry; Fields, Gregg B; Sagi, Irit; Havenith, Martina

    2012-01-01

    Solvent dynamics can play a major role in enzyme activity, but obtaining an accurate, quantitative picture of solvent activity during catalysis is quite challenging. Here, we combine terahertz spectroscopy and X-ray absorption analyses to measure changes in the coupled water-protein motions during peptide hydrolysis by a zinc-dependent human metalloprotease. These changes were tightly correlated with rearrangements at the active site during the formation of productive enzyme-substrate intermediates and were different from those in an enzyme–inhibitor complex. Molecular dynamics simulations showed a steep gradient of fast-to-slow coupled protein-water motions around the protein, active site and substrate. Our results show that water retardation occurs before formation of the functional Michaelis complex. We propose that the observed gradient of coupled protein-water motions may assist enzyme-substrate interactions through water-polarizing mechanisms that are remotely mediated by the catalytic metal ion and the enzyme active site. PMID:21926991

  2. Correlated structural kinetics and retarded solvent dynamics at the metalloprotease active site

    SciTech Connect

    Grossman, Moran; Born, Benjamin; Heyden, Matthias; Tworowski, Dmitry; Fields, Gregg B.; Sagi, Irit; Havenith, Martina

    2011-09-18

    Solvent dynamics can play a major role in enzyme activity, but obtaining an accurate, quantitative picture of solvent activity during catalysis is quite challenging. Here, we combine terahertz spectroscopy and X-ray absorption analyses to measure changes in the coupled water-protein motions during peptide hydrolysis by a zinc-dependent human metalloprotease. These changes were tightly correlated with rearrangements at the active site during the formation of productive enzyme-substrate intermediates and were different from those in an enzyme–inhibitor complex. Molecular dynamics simulations showed a steep gradient of fast-to-slow coupled protein-water motions around the protein, active site and substrate. Our results show that water retardation occurs before formation of the functional Michaelis complex. We propose that the observed gradient of coupled protein-water motions may assist enzyme-substrate interactions through water-polarizing mechanisms that are remotely mediated by the catalytic metal ion and the enzyme active site.

  3. Active Site Metal Occupancy and Cyclic Di-GMP Phosphodiesterase Activity of Thermotoga maritima HD-GYP.

    PubMed

    Miner, Kyle D; Kurtz, Donald M

    2016-02-16

    HD-GYPs make up a subclass of the metal-dependent HD phosphohydrolase superfamily and catalyze conversion of cyclic di(3',5')-guanosine monophosphate (c-di-GMP) to 5'-phosphoguanylyl-(3'→5')-guanosine (pGpG) and GMP. Until now, the only reported crystal structure of an HD-GYP that also exhibits c-di-GMP phosphodiesterase activity contains a His/carboxylate ligated triiron active site. However, other structural and phylogenetic correlations indicate that some HD-GYPs contain dimetal active sites. Here we provide evidence that an HD-GYP c-di-GMP phosphodiesterase, TM0186, from Thermotoga maritima can accommodate both di- and trimetal active sites. We show that an as-isolated iron-containing TM0186 has an oxo/carboxylato-bridged diferric site, and that the reduced (diferrous) form is necessary and sufficient to catalyze conversion of c-di-GMP to pGpG, but that conversion of pGpG to GMP requires more than two metals per active site. Similar c-di-GMP phosphodiesterase activities were obtained with divalent iron or manganese. On the basis of activity correlations with several putative metal ligand residue variants and molecular dynamics simulations, we propose that TM0186 can accommodate both di- and trimetal active sites. Our results also suggest that a Glu residue conserved in a subset of HD-GYPs is required for formation of the trimetal site and can also serve as a labile ligand to the dimetal site. Given the anaerobic growth requirement of T. maritima, we suggest that this HD-GYP can function in vivo with either divalent iron or manganese occupying di- and trimetal sites. PMID:26786892

  4. Formation of target-specific binding sites in enzymes: solid-phase molecular imprinting of HRP.

    PubMed

    Czulak, J; Guerreiro, A; Metran, K; Canfarotta, F; Goddard, A; Cowan, R H; Trochimczuk, A W; Piletsky, S

    2016-06-01

    Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates. PMID:27174700

  5. Molecular emission in chemically active protostellar outflows

    NASA Astrophysics Data System (ADS)

    Lefloch, B.

    2011-12-01

    Protostellar outflows play an important role in the dynamical and chemical evolution of cloud through shocks. The Herschel Space Observatory (HSO) brings new insight both on the molecular content and the physical conditions in protostellar shocks through high spectral and angular resolution studies of the emission of major gas cooling agents and hydrides. The Herschel/CHESS key-program is carrying out an in depth study of the prototypical shock region L1157-B1. Analysis of the line profiles detected allows to constrain the formation/destruction route of various molecular species, in relation with the predictions of MHD shock models. The Herschel/WISH key-program investigates the properties and origin of water emission in a broad sample of protostellar outflows and envelopes. Implications of the first results for future studies on mass-loss phenomena are discussed.

  6. Coarse-grained molecular simulation of epidermal growth factor receptor protein tyrosine kinase multi-site self-phosphorylation.

    PubMed

    Koland, John G

    2014-01-01

    Upon the ligand-dependent dimerization of the epidermal growth factor receptor (EGFR), the intrinsic protein tyrosine kinase (PTK) activity of one receptor monomer is activated, and the dimeric receptor undergoes self-phosphorylation at any of eight candidate phosphorylation sites (P-sites) in either of the two C-terminal (CT) domains. While the structures of the extracellular ligand binding and intracellular PTK domains are known, that of the ∼225-amino acid CT domain is not, presumably because it is disordered. Receptor phosphorylation on CT domain P-sites is critical in signaling because of the binding of specific signaling effector molecules to individual phosphorylated P-sites. To investigate how the combination of conventional substrate recognition and the unique topological factors involved in the CT domain self-phosphorylation reaction lead to selectivity in P-site phosphorylation, we performed coarse-grained molecular simulations of the P-site/catalytic site binding reactions that precede EGFR self-phosphorylation events. Our results indicate that self-phosphorylation of the dimeric EGFR, although generally believed to occur in trans, may well occur with a similar efficiency in cis, with the P-sites of both receptor monomers being phosphorylated to a similar extent. An exception was the case of the most kinase-proximal P-site-992, the catalytic site binding of which occurred exclusively in cis via an intramolecular reaction. We discovered that the in cis interaction of P-site-992 with the catalytic site was facilitated by a cleft between the N-terminal and C-terminal lobes of the PTK domain that allows the short CT domain sequence tethering P-site-992 to the PTK core to reach the catalytic site. Our work provides several new mechanistic insights into the EGFR self-phosphorylation reaction, and demonstrates the potential of coarse-grained molecular simulation approaches for investigating the complexities of self-phosphorylation in molecules such as EGFR

  7. Detection of DNA methyltransferase activity using allosteric molecular beacons.

    PubMed

    Zhang, Weiting; Zu, Xiaolong; Song, Yanling; Zhu, Zhi; Yang, Chaoyong James

    2016-01-21

    Abnormal DNA methylation patterns caused by altered DNA methyltransferase (MTase) activity are closely associated with cancer. Herein, using DNA adenine methylation methyltransferase (Dam MTase) as a model analyte, we designed an allosteric molecular beacon (aMB) for sensitive detection of Dam MTase activity. When the specific site in an aMB is methylated by Dam MTase, the probe can be cut by the restriction nuclease DpnI to release a fluorophore labeled aptamer specific for streptavidin (SA) which will bind to SA beads to generate highly fluorescent beads for easy signal readout by a microscope or flow cytometer. However, aMBs maintain a hairpin structure without the binding ability to SA beads in the absence of Dam MTase, leading to weakly fluorescent SA beads. Unlike the existing signal amplified assays, our method is simpler and more convenient. The high performance of the aptamer and the easy bead separation process make this probe superior to other methods for the detection of MTase in complex biological systems. Overall, the proposed method with a detection limit of 0.57 U mL(-1) for Dam MTase shows great potential for further applications in the detection of other MTases, screening of MTase inhibitors, and early diagnosis of cancer. PMID:26478921

  8. Molecular architecture of the ribosome-bound Hepatitis C Virus internal ribosomal entry site RNA.

    PubMed

    Yamamoto, Hiroshi; Collier, Marianne; Loerke, Justus; Ismer, Jochen; Schmidt, Andrea; Hilal, Tarek; Sprink, Thiemo; Yamamoto, Kaori; Mielke, Thorsten; Bürger, Jörg; Shaikh, Tanvir R; Dabrowski, Marylena; Hildebrand, Peter W; Scheerer, Patrick; Spahn, Christian M T

    2015-12-14

    Internal ribosomal entry sites (IRESs) are structured cis-acting RNAs that drive an alternative, cap-independent translation initiation pathway. They are used by many viruses to hijack the translational machinery of the host cell. IRESs facilitate translation initiation by recruiting and actively manipulating the eukaryotic ribosome using only a subset of canonical initiation factor and IRES transacting factors. Here we present cryo-EM reconstructions of the ribosome 80S- and 40S-bound Hepatitis C Virus (HCV) IRES. The presence of four subpopulations for the 80S•HCV IRES complex reveals dynamic conformational modes of the complex. At a global resolution of 3.9 Å for the most stable complex, a derived atomic model reveals a complex fold of the IRES RNA and molecular details of its interaction with the ribosome. The comparison of obtained structures explains how a modular architecture facilitates mRNA loading and tRNA binding to the P-site. This information provides the structural foundation for understanding the mechanism of HCV IRES RNA-driven translation initiation. PMID:26604301

  9. Active Sites Environmental Monitoring Program FY 1996 annual report

    SciTech Connect

    Morrissey, C.M.; Marshall, D.S.; Cunningham, G.R.

    1997-11-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from October 1995 through September 1996. The Radioactive Solid Waste Operations Group (RSWOG) of the Waste Management and Remedial Action Division (WMRAD) and the Environmental Sciences Division (ESD) at Oak Ridge National Laboratory (ORNL) established ASEMP in 1989. The purpose of the program is to provide early detection and performance monitoring at active low-level waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 North as required by Chapters 2 and 3 of US Department of Energy Order 5820.2A.

  10. Active sites environmental monitoring Program - Program Plan: Revision 2

    SciTech Connect

    Morrissey, C.M.; Hicks, D.S.; Ashwood, T.L.; Cunningham, G.R.

    1994-05-01

    The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of active low-level-waste (LLW) and transuranic (TRU) waste facilities at Oak Ridge National Laboratory (ORNL). Several changes have recently occurred in regard to the sites that are currently used for waste storage and disposal. These changes require a second set of revisions to the ASEMP program plan. This document incorporates those revisions. This program plan presents the organization and procedures for monitoring the active sites. The program plan also provides internal reporting levels to guide the evaluation of monitoring results.

  11. Computational mutagenesis reveals the role of active-site tyrosine in stabilising a boat conformation for the substrate: QM/MM molecular dynamics studies of wild-type and mutant xylanases.

    PubMed

    Soliman, Mahmoud E S; Ruggiero, Giuseppe D; Pernía, J Javier Ruiz; Greig, Ian R; Williams, Ian H

    2009-02-01

    Molecular dynamics simulations have been performed for non-covalent complexes of phenyl beta-xylobioside with the retaining endo-beta-1,4-xylanase from B. circulans (BCX) and its Tyr69Phe mutant using a hybrid QM/MM methodology. A trajectory initiated for the wild-type enzyme-substrate complex with the proximal xylose ring bound at the -1 subsite (adjacent to the scissile glycosidic bond) in the (4)C(1) chair conformation shows spontaneous transformation to the (2,5)B boat conformation, and potential of mean force calculations indicate that the boat is approximately 30 kJ mol(-1) lower in free energy than the chair. Analogous simulations for the mutant lacking one oxygen atom confirm the key role of Tyr69 in stabilizing the boat in preference to the (4)C(1) chair conformation, with a relative free energy difference of about 20 kJ mol(-1), by donating a hydrogen bond to the endocyclic oxygen of the proximal xylose ring. QM/MM MD simulations for phenyl beta-xyloside in water, with and without a propionate/propionic acid pair to mimic the catalytic glutamate/glutamic acid pair of the enzyme, show the (4)C(1) chair to be stable, although a hydrogen bond between the OH group at C2 of xylose and the propionate moiety seems to provide some stabilization for the (2,5)B conformation. PMID:19156310

  12. Failure of origin activation in response to fork stalling leads to chromosomal instability at fragile sites.

    PubMed

    Ozeri-Galai, Efrat; Lebofsky, Ronald; Rahat, Ayelet; Bester, Assaf C; Bensimon, Aaron; Kerem, Batsheva

    2011-07-01

    Perturbed DNA replication in early stages of cancer development induces chromosomal instability preferentially at fragile sites. However, the molecular basis for this instability is unknown. Here, we show that even under normal growth conditions, replication fork progression along the fragile site, FRA16C, is slow and forks frequently stall at AT-rich sequences, leading to activation of additional origins to enable replication completion. Under mild replication stress, the frequency of stalling at AT-rich sequences is further increased. Strikingly, unlike in the entire genome, in the FRA16C region additional origins are not activated, suggesting that all potential origins are already activated under normal conditions. Thus, the basis for FRA16C fragility is replication fork stalling at AT-rich sequences and inability to activate additional origins under replication stress. Our results provide a mechanism explaining the replication stress sensitivity of fragile sites and thus, the basis for genomic instability during early stages of cancer development. PMID:21726815

  13. Molecular aspects of vitamin D anticancer activity.

    PubMed

    Picotto, Gabriela; Liaudat, Ana C; Bohl, Luciana; Tolosa de Talamoni, Nori

    2012-10-01

    Environment may influence the development and prevention of cancer. Calcitriol has been associated with calcium homeostasis regulation. Many epidemiological, biochemical, and genetic studies have shown non-classic effects of vitamin D, such as its involvement in the progression of different cancers. Although vitamin D induces cellular arrest, triggers apoptotic pathways, inhibits angiogenesis, and alters cellular adhesion, the precise mechanisms of its action are still not completely established. This article will present a revision about the molecular aspects proposed to be involved in the anticancer action of calcitriol. Adequate levels of vitamin D to prevent cancer development will also be discussed. PMID:22963190

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

    PubMed

    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

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

    PubMed

    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

  16. Conformational coupling, bridge helix dynamics and active site dehydration in catalysis by RNA polymerase

    PubMed Central

    Seibold, Steve A.; Singh, Badri Nath; Zhang, Chunfen; Kireeva, Maria; Domecq, Céline; Bouchard, Annie; Nazione, Anthony M.; Feig, Michael; Cukier, Robert I.; Coulombe, Benoit; Kashlev, Mikhail; Hampsey, Michael; Burton, Zachary F.

    2010-01-01

    Molecular dynamics simulation of Thermus thermophilus (Tt) RNA polymerase (RNAP) in a catalytic conformation demonstrates that the active site dNMP-NTP base pair must be substantially dehydrated to support full active site closing and optimum conditions for phosphodiester bond synthesis. In silico mutant β R428A RNAP, which was designed based on substitutions at the homologous position (Rpb2 R512) of Saccharomyces cerevisiae (Sc) RNAP II, was used as a reference structure to compare to Tt RNAP in simulations. Long range conformational coupling linking a dynamic segment of the bridge α-helix, the extended fork loop, the active site, and the trigger loop-trigger helix is apparent and adversely affected in β R428A RNAP. Furthermore, bridge helix bending is detected in the catalytic structure, indicating that bridge helix dynamics may regulate phosphodiester bond synthesis as well as translocation. An active site “latch” assembly that includes a key trigger helix residue Tt β’ H1242 and highly conserved active site residues β E445 and R557 appears to help regulate active site hydration/dehydration. The potential relevance of these observations in understanding RNAP and DNAP induced fit and fidelity is discussed. PMID:20478425

  17. Mimicking enzymatic active sites on surfaces for energy conversion chemistry.

    PubMed

    Gutzler, Rico; Stepanow, Sebastian; Grumelli, Doris; Lingenfelder, Magalí; Kern, Klaus

    2015-07-21

    Metal-organic supramolecular chemistry on surfaces has matured to a point where its underlying growth mechanisms are well understood and structures of defined coordination environments of metal atoms can be synthesized in a controlled and reproducible procedure. With surface-confined molecular self-assembly, scientists have a tool box at hand which can be used to prepare structures with desired properties, as for example a defined oxidation number and spin state of the transition metal atoms within the organic matrix. From a structural point of view, these coordination sites in the supramolecular structure resemble the catalytically active sites of metallo-enzymes, both characterized by metal centers coordinated to organic ligands. Several chemical reactions take place at these embedded metal ions in enzymes and the question arises whether these reactions also take place using metal-organic networks as catalysts. Mimicking the active site of metal atoms and organic ligands of enzymes in artificial systems is the key to understanding the selectivity and efficiency of enzymatic reactions. Their catalytic activity depends on various parameters including the charge and spin configuration in the metal ion, but also on the organic environment, which can stabilize intermediate reaction products, inhibits catalytic deactivation, and serves mostly as a transport channel for the reactants and products and therefore ensures the selectivity of the enzyme. Charge and spin on the transition metal in enzymes depend on the one hand on the specific metal element, and on the other hand on its organic coordination environment. These two parameters can carefully be adjusted in surface confined metal-organic networks, which can be synthesized by virtue of combinatorial mixing of building synthons. Different organic ligands with varying functional groups can be combined with several transition metals and spontaneously assemble into ordered networks. The catalytically active metal

  18. A molecular dynamics examination on mutation-induced catalase activity in coral allene oxide synthase.

    PubMed

    De Luna, Phil; Bushnell, Eric A C; Gauld, James W

    2013-11-27

    Coral allene oxide synthase (cAOS) catalyzes the formation of allene oxides from fatty acid hydroperoxides. Interestingly, its active site differs from that of catalase by only a single residue yet is incapable of catalase activity. That is, it is unable to catalyze the decomposition of hydrogen peroxide to molecular oxygen and water. However, the single active-site mutation T66V allows cAOS to exhibit catalase activity. We have performed a series of molecular dynamics (MD) simulations in order to gain insights into the differences in substrate (8R-hydroperoxyeicosatetraenoic) and H2O2 active site binding between wild-type cAOS and the T66V mutant cAOS. It is observed that in wild-type cAOS the active site Thr66 residue consistently forms a strong hydrogen-bonding interaction with H2O2 (catalase substrate) and, importantly, with the aid of His67 helps to pull H2O2 away from the heme Fe center. In contrast, in the T66V-cAOS mutant the H2O2 is much closer to the heme's Fe center and now forms a consistent Fe···O2H2 interaction. In addition, the His67···H2O2 distance shortens considerably, increasing the likelihood of a Cpd I intermediate and hence exhibiting catalase activity. PMID:24164352

  19. Comparative molecular modelling of biologically active sterols

    NASA Astrophysics Data System (ADS)

    Baran, Mariusz; Mazerski, Jan

    2015-04-01

    Membrane sterols are targets for a clinically important antifungal agent - amphotericin B. The relatively specific antifungal action of the drug is based on a stronger interaction of amphotericin B with fungal ergosterol than with mammalian cholesterol. Conformational space occupied by six sterols has been defined using the molecular dynamics method to establish if the conformational features correspond to the preferential interaction of amphotericin B with ergosterol as compared with cholesterol. The compounds studied were chosen on the basis of structural features characteristic for cholesterol and ergosterol and on available experimental data on the ability to form complexes with the antibiotic. Statistical analysis of the data obtained has been performed. The results show similarity of the conformational spaces occupied by all the sterols tested. This suggests that the conformational differences of sterol molecules are not the major feature responsible for the differential sterol - drug affinity.

  20. The Three Mycobacterium tuberculosis Antigen 85 Isoforms Have Unique Substrates and Activities Determined by Non-active Site Regions*

    PubMed Central

    Backus, Keriann M.; Dolan, Michael A.; Barry, Conor S.; Joe, Maju; McPhie, Peter; Boshoff, Helena I. M.; Lowary, Todd L.; Davis, Benjamin G.; Barry, Clifton E.

    2014-01-01

    The three isoforms of antigen 85 (A, B, and C) are the most abundant secreted mycobacterial proteins and catalyze transesterification reactions that synthesize mycolated arabinogalactan, trehalose monomycolate (TMM), and trehalose dimycolate (TDM), important constituents of the outermost layer of the cellular envelope of Mycobacterium tuberculosis. These three enzymes are nearly identical at the active site and have therefore been postulated to exist to evade host immunity. Distal to the active site is a second putative carbohydrate-binding site of lower homology. Mutagenesis of the three isoforms at this second site affected both substrate selectivity and overall catalytic activity in vitro. Using synthetic and natural substrates, we show that these three enzymes exhibit unique selectivity; antigen 85A more efficiently mycolates TMM to form TDM, whereas C (and to a lesser extent B) has a higher rate of activity using free trehalose to form TMM. This difference in substrate selectivity extends to the hexasaccharide fragment of cell wall arabinan. Mutation of secondary site residues from the most active isoform (C) into those present in A or B partially interconverts this substrate selectivity. These experiments in combination with molecular dynamics simulations reveal that differences in the N-terminal helix α9, the adjacent Pro216–Phe228 loop, and helix α5 are the likely cause of changes in activity and substrate selectivity. These differences explain the existence of three isoforms and will allow for future work in developing inhibitors. PMID:25028517

  1. Active site - a site of binding of affinity inhibitors in baker's yeast inorganic pyrophosphatase

    SciTech Connect

    Svyato, I.E.; Sklyankina, V.A.; Avaeva, S.M.

    1986-03-20

    The interaction of the enzyme-substrate complex with methyl phosphate, O-phosphoethanolamine, O-phosphopropanolamine, N-acetylphosphoserine, and phosphoglyolic acid, as well as pyrophosphatase, modified by monoesters of phosphoric acid, with pyrophosphate and tripolyphosphate, was investigated. It was shown that the enzyme containing the substrate in the active site does not react with monophosphates, but modified pyrophosphatase entirely retains the ability to bind polyanions to the regulatory site. It is concluded that the inactivation of baker's yeast inorganic pyrophosphatase by monoesters of phosphoric acid, which are affinity inhibitors of it, is the result of modification of the active site of the enzyme.

  2. Computational investigation of stoichiometric effects, binding site heterogeneities, and selectivities of molecularly imprinted polymers.

    PubMed

    Terracina, Jacob J; Bergkvist, Magnus; Sharfstein, Susan T

    2016-06-01

    A series of quantum mechanical (QM) computational optimizations of molecularly imprinted polymer (MIP) systems were used to determine optimal monomer-to-target ratios. Imidazole- and xanthine-derived target molecules were studied. The investigation included both small-scale models (3-7 molecules) and larger-scale models (15-35 molecules). The optimal ratios differed between the small and larger scales. For the larger models containing multiple targets, binding-site surface area analysis was used to quantify the heterogeneity of these sites. The more fully surrounded sites had greater binding energies. No discretization of binding modes was seen, furthering arguments for continuous affinity distribution models. Molecular mechanical (MM) docking was then used to measure the selectivities of the QM-optimized binding sites. Selectivity was also shown to improve as binding sites become more fully encased by the monomers. For internal sites, docking consistently showed selectivity favoring the molecules that had been imprinted via QM geometry optimizations. The computationally imprinted sites were shown to exhibit size-, shape-, and polarity-based selectivity. Here we present a novel approach to investigate the selectivity and heterogeneity of imprinted polymer binding sites, by applying the rapid orientation screening of MM docking to the highly accurate QM-optimized geometries. Modeling schemes were designed such that no computing clusters or other specialized modeling equipment would be required. Improving the in silico analysis of MIP system properties will ultimately allow for the production of more sensitive and selective polymers. PMID:27207254

  3. Molecular mechanisms of asymmetric RAF dimer activation.

    PubMed

    Jambrina, Pablo G; Bohuszewicz, Olga; Buchete, Nicolae-Viorel; Kolch, Walter; Rosta, Edina

    2014-08-01

    Protein phosphorylation is one of the most common post-translational modifications in cell regulatory mechanisms. Dimerization plays also a crucial role in the kinase activity of many kinases, including RAF, CDK2 (cyclin-dependent kinase 2) and EGFR (epidermal growth factor receptor), with heterodimers often being the most active forms. However, the structural and mechanistic details of how phosphorylation affects the activity of homo- and hetero-dimers are largely unknown. Experimentally, synthesizing protein samples with fully specified and homogeneous phosphorylation states remains a challenge for structural biology and biochemical studies. Typically, multiple changes in phosphorylation lead to activation of the same protein, which makes structural determination methods particularly difficult. It is also not well understood how the occurrence of phosphorylation and dimerization processes synergize to affect kinase activities. In the present article, we review available structural data and discuss how MD simulations can be used to model conformational transitions of RAF kinase dimers, in both their phosphorylated and unphosphorylated forms. PMID:25109958

  4. A small ribozyme with dual-site kinase activity

    PubMed Central

    Biondi, Elisa; Maxwell, Adam W.R.; Burke, Donald H.

    2012-01-01

    Phosphoryl transfer onto backbone hydroxyls is a recognized catalytic activity of nucleic acids. We find that kinase ribozyme K28 possesses an unusually complex active site that promotes (thio)phosphorylation of two residues widely separated in primary sequence. After allowing the ribozyme to radiolabel itself by phosphoryl transfer from [γ-32P]GTP, DNAzyme-mediated cleavage yielded two radiolabeled cleavage fragments, indicating phosphorylation sites within each of the two cleavage fragments. These sites were mapped by alkaline digestion and primer extension pausing. Enzymatic digestion and mutational analysis identified nucleotides important for activity and established the active structure as being a constrained pseudoknot with unusual connectivity that may juxtapose the two reactive sites. Nuclease sensitivities for nucleotides near the pseudoknot core were altered in the presence of GTPγS, indicating donor-induced folding. The 5′ target site was more strongly favored in full-length ribozyme K28 (128 nt) than in truncated RNAs (58 nt). Electrophoretic mobilities of self-thiophosphorylated products on organomercurial gels are distinct from the 5′ mono-thiophosphorylated product produced by reaction with polynucleotide kinase, potentially indicating simultaneous labeling of both sites within individual RNA strands. Our evidence supports a single, compact structure with local dynamics, rather than global rearrangement, as being responsible for dual-site phosphorylation. PMID:22618879

  5. Molecular mechanisms regulating NLRP3 inflammasome activation

    PubMed Central

    Jo, Eun-Kyeong; Kim, Jin Kyung; Shin, Dong-Min; Sasakawa, Chihiro

    2016-01-01

    Inflammasomes are multi-protein signaling complexes that trigger the activation of inflammatory caspases and the maturation of interleukin-1β. Among various inflammasome complexes, the NLRP3 inflammasome is best characterized and has been linked with various human autoinflammatory and autoimmune diseases. Thus, the NLRP3 inflammasome may be a promising target for anti-inflammatory therapies. In this review, we summarize the current understanding of the mechanisms by which the NLRP3 inflammasome is activated in the cytosol. We also describe the binding partners of NLRP3 inflammasome complexes activating or inhibiting the inflammasome assembly. Our knowledge of the mechanisms regulating NLRP3 inflammasome signaling and how these influence inflammatory responses offers further insight into potential therapeutic strategies to treat inflammatory diseases associated with dysregulation of the NLRP3 inflammasome. PMID:26549800

  6. Active Site Inhibitors Protect Protein Kinase C from Dephosphorylation and Stabilize Its Mature Form*

    PubMed Central

    Gould, Christine M.; Antal, Corina E.; Reyes, Gloria; Kunkel, Maya T.; Adams, Ryan A.; Ziyar, Ahdad; Riveros, Tania; Newton, Alexandra C.

    2011-01-01

    Conformational changes acutely control protein kinase C (PKC). We have previously shown that the autoinhibitory pseudosubstrate must be removed from the active site in order for 1) PKC to be phosphorylated by its upstream kinase phosphoinositide-dependent kinase 1 (PDK-1), 2) the mature enzyme to bind and phosphorylate substrates, and 3) the mature enzyme to be dephosphorylated by phosphatases. Here we show an additional level of conformational control; binding of active site inhibitors locks PKC in a conformation in which the priming phosphorylation sites are resistant to dephosphorylation. Using homogeneously pure PKC, we show that the active site inhibitor Gö 6983 prevents the dephosphorylation by pure protein phosphatase 1 (PP1) or the hydrophobic motif phosphatase, pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP). Consistent with results using pure proteins, treatment of cells with the competitive inhibitors Gö 6983 or bisindolylmaleimide I, but not the uncompetitive inhibitor bisindolylmaleimide IV, prevents the dephosphorylation and down-regulation of PKC induced by phorbol esters. Pulse-chase analyses reveal that active site inhibitors do not affect the net rate of priming phosphorylations of PKC; rather, they inhibit the dephosphorylation triggered by phorbol esters. These data provide a molecular explanation for the recent studies showing that active site inhibitors stabilize the phosphorylation state of protein kinases B/Akt and C. PMID:21715334

  7. Binding site identification and role of permanent water molecule of PIM-3 kinase: A molecular dynamics study.

    PubMed

    Ul-Haq, Zaheer; Gul, Sana; Usmani, Saman; Wadood, Abdul; Khan, Waqasuddin

    2015-11-01

    The kinome is a protein kinase complement of the human genome, categorized as serine/threonine and tyrosine kinases. These kinases catalyze phosphorylation reaction by using ATP as phosphoryl donor. Proviral Integration Site for Moloney Murine Leukemia Virus (PIM) kinase encodes serine/threonine protein kinases that recognized as proto-oncogene, responsible for rapid growth of cancerous cells. It is implicated in cell survival and function via cell cycle progression and its metabolism. PIM-3, sub-member of PIM kinases is a proto-oncogene, its overexpression inhibits apoptosis, and results in progression of hepatocellular carcinoma. PIM-3 is considered as a promising drug target but attempts to develop its specific inhibitors is slowed down due to the lack of 3D structure by any experimental technique. In silico techniques generally facilitate scientist to explore hidden structural features in order to improve drug discovery. In the present study, homology modeling, molecular docking and MD simulation techniques were utilized to explore the structure and dynamics of PIM-3 kinase. Induction of water molecules during molecular docking simulation explored differences in the hinge region between PIM-1 and PIM-3 kinases that may be responsible for specificity. Furthermore, role of water molecules in the active site was also explored via radial distribution function (RDF) after a 10 ns molecular dynamics (MD) simulations. Generated RDF plots exhibited the importance of water for inhibitor binding through their bridging capability that links the ligand with binding site residues. PMID:26529487

  8. Enhanced Enzyme Kinetic Stability by Increasing Rigidity within the Active Site*

    PubMed Central

    Xie, Yuan; An, Jiao; Yang, Guangyu; Wu, Geng; Zhang, Yong; Cui, Li; Feng, Yan

    2014-01-01

    Enzyme stability is an important issue for protein engineers. Understanding how rigidity in the active site affects protein kinetic stability will provide new insight into enzyme stabilization. In this study, we demonstrated enhanced kinetic stability of Candida antarctica lipase B (CalB) by mutating the structurally flexible residues within the active site. Six residues within 10 Å of the catalytic Ser105 residue with a high B factor were selected for iterative saturation mutagenesis. After screening 2200 colonies, we obtained the D223G/L278M mutant, which exhibited a 13-fold increase in half-life at 48 °C and a 12 °C higher T5015, the temperature at which enzyme activity is reduced to 50% after a 15-min heat treatment. Further characterization showed that global unfolding resistance against both thermal and chemical denaturation also improved. Analysis of the crystal structures of wild-type CalB and the D223G/L278M mutant revealed that the latter formed an extra main chain hydrogen bond network with seven structurally coupled residues within the flexible α10 helix that are primarily involved in forming the active site. Further investigation of the relative B factor profile and molecular dynamics simulation confirmed that the enhanced rigidity decreased fluctuation of the active site residues at high temperature. These results indicate that enhancing the rigidity of the flexible segment within the active site may provide an efficient method for improving enzyme kinetic stability. PMID:24448805

  9. Dashboard applications to monitor experiment activities at sites

    NASA Astrophysics Data System (ADS)

    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.

  10. Architecture and active site of particulate methane monooxygenase

    PubMed Central

    Culpepper, Megen A.; Rosenzweig, Amy C.

    2012-01-01

    Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria, organisms that live on methane gas as their sole carbon source. Understanding pMMO function has important implications for bioremediation applications and for the development of new, environmentally friendly catalysts for the direct conversion of methane to methanol. Crystal structures of pMMOs from three different methanotrophs reveal a trimeric architecture, consisting of three copies each of the pmoB, pmoA, and pmoC subunits. There are three distinct metal centers in each protomer of the trimer, mononuclear and dinuclear copper sites in the periplasmic regions of pmoB and a mononuclear site within the membrane that can be occupied by copper or zinc. Various models for the pMMO active site have been proposed within these structural constraints, including dicopper, tricopper, and diiron centers. Biochemical and spectroscopic data on pMMO and recombinant soluble fragments, denoted spmoB proteins, indicate that the active site involves copper and is located at the site of the dicopper center in the pmoB subunit. Initial spectroscopic evidence for O2 binding at this site has been obtained. Despite these findings, questions remain about the active site identity and nuclearity and will be the focus of future studies. PMID:22725967

  11. Computational study on the roles of amino acid residues in the active site formation mechanism of blue-light photoreceptors

    NASA Astrophysics Data System (ADS)

    Sato, Ryuma; Kitoh-Nishioka, Hirotaka; Ando, Koji; Yamato, Takahisa

    2015-07-01

    To examine the functional roles of the active site methionine (M-site) and glutamic acid (E-site) residues of blue-light photoreceptors, we performed in silico mutation at the M-site in a systematic manner and focused on the hydrogen bonding between the E-site and the substrate: the cyclobutane-pyrimidine dimer (CPD). Fragment molecular orbital calculations with electron correlations demonstrated that substitution of the M-site methionine with either alanine or glutamine always destabilizes the interaction energy between the E-site and the CPD by more than 12.0 kcal/mol, indicating that the methionine and glutamic acid residues cooperatively facilitate the enzymatic reaction in the active site.

  12. Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site.

    PubMed

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-04-20

    Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2domains reveal that the (HhH)2domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide. PMID:26908655

  13. Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site

    PubMed Central

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-01-01

    Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2 domains reveal that the (HhH)2 domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2 domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2 domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2 domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide. PMID:26908655

  14. Expansion of access tunnels and active-site cavities influence activity of haloalkane dehalogenases in organic cosolvents.

    PubMed

    Stepankova, Veronika; Khabiri, Morteza; Brezovsky, Jan; Pavelka, Antonin; Sykora, Jan; Amaro, Mariana; Minofar, Babak; Prokop, Zbynek; Hof, Martin; Ettrich, Rudiger; Chaloupkova, Radka; Damborsky, Jiri

    2013-05-10

    The use of enzymes for biocatalysis can be significantly enhanced by using organic cosolvents in the reaction mixtures. Selection of the cosolvent type and concentration range for an enzymatic reaction is challenging and requires extensive empirical testing. An understanding of protein-solvent interaction could provide a theoretical framework for rationalising the selection process. Here, the behaviour of three model enzymes (haloalkane dehalogenases) was investigated in the presence of three representative organic cosolvents (acetone, formamide, and isopropanol). Steady-state kinetics assays, molecular dynamics simulations, and time-resolved fluorescence spectroscopy were used to elucidate the molecular mechanisms of enzyme-solvent interactions. Cosolvent molecules entered the enzymes' access tunnels and active sites, enlarged their volumes with no change in overall protein structure, but surprisingly did not act as competitive inhibitors. At low concentrations, the cosolvents either enhanced catalysis by lowering K(0.5) and increasing k(cat), or caused enzyme inactivation by promoting substrate inhibition and decreasing k(cat). The induced activation and inhibition of the enzymes correlated with expansion of the active-site pockets and their occupancy by cosolvent molecules. The study demonstrates that quantitative analysis of the proportions of the access tunnels and active-sites occupied by organic solvent molecules provides the valuable information for rational selection of appropriate protein-solvent pair and effective cosolvent concentration. PMID:23564727

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

    SciTech Connect

    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.

  16. An active-site peptide from pepsin C

    PubMed Central

    Kay, J.; Ryle, A. P.

    1971-01-01

    Porcine pepsin C is inactivated rapidly and irreversibly by diazoacetyl-dl-norleucine methyl ester in the presence of cupric ions at pH values above 4.5. The inactivation is specific in that complete inactivation accompanies the incorporation of 1mol of inhibitor residue/mol of enzyme and evidence has been obtained to suggest that the reaction occurs with an active site residue. The site of reaction is the β-carboxyl group of an aspartic acid residue in the sequence Ile-Val-Asp-Thr. This sequence is identical with the active-site sequence in pepsin and the significance of this in terms of the different activities of the two enzymes is discussed. PMID:4942834

  17. A molecular model for adsorption of water on activated carbon: Comparison of simulation and experiment

    SciTech Connect

    McCallum, C.L.; McGrother, S.C.; Bandosz, T.J.; Mueller, E.A.; Gubbins, K.E.

    1999-01-19

    Experimental and molecular simulation results are presented for the adsorption of water onto activated carbons. The pore size distribution for the carbon studied was determined from nitrogen adsorption data using density functional theory, and the density of acidic and basic surface sites was found using Boehm and potentiometric titration. The total surface site density was 0.675 site/nm{sup 2}. Water adsorption was measured for relative pressures P/P{sub 0} down to 10{sup {minus}3}. A new molecular model for the water/activated carbon system is presented, which the authors term the effective single group model, and grand canonical Monte Carlo simulations are reported for the range of pressures covered in the experiments. A comparison of these simulations with the experiments show generally good agreement, although some discrepancies are attributed to the simplification of using a single surface group species, while those at high pressure are believed to arise from uncertainties in the pore size distribution. The simulation results throw new light on the adsorption mechanism for water at low pressures. The influence of varying both the density of surface sites and the size of the graphite microcrystals is studied using molecular simulation.

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

    PubMed

    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

  19. Staphylococcal SplB serine protease utilizes a novel molecular mechanism of activation.

    PubMed

    Pustelny, Katarzyna; Zdzalik, Michal; Stach, Natalia; Stec-Niemczyk, Justyna; Cichon, Przemyslaw; Czarna, Anna; Popowicz, Grzegorz; Mak, Pawel; Drag, Marcin; Salvesen, Guy S; Wladyka, Benedykt; Potempa, Jan; Dubin, Adam; Dubin, Grzegorz

    2014-05-30

    Staphylococcal SplB protease belongs to the chymotrypsin family. Chymotrypsin zymogen is activated by proteolytic processing at the N terminus, resulting in significant structural rearrangement at the active site. Here, we demonstrate that the molecular mechanism of SplB protease activation differs significantly and we characterize the novel mechanism in detail. Using peptide and protein substrates we show that the native signal peptide, or any N-terminal extension, has an inhibitory effect on SplB. Only precise N-terminal processing releases the full proteolytic activity of the wild type analogously to chymotrypsin. However, comparison of the crystal structures of mature SplB and a zymogen mimic show no rearrangement at the active site whatsoever. Instead, only the formation of a unique hydrogen bond network, distant form the active site, by the new N-terminal glutamic acid of mature SplB is observed. The importance of this network and influence of particular hydrogen bond interactions at the N terminus on the catalytic process is demonstrated by evaluating the kinetics of a series of mutants. The results allow us to propose a consistent model where changes in the overall protein dynamics rather than structural rearrangement of the active site are involved in the activation process. PMID:24713703

  20. Molecular Determinants of GS-9620-Dependent TLR7 Activation

    PubMed Central

    Rebbapragada, Indrani; Birkus, Gabriel; Perry, Jason; Xing, Weimei; Kwon, HyockJoo; Pflanz, Stefan

    2016-01-01

    GS-9620 is an orally administered agonist of Toll-like receptor (TLR)7 currently being evaluated in clinical studies for the treatment of chronic HBV and HIV patients. GS-9620 has shown antiviral efficacy in preclinical models of chronic hepadnavirus infection in woodchuck as well as chimpanzee. However, the molecular determinants of GS-9620-dependent activation of TLR7 are not well defined. The studies presented here elucidate GS-9620 subcellular distribution and characterize its molecular interactions with human TLR7 using structure-guided mutational analysis. Based on our results we present a molecular model of TLR7 bound to GS-9620. We also determine that several coding SNPs had no effect on GS-9620-dependent TLR7 activation. In addition, our studies provide evidence that TLR7 exists in a ligand-independent oligomeric state and that, TLR7 activation by GS-9620 is likely associated with compound-induced conformational changes. Finally, we demonstrate that activation of NF-κB and Akt pathways in primary plasmacytoid dendritic cells occur as immediate downstream cellular responses to GS-9620 stimulation. The data presented here further our understanding of the molecular parameters governing TLR7 activation by GS-9620, and more generally by nucleos/tide-related ligands. PMID:26784926

  1. Impact of Binding Site Comparisons on Medicinal Chemistry and Rational Molecular Design.

    PubMed

    Ehrt, Christiane; Brinkjost, Tobias; Koch, Oliver

    2016-05-12

    Modern rational drug design not only deals with the search for ligands binding to interesting and promising validated targets but also aims to identify the function and ligands of yet uncharacterized proteins having impact on different diseases. Additionally, it contributes to the design of inhibitors with distinct selectivity patterns and the prediction of possible off-target effects. The identification of similarities between binding sites of various proteins is a useful approach to cope with those challenges. The main scope of this perspective is to describe applications of different protein binding site comparison approaches to outline their applicability and impact on molecular design. The article deals with various substantial application domains and provides some outstanding examples to show how various binding site comparison methods can be applied to promote in silico drug design workflows. In addition, we will also briefly introduce the fundamental principles of different protein binding site comparison methods. PMID:27046190

  2. Molecular Dynamics Simulations of Solvation and Kink Site Formation at the {001} Barite-Water Interface.

    SciTech Connect

    Stack, Andrew G

    2009-09-01

    Solvation and kink site formation on step edges are known to be controlling parameters in crystal growth and dissolution. However, links from classical crystal growth models to specific reactions at the mineral-water interface have remained elusive. Molecular dynamics is used here to examine the water structure on barium surface sites and kink site formation enthalpies for material adsorbed to and removed from the step parallel to the [120] direction on the {001} barite-water interface. The bariums at the interface are shown to be coordinatively unsaturated with respect to water, and it is suggested that this is due to a steric hindrance from the nature of the interface. Kink site detachment energies that include hydration energies are endothermic for barium and exothermic for sulfate. The implications and problems of using these parameters in a crystal growth model are discussed.

  3. Coexistence and competition of on-site and intersite Coulomb interactions in Mott-molecular-dimers

    NASA Astrophysics Data System (ADS)

    Juliano, R. C.; de Arruda, A. S.; Craco, L.

    2016-02-01

    We reveal the interplay between on-site (U) and intersite (V) Coulomb interactions in the extended two-site Hubbard model. Due to its atomic-like form quantum correlations intrinsic to Mott-molecular-dimers are exactly computed. Our results for physical quantities such as double occupancy and specific heat are consistent with those obtained for the one-band Hubbard model, suggesting that a two-site dimer model is able to capture the essential thermodynamic properties of strongly interacting electron systems. It is noted that intersite Coulomb interactions promote the formation of doublons, which compete with the spin-singlet state induced by the on-site Coulomb repulsion. Our results are expected to be relevant for understanding electronic and thermodynamical properties of interacting electrons in systems with strongly coupled magnetic atoms.

  4. DNA damage processing by human 8-oxoguanine-DNA glycosylase mutants with the occluded active site.

    PubMed

    Lukina, Maria V; Popov, Alexander V; Koval, Vladimir V; Vorobjev, Yuri N; Fedorova, Olga S; Zharkov, Dmitry O

    2013-10-01

    8-Oxoguanine-DNA glycosylase (OGG1) removes premutagenic lesion 8-oxoguanine (8-oxo-G) from DNA and then nicks the nascent abasic (apurinic/apyrimidinic) site by β-elimination. Although the structure of OGG1 bound to damaged DNA is known, the dynamic aspects of 8-oxo-G recognition are not well understood. To comprehend the mechanisms of substrate recognition and processing, we have constructed OGG1 mutants with the active site occluded by replacement of Cys-253, which forms a wall of the base-binding pocket, with bulky leucine or isoleucine. The conformational dynamics of OGG1 mutants were characterized by single-turnover kinetics and stopped-flow kinetics with fluorescent detection. Additionally, the conformational mobility of wild type and the mutant OGG1 substrate complex was assessed using molecular dynamics simulations. Although pocket occlusion distorted the active site and greatly decreased the catalytic activity of OGG1, it did not fully prevent processing of 8-oxo-G and apurinic/apyrimidinic sites. Both mutants were notably stimulated in the presence of free 8-bromoguanine, indicating that this base can bind to the distorted OGG1 and facilitate β-elimination. The results agree with the concept of enzyme plasticity, suggesting that the active site of OGG1 is flexible enough to compensate partially for distortions caused by mutation. PMID:23955443

  5. Substrate conformational transitions in the active site of chorismate mutase: Their role in the catalytic mechanism

    PubMed Central

    Guo, Hong; Cui, Qiang; Lipscomb, William N.; Karplus, Martin

    2001-01-01

    Chorismate mutase acts at the first branch-point of aromatic amino acid biosynthesis and catalyzes the conversion of chorismate to prephenate. The results of molecular dynamics simulations of the substrate in solution and in the active site of chorismate mutase are reported. Two nonreactive conformers of chorismate are found to be more stable than the reactive pseudodiaxial chair conformer in solution. It is shown by QM/MM molecular dynamics simulations, which take into account the motions of the enzyme, that when these inactive conformers are bound to the active site, they are rapidly converted to the reactive chair conformer. This result suggests that one contribution of the enzyme is to bind the more prevalent nonreactive conformers and transform them into the active form in a step before the chemical reaction. The motion of the reactive chair conformer in the active site calculated by using the QM/MM potential generates transient structures that are closer to the transition state than is the stable CHAIR conformer. PMID:11481470

  6. Active Site and Remote Contributions to Catalysis in Methylthioadenosine Nucleosidases

    PubMed Central

    Thomas, Keisha; Cameron, Scott A.; Almo, Steven C.; Burgos, Emmanuel S.; Gulab, Shivali A.; Schramm, Vern L.

    2015-01-01

    5′-Methylthioadenosine/S-adenosyl-L-homocysteine nucleosidases (MTANs) catalyze the hydrolysis of 5′-methylthioadenosine to adenine and 5-methylthioribose. The amino acid sequences of the MTANs from Vibrio cholerae (VcMTAN) and Escherichia coli (EcMTAN) are 60% identical and 75% similar. Protein structure folds and kinetic properties are similar. However, binding of transition-state analogues is dominated by favorable entropy in VcMTAN and by enthalpy in EcMTAN. Catalytic sites of VcMTAN and EcMTAN in contact with reactants differ by two residues; Ala113 and Val153 in VcMTAN are Pro113 and Ile152, respectively, in EcMTAN. We mutated the VcMTAN catalytic site residues to match those of EcMTAN in anticipation of altering its properties toward EcMTAN. Inhibition of VcMTAN by transition-state analogues required filling both active sites of the homodimer. However, in the Val153Ile mutant or double mutants, transition-state analogue binding at one site caused complete inhibition. Therefore, a single amino acid, Val153, alters the catalytic site cooperativity in VcMTAN. The transition-state analogue affinity and thermodynamics in mutant VcMTAN became even more unlike those of EcMTAN, the opposite of expectations from catalytic site similarity; thus, catalytic site contacts in VcMTAN are unable to recapitulate the properties of EcMTAN. X-ray crystal structures of EcMTAN, VcMTAN, and a multiple-site mutant of VcMTAN most closely resembling EcMTAN in catalytic site contacts show no major protein conformational differences. The overall protein architectures of these closely related proteins are implicated in contributing to the catalytic site differences. PMID:25806409

  7. Counting Active Sites on Titanium Oxide-Silica Catalysts for Hydrogen Peroxide Activation through In Situ Poisoning with Phenylphosphonic Acid

    SciTech Connect

    Eaton, Todd R.; Boston, Andrew M.; Thompson, Anthony B.; Gray, Kimberly A.; Notestein, Justin M.

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  9. Decolorization / deodorization of zein via activated carbons and molecular sieves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective is to evaluate a series of granular media consisting of activated carbons and molecular sieves in a batch process for the purpose of clarifying and removal of color and odor components from yellow zein dispersed in an aqueous alcohol medium. The major contributors of yellow zein is du...

  10. Threshold occupancy and specific cation binding modes in the hammerhead ribozyme active site are required for active conformation

    PubMed Central

    Lee, Tai-Sung; Giambaşu, George M.; Sosa, Carlos P.; Martick, Monika; Scott, William G.; York, Darrin M.

    2009-01-01

    The relationship between formation of active in-line attack conformations and monovalent (Na+) and divalent (Mg2+) metal ion binding in the hammerhead ribozyme has been explored with molecular dynamics simulations. To stabilize repulsions between negatively charged groups, different requirements of threshold occupancy of metal ions were observed in the reactant and activated precursor states both in the presence or absence of a Mg2+ in the active site. Specific bridging coordination patterns of the ions are correlated with the formation of active in-line attack conformations and can be accommodated in both cases. Furthermore, simulation results suggest that the hammerhead ribozyme folds to form an electronegative recruiting pocket that attracts high local concentrations of positive charge. The present simulations help to reconcile experiments that probe the metal ion sensitivity of hammerhead ribozyme catalysis and support the supposition that Mg2+, in addition to stabilizing active conformations, plays a specific chemical role in catalysis. PMID:19265710

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

    ERIC Educational Resources Information Center

    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…

  12. A facile ionic liquid promoted synthesis, cholinesterase inhibitory activity and molecular modeling study of novel highly functionalized spiropyrrolidines.

    PubMed

    Almansour, Abdulrahman I; Kumar, Raju Suresh; Arumugam, Natarajan; Basiri, Alireza; Kia, Yalda; Ali, Mohamed Ashraf; Farooq, Mehvish; Murugaiyah, Vikneswaran

    2015-01-01

    A series of novel dimethoxyindanone embedded spiropyrrolidines were synthesized in ionic liquid, [bmim]Br and were evaluated for their inhibitory activities towards cholinesterases. Among the spiropyrrolidines, compound 4f exhibited the most potent activity with an IC50 value of 1.57 µM against acethylcholinesterase (AChE). Molecular docking simulation for the most active compound was employed with the aim of disclosing its binding mechanism to the active site of AChE receptor. PMID:25642838

  13. Tuned by metals: the TET peptidase activity is controlled by 3 metal binding sites

    PubMed Central

    Colombo, Matteo; Girard, Eric; Franzetti, Bruno

    2016-01-01

    TET aminopeptidases are dodecameric particles shared in the three life domains involved in various biological processes, from carbon source provider in archaea to eye-pressure regulation in humans. Each subunit contains a dinuclear metal site (M1 and M2) responsible for the enzyme catalytic activity. However, the role of each metal ion is still uncharacterized. Noteworthy, while mesophilic TETs are activated by Mn2+, hyperthermophilic TETs prefers Co2+. Here, by means of anomalous x-ray crystallography and enzyme kinetics measurements of the TET3 aminopeptidase from the hyperthermophilic organism Pyrococcus furiosus (PfTET3), we show that M2 hosts the catalytic activity of the enzyme, while M1 stabilizes the TET3 quaternary structure and controls the active site flexibility in a temperature dependent manner. A new third metal site (M3) was found in the substrate binding pocket, modulating the PfTET3 substrate preferences. These data show that TET activity is tuned by the molecular interplay among three metal sites. PMID:26853450

  14. Active sites environmental monitoring program. Annual report FY 1992

    SciTech Connect

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

    1994-04-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) at ORNL from October 1991 through September 1992. Solid Waste Operations and the Environmental Sciences Division established ASEMP in 1989 to provide early detection and performance monitoring at active low-level waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 as required by Chapter 2 and 3 of US Department of Energy Order 5820.2A. The Interim Waste Management Facility (IWMF) began operation in December 1991. Monitoring results from the tumulus and IWMF disposal pads continue to indicate that no LLW is leaching from the storage vaults. Storm water falling on the IWMF active pad was collected and transported to the Process Waste Treatment Plant while operators awaited approval of the National Pollutant Discharge Elimination System (NPDES) permit. Several of the recent samples collected from the active IWMF pad had pH levels above the NPDES limit of 9.0 because of alkali leached from the concrete. The increase in gross beta activity has been slight; only 1 of the 21 samples collected contained activity above the 5.0 Bq/L action level. Automated sample-collection and flow-measurement equipment has been installed at IWMF and is being tested. The flume designed to electronically measure flow from the IWMF pads and underpads is too large to be of practical value for measuring most flows at this site. Modification of this system will be necessary. A CO{sub 2} bubbler system designed to reduce the pH of water from the pads is being tested at IWMF.

  15. Purification, molecular cloning, and expression of the mammalian sigma1-binding site.

    PubMed Central

    Hanner, M; Moebius, F F; Flandorfer, A; Knaus, H G; Striessnig, J; Kempner, E; Glossmann, H

    1996-01-01

    Sigma-ligands comprise several chemically unrelated drugs such as haloperidol, pentazocine, and ditolylguanidine, which bind to a family of low molecular mass proteins in the endoplasmic reticulum. These so-called sigma-receptors are believed to mediate various pharmacological effects of sigma-ligands by as yet unknown mechanisms. Based on their opposite enantioselectivity for benzomorphans and different molecular masses, two subtypes are differentiated. We purified the sigma1-binding site as a single 30-kDa protein from guinea pig liver employing the benzomorphan(+)[3H]pentazocine and the arylazide (-)[3H]azidopamil as specific probes. The purified (+)[3H]pentazocine-binding protein retained its high affinity for haloperidol, pentazocine, and ditolylguanidine. Partial amino acid sequence obtained after trypsinolysis revealed no homology to known proteins. Radiation inactivation of the pentazocine-labeled sigma1-binding site yielded a molecular mass of 24 +/- 2 kDa. The corresponding cDNA was cloned using degenerate oligonucleotides and cDNA library screening. Its open reading frame encoded a 25.3-kDa protein with at least one putative transmembrane segment. The protein expressed in yeast cells transformed with the cDNA showed the pharmacological characteristics of the brain and liver sigma1-binding site. The deduced amino acid sequence was structurally unrelated to known mammalian proteins but it shared homology with fungal proteins involved in sterol synthesis. Northern blots showed high densities of the sigma1-binding site mRNA in sterol-producing tissues. This is also in agreement with the known ability of sigma1-binding sites to interact with steroids, such as progesterone. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8755605

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

    PubMed

    Beke-Somfai, Tamás; Lincoln, Per; Nordén, Bengt

    2013-02-01

    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. PMID:23345443

  17. CO Oxidation on Au/TiO2: Condition-Dependent Active Sites and Mechanistic Pathways.

    PubMed

    Wang, Yang-Gang; Cantu, David C; Lee, Mal-Soon; Li, Jun; Glezakou, Vassiliki-Alexandra; Rousseau, Roger

    2016-08-24

    We present results of ab initio electronic structure and molecular dynamics simulations (AIMD), as well as a microkinetic model of CO oxidation catalyzed by TiO2 supported Au nanocatalysts. A coverage-dependent microkinetic analysis, based on energetics obtained with density functional methods, shows that the dominant kinetic pathway, activated oxygen species, and catalytic active sites are all strongly depended on both temperature and oxygen partial pressure. Under oxidizing conditions and T < 400 K, the prevalent pathway involves a dynamic single atom catalytic mechanism. This reaction is catalyzed by a transient Au-CO species that migrates from the Au-cluster onto a surface oxygen adatom. It subsequently reacts with the TiO2 support via a Mars van Krevelen mechanism to form CO2 and finally the Au atom reintegrates back into the gold cluster to complete the catalytic cycle. At 300 ≤ T ≤ 600 K, oxygen-bound single Oad-Au(+)-CO sites and the perimeter Au-sites of the nanoparticle work in tandem to optimally catalyze the reaction. Above 600 K, a variety of alternate pathways associated with both single-atom and the perimeter sites of the Au nanoparticle are found to be active. Under low oxygen pressures, Oad-Au(+)-CO species can be a source of catalyst deactivation and the dominant pathway involves only Au-perimeter sites. A detailed comparison of the current model and the existing literature resolves many apparent inconsistencies in the mechanistic interpretations. PMID:27480512

  18. Probing the promiscuous active site of myo-inositol dehydrogenase using synthetic substrates, homology modeling, and active site modification.

    PubMed

    Daniellou, Richard; Zheng, Hongyan; Langill, David M; Sanders, David A R; Palmer, David R J

    2007-06-26

    The active site of myo-inositol dehydrogenase (IDH, EC 1.1.1.18) from Bacillus subtilis recognizes a variety of mono- and disaccharides, as well as 1l-4-O-substituted inositol derivatives. It catalyzes the NAD+-dependent oxidation of the axial alcohol of these substrates with comparable kinetic constants. We have found that 4-O-p-toluenesulfonyl-myo-inositol does not act as a substrate for IDH, in contrast to structurally similar compounds such as those bearing substituted benzyl substituents in the same position. X-ray crystallographic analysis of 4-O-p-toluenesulfonyl-myo-inositol and 4-O-(2-naphthyl)methyl-myo-inositol, which is a substrate for IDH, shows a distinct difference in the preferred conformation of the aryl substituent. Conformational analysis of known substrates of IDH suggests that this conformational difference may account for the difference in reactivity of 4-O-p-toluenesulfonyl-myo-inositol in the presence of IDH. A sequence alignment of IDH with the homologous glucose-fructose oxidoreductase allowed the construction of an homology model of inositol dehydrogenase, to which NADH and 4-O-benzyl-scyllo-inosose were docked and the active site energy minimized. The active site model is consistent with all experimental results and suggests that a conserved tyrosine-glycine-tyrosine motif forms the hydrophobic pocket adjoining the site of inositol recognition. Y233F and Y235F retain activity, while Y233R and Y235R do not. A histidine-aspartate pair, H176 and D172, are proposed to act as a dyad in which H176 is the active site acid/base. The enzyme is inactivated by diethyl pyrocarbonate, and the mutants H176A and D172N show a marked loss of activity. Kinetic isotope effect experiments with D172N indicate that chemistry is rate-determining for this mutant. PMID:17539607

  19. Molecular Mechanism of Active Zone Organization at Vertebrate Neuromuscular Junctions

    PubMed Central

    Nishimune, Hiroshi

    2013-01-01

    Organization of presynaptic active zones is essential for development, plasticity, and pathology of the nervous system. Recent studies indicate a trans-synaptic molecular mechanism that organizes the active zones by connecting the pre- and the postsynaptic specialization. The presynaptic component of this trans-synaptic mechanism is comprised of cytosolic active zone proteins bound to the cytosolic domains of voltage-dependent calcium channels (P/Q-, N-, and L-type) on the presynaptic membrane. The postsynaptic component of this mechanism is the synapse organizer (laminin β2) that is expressed by the postsynaptic cell and accumulates specifically on top of the postsynaptic specialization. The pre- and the postsynaptic components interact directly between the extracellular domains of calcium channels and laminin β2 to anchor the presynaptic protein complex in front of the postsynaptic specialization. Hence, the presynaptic calcium channel functions as a scaffolding protein for active zone organization and as an ion-conducting channel for synaptic transmission. In contrast to the requirement of calcium influx for synaptic transmission, the formation of the active zone does not require the calcium influx through the calcium channels. Importantly, the active zones of adult synapses are not stable structures and require maintenance for their integrity. Furthermore, aging or diseases of the central and peripheral nervous system impair the active zones. This review will focus on the molecular mechanisms that organize the presynaptic active zones and summarize recent findings at the neuromuscular junctions and other synapses. PMID:22135013

  20. Molecular docking characterizes substrate-binding sites and efflux modulation mechanisms within P-glycoprotein.

    PubMed

    Ferreira, Ricardo J; Ferreira, Maria-José U; dos Santos, Daniel J V A

    2013-07-22

    P-Glycoprotein (Pgp) is one of the best characterized ABC transporters, often involved in the multidrug-resistance phenotype overexpressed by several cancer cell lines. Experimental studies contributed to important knowledge concerning substrate polyspecificity, efflux mechanism, and drug-binding sites. This information is, however, scattered through different perspectives, not existing a unifying model for the knowledge available for this transporter. Using a previously refined structure of murine Pgp, three putative drug-binding sites were hereby characterized by means of molecular docking. The modulator site (M-site) is characterized by cross interactions between both Pgp halves herein defined for the first time, having an important role in impairing conformational changes leading to substrate efflux. Two other binding sites, located next to the inner leaflet of the lipid bilayer, were identified as the substrate-binding H and R sites by matching docking and experimental results. A new classification model with the ability to discriminate substrates from modulators is also proposed, integrating a vast number of theoretical and experimental data. PMID:23802684

  1. Site-specific basicities regulate molecular recognition in receptor binding: in silico docking of thyroid hormones.

    PubMed

    Tóth, Gergő; Baska, Ferenc; Schretner, András; Rácz, Akos; Noszál, Béla

    2013-09-01

    Interactions between thyroid hormone α and β receptors and the eight protonation microspecies of each of the main thyroid hormones (thyroxine, liothyronine, and reverse liothyronine) were investigated and quantitated by molecular modeling. Flexible docking of the various protonation forms of thyroid hormones and high-affinity thyromimetics to the two thyroid receptors was carried out. In this method the role of the ionization state of each basic site could be studied in the composite process of molecular recognition. Our results quantitate at the molecular level how the ionization state and the charge distribution influence the protein binding. The anionic form of the carboxyl group (i.e., carboxylate site) is essential for protein binding, whereas the protonated form of amino group worsens the binding. The protonation state of the phenolate plays a less important role in the receptor affinity; its protonation, however, alters the electron density and the concomitant stacking propensity of the aromatic rings, resulting in a different binding score. The combined results of docking and microspeciation studies show that microspecies with the highest concentration at the pH of blood are not the strongest binding ones. The calculated binding free energy values can be well interpreted in terms of the interactions between the actual sites of the microspecies and the receptor amino acids. Our docking results were validated and compared with biological data from the literature. Since the thyroid hormone receptors influence several physiologic functions, such as metabolic rate, cholesterol and triglyceride levels, and heart frequency, our binding results provide a molecular basis for drug design and development in related therapeutic indications. PMID:23907234

  2. Cyclic silicate active site and stereochemical match for apatite nucleation on pseudowollastonite bioceramic-bone interfaces.

    PubMed

    Sahai, Nita; Anseau, Michel

    2005-10-01

    Hydroxyapatite (Ca5(PO4)3(OH)) forms on pseudowollastonite (psW) (alpha-CaSiO3) in vitro in simulated body fluid, human parotid saliva and cell-culture medium, and in vivo in implanted rat tibias. We used crystallographic constraints with ab initio molecular orbital calculations to identify the active site and reaction mechanism for heterogeneous nucleation of the earliest calcium phosphate oligomer/phase. The active site is the planar, cyclic, silicate trimer (Si3O9) on the (001) face of psW. The trimer has three silanol groups (>SiOH) arranged at 60 degrees from each other, providing a stereochemical match for O atoms bonded to Ca2+ on the (001) face of hydroxyapatite. Calcium phosphate nucleation is modeled in steps as hydrolysis of surface Ca-O bonds with leaching of Ca2+ into solution, protonation of the surface Si-O groups to form silanols, calcium sorption as an inner-sphere surface complex and, attachment of HPO4(2-). Our model explains the experimental solution and high resolution transmission electron microscopy data for epitaxial hydroxyapatite growth on psW in vitro and in vivo. We propose that the cyclic silicate trimer is the universal active site for heterogeneous, stereochemically promoted nucleation on silicate-based bioactive ceramics. A critical active site-density and a point of zero charge of the bioceramic less than physiological pH are required for bioactivity. PMID:15949543

  3. Wobble Pairs of the HDV Ribozyme Play Specific Roles in Stabilization of Active Site Dynamics

    PubMed Central

    Sripathi, Kamali N.; Banáš, Pavel; Reblova, Kamila; Šponer, Jiři; Otyepka, Michal

    2015-01-01

    The hepatitis delta virus (HDV) is the only known human pathogen whose genome contains a catalytic RNA motif (ribozyme). The overall architecture of the HDV ribozyme is that of a double-nested pseudoknot, with two GU pairs flanking the active site. Although extensive studies have shown that mutation of either wobble results in decreased catalytic activity, little work has focused on linking these mutations to specific structural effects on catalytic fitness. Here we use molecular dynamics simulations based on an activated structure to probe the active site dynamics as a result of wobble pair mutations. In both wild-type and mutant ribozymes, the in-line fitness of the active site (as a measure of catalytic proficiency) strongly depends on the presence of a C75(N3H3+)N1(O5′) hydrogen bond, which positions C75 as the general acid for the reaction. Our mutational analyses show that each GU wobble supports catalytically fit conformations in distinct ways; the reverse G25U20 wobble promotes high in-line fitness, high occupancy of the C75(N3H3+)G1(O5′) general-acid hydrogen bond and stabilization of the G1U37 wobble, while the G1U37 wobble acts more locally by stabilizing high in-line fitness and the C75(N3H3+)G1(O5′) hydrogen bond. We also find that stable type I A-minor and P1.1 hydrogen bonding above and below the active site, respectively, prevent local structural disorder from spreading and disrupting global conformation. Taken together, our results define specific, often redundant architectural roles for several structural motifs of the HDV ribozyme active site, expanding the known roles of these motifs within all HDV-like ribozymes and other structured RNAs. PMID:25631765

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

    PubMed

    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

  5. Active-Site-Accessible, Porphyrinic Metal;#8722;Organic Framework Materials

    SciTech Connect

    Farha, Omar K.; Shultz, Abraham M.; Sarjeant, Amy A.; Nguyen, SonBinh T.; Hupp, Joseph T.

    2012-02-06

    On account of their structural similarity to cofactors found in many metallo-enzymes, metalloporphyrins are obvious potential building blocks for catalytically active, metal-organic framework (MOF) materials. While numerous porphyrin-based MOFs have already been described, versions featuring highly accessible active sites and permanent microporosity are remarkably scarce. Indeed, of the more than 70 previously reported porphyrinic MOFs, only one has been shown to be both permanently microporous and contain internally accessible active sites for chemical catalysis. Attempts to generalize the design approach used in this single successful case have failed. Reported here, however, is the synthesis of an extended family of MOFs that directly incorporate a variety of metalloporphyrins (specifically Al{sup 3+}, Zn{sup 2+}, Pd{sup 2+}, Mn{sup 3+}, and Fe{sup 3+} complexes). These robust porphyrinic materials (RPMs) feature large channels and readily accessible active sites. As an illustrative example, one of the manganese-containing RPMs is shown to be catalytically competent for the oxidation of alkenes and alkanes.

  6. Nest predation increases with parental activity: Separating nest site and parental activity effects

    USGS Publications Warehouse

    Martin, T.E.; Scott, J.; Menge, C.

    2000-01-01

    Alexander Skutch hypothesized that increased parental activity can increase the risk of nest predation. We tested this hypothesis using ten open-nesting bird species in Arizona, USA. Parental activity was greater during the nestling than incubation stage because parents visited the nest frequently to feed their young during the nestling stage. However, nest predation did not generally increase with parental activity between nesting stages across the ten study species. Previous investigators have found similar results. We tested whether nest site effects might yield higher predation during incubation because the most obvious sites are depredated most rapidly. We conducted experiments using nest sites from the previous year to remove parental activity. Our results showed that nest sites have highly repeatable effects on nest predation risk; poor nest sites incurred rapid predation and caused predation rates to be greater during the incubation than nestling stage. This pattern also was exhibited in a bird species with similar (i.e. controlled) parental activity between nesting stages. Once nest site effects are taken into account, nest predation shows a strong proximate increase with parental activity during the nestling stage within and across species. Parental activity and nest sites exert antagonistic influences on current estimates of nest predation between nesting stages and both must be considered in order to understand current patterns of nest predation, which is an important source of natural selection.

  7. Nest predation increases with parental activity: separating nest site and parental activity effects.

    PubMed Central

    Martin, T E; Scott, J; Menge, C

    2000-01-01

    Alexander Skutch hypothesized that increased parental activity can increase the risk of nest predation. We tested this hypothesis using ten open-nesting bird species in Arizona, USA. Parental activity was greater during the nestling than incubation stage because parents visited the nest frequently to feed their young during the nestling stage. However, nest predation did not generally increase with parental activity between nesting stages across the ten study species. Previous investigators have found similar results. We tested whether nest site effects might yield higher predation during incubation because the most obvious sites are depredated most rapidly. We conducted experiments using nest sites from the previous year to remove parental activity. Our results showed that nest sites have highly repeatable effects on nest predation risk; poor nest sites incurred rapid predation and caused predation rates to be greater during the incubation than nestling stage. This pattern also was exhibited in a bird species with similar (i.e. controlled) parental activity between nesting stages. Once nest site effects are taken into account, nest predation shows a strong proximate increase with parental activity during the nestling stage within and across species. Parental activity and nest sites exert antagonistic influences on current estimates of nest predation between nesting stages and both must be considered in order to understand current patterns of nest predation, which is an important source of natural selection. PMID:11413645

  8. Active site density and reactivity for the photocatalytic dehydrogenation of 2-propanol on TiO 2 (110)

    NASA Astrophysics Data System (ADS)

    Brinkley, David; Engel, Thomas

    1998-10-01

    By combining molecular beam experiments at 350 K with low temperature experiments on an immobile adlayer, we determine the fraction of surface sites active for the photocatalytic dehydrogenation of 2-propanol to acetone to be 0.08 on a fully oxidized surface. We also find that the reaction probability at these sites is essentially unity. The presence of oxygen vacancies (≈0.1 monolayer) increases the fraction of active sites to 0.15 without changing the reaction probability, reaction kinetics, or product distribution. From this, we conclude that only sites which can bind O 2 strongly are active for the reaction. Candidates for such sites include steps and kinks on fully oxidized surfaces and oxygen lattice vacancies on reduced surfaces.

  9. Nucleolar protein B23 has molecular chaperone activities.

    PubMed Central

    Szebeni, A.; Olson, M. O.

    1999-01-01

    Protein B23 is an abundant, multifunctional nucleolar phosphoprotein whose activities are proposed to play a role in ribosome assembly. Szebeni et al. (1997) showed stimulation of nuclear import in vitro by protein B23 and suggested that this effect was due to a molecular chaperone-like activity. Protein B23 was tested for chaperone activities using several protein substrates. The temperature-dependent and -independent aggregation of the HIV-1 Rev protein was measured using a zero angle light scattering (turbidity) assay. Protein B23 inhibited the aggregation of the Rev protein, with the amount of inhibition proportional to the concentration of B23 added. This activity was saturable with nearly complete inhibition when the molar ratio of B23:Rev was slightly above one. Protein B23 also protected liver alcohol dehydrogenase (LADH), carboxypeptidase A, citrate synthase, and rhodanese from aggregation during thermal denaturation and preserved the enzyme activity of LADH under these conditions. In addition, protein B23 was able to promote the restoration of activity of LADH previously denatured with guanidine-HCl. Protein B23 preferentially bound denatured substrates and exposed hydrophobic regions when complexed with denatured proteins. Thus, by several criteria, protein B23 behaves like a molecular chaperone; these activities may be related to its role in ribosome biogenesis. PMID:10211837

  10. Insight into the mechanism of polyphenols on the activity of HMGR by molecular docking.

    PubMed

    Islam, Barira; Sharma, Charu; Adem, Abdu; Aburawi, Elhadi; Ojha, Shreesh

    2015-01-01

    Statins are hypolipidemic drugs that are effective in the treatment of hypercholesterolemia by attenuating cholesterol synthesis in the liver via competitive inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Recently, dietary changes associated with drug therapy have garnered attention as novel drugs to mitigate or ameliorate hypercholesterolemia. The present study was undertaken to observe different dietary polyphenols that can bind to the active site of HMGR and inhibit it. Results from the 12 dietary polyphenols tested reveal that polyphenols can bind to HMGR and block the binding of nicotinamide adenine dinucleotide phosphate (NADP(+)). We observed that the rigidity of phenolic rings prevents the polyphenols from docking to the enzyme activity site. The presence of an ester linkage between the phenolic rings in (-)-epigallocatechin-3-gallate (EGCG) and the alkyl chain in curcumin allows them to orient in the active site of the HMGR and bind to the catalytic residues. EGCG and curcumin showed binding to the active site residues with a low GRID score, which may be a potential inhibitor of HMGR. Kaempferol showed binding to HMG-CoA, but with low binding affinity. These observations provide a rationale for the consistent hypolipidemic effect of EGCG and curcumin, which has been previously reported in several epidemiological and animal studies. Therefore, this study substantiates the mechanism of polyphenols on the activity of HMGR by molecular docking and provides the impetus for drug design involving further structure-function relationship studies. PMID:26357462

  11. Insight into the mechanism of polyphenols on the activity of HMGR by molecular docking

    PubMed Central

    Islam, Barira; Sharma, Charu; Adem, Abdu; Aburawi, Elhadi; Ojha, Shreesh

    2015-01-01

    Statins are hypolipidemic drugs that are effective in the treatment of hypercholesterolemia by attenuating cholesterol synthesis in the liver via competitive inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Recently, dietary changes associated with drug therapy have garnered attention as novel drugs to mitigate or ameliorate hypercholesterolemia. The present study was undertaken to observe different dietary polyphenols that can bind to the active site of HMGR and inhibit it. Results from the 12 dietary polyphenols tested reveal that polyphenols can bind to HMGR and block the binding of nicotinamide adenine dinucleotide phosphate (NADP+). We observed that the rigidity of phenolic rings prevents the polyphenols from docking to the enzyme activity site. The presence of an ester linkage between the phenolic rings in (–)-epigallocatechin-3-gallate (EGCG) and the alkyl chain in curcumin allows them to orient in the active site of the HMGR and bind to the catalytic residues. EGCG and curcumin showed binding to the active site residues with a low GRID score, which may be a potential inhibitor of HMGR. Kaempferol showed binding to HMG-CoA, but with low binding affinity. These observations provide a rationale for the consistent hypolipidemic effect of EGCG and curcumin, which has been previously reported in several epidemiological and animal studies. Therefore, this study substantiates the mechanism of polyphenols on the activity of HMGR by molecular docking and provides the impetus for drug design involving further structure–function relationship studies. PMID:26357462

  12. Evolution of a designed retro-aldolase leads to complete active site remodeling

    PubMed Central

    Giger, Lars; Caner, Sami; Obexer, Richard; Kast, Peter; Baker, David; Ban, Nenad; Hilvert, Donald

    2013-01-01

    Evolutionary advances are often fueled by unanticipated innovation. Directed evolution of a computationally designed enzyme suggests that dramatic molecular changes can also drive the optimization of primitive protein active sites. The specific activity of an artificial retro-aldolase was boosted >4,400 fold by random mutagenesis and screening, affording catalytic efficiencies approaching those of natural enzymes. However, structural and mechanistic studies reveal that the engineered catalytic apparatus, consisting of a reactive lysine and an ordered water molecule, was unexpectedly abandoned in favor of a new lysine residue in a substrate binding pocket created during the optimization process. Structures of the initial in silico design, a mechanistically promiscuous intermediate, and one of the most evolved variants highlight the importance of loop mobility and supporting functional groups in the emergence of the new catalytic center. Such internal competition between alternative reactive sites may have characterized the early evolution of many natural enzymes. PMID:23748672

  13. Computation of Rate Constants for Diffusion of Small Ligands to and from Buried Protein Active Sites.

    PubMed

    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

  14. Active Sites Environmental Monitoring Program. FY 1993: Annual report

    SciTech Connect

    Morrissey, C.M.; Ashwood, T.L.; Hicks, D.S.; Marsh, J.D.

    1994-08-01

    This report continues a series of annual and semiannual reports that present the results of the Active Sites Environmental Monitoring Program (ASEMP) monitoring activities. The report details monitoring data for fiscal year (FY) 1993 and is divided into three major areas: SWSA 6 [including tumulus pads, Interim Waste Management Facility (IWMF), and other sites], the low-level Liquid-Waste Solidification Project (LWSP), and TRU-waste storage facilities in SWSA 5 N. The detailed monitoring methodology is described in the second revision of the ASEMP program plan. This report also presents a summary of the methodology used to gather data for each major area along with the results obtained during FY 1993.

  15. Active sites in char gasification: Final technical report

    SciTech Connect

    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.

  16. Cellular and Molecular Mechanisms Underpinning Macrophage Activation during Remyelination

    PubMed Central

    Lloyd, Amy F.; Miron, Veronique E.

    2016-01-01

    Remyelination is an example of central nervous system (CNS) regeneration, whereby myelin is restored around demyelinated axons, re-establishing saltatory conduction and trophic/metabolic support. In progressive multiple sclerosis, remyelination is limited or fails altogether which is considered to contribute to axonal damage/loss and consequent disability. Macrophages have critical roles in both CNS damage and regeneration, such as remyelination. This diverse range in functions reflects the ability of macrophages to acquire tissue microenvironment-specific activation states. This activation is dynamically regulated during efficient regeneration, with a switch from pro-inflammatory to inflammation-resolution/pro-regenerative phenotypes. Although, some molecules and pathways have been implicated in the dynamic activation of macrophages, such as NFκB, the cellular and molecular mechanisms underpinning plasticity of macrophage activation are unclear. Identifying mechanisms regulating macrophage activation to pro-regenerative phenotypes may lead to novel therapeutic strategies to promote remyelination in multiple sclerosis. PMID:27446913

  17. A three-dimensional model of mammalian tyrosinase active site accounting for loss of function mutations.

    PubMed

    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

  18. Donor site healing dynamics: molecular, histological, and noninvasive imaging assessment in a porcine model.

    PubMed

    Mauskar, Neil A; Sood, Subeena; Travis, Taryn E; Matt, Sarah E; Mino, Matthew J; Burnett, Mary-Susan; Moffatt, Lauren T; Fidler, Philip; Epstein, Stephen E; Jordan, Marion H; Shupp, Jeffrey W

    2013-01-01

    Understanding the physiology of donor site healing will lead to advances in how these wounds are treated and may ultimately allow faster healing, more frequent autografting, and more effective care of the burn-injured patient. Unfortunately, a paucity of data exists regarding perfusion metrics over the course of donor site healing. Furthermore, there are no studies that interrelate indices of perfusion with the molecular and cellular processes of donor site healing. Male Duroc pigs were anesthetized and donor site wounds were created using a Zimmer dermatome at a depth of 0.060 inch (1.52 mm). Digital photographs, laser Doppler images, and punch biopsies were obtained before and after excision and on days 2, 4, 7, 9, 11, 14, and 16 until wounds were healed. RNA isolation was performed and quantitative polymerase chain reaction was used to examine differential gene expression over the time course. Formalin-fixed biopsies were embedded in paraffin, sectioned, stained, and examined. Wound surfaces were 83% re-epithelialized by day 16. Perfusion peaked on day 2 then declined, but it remained significantly elevated compared to before excision (P < .05). From day 9 onward, mean perfusion units were not significantly different from baseline (P < .05). Twenty-two representative genes were selected for examination. RNA expression of collagen, tenascin-cytoactin, inflammatory cytokines, remodeling enzymes, growth factors, and Wnt was increased. Inflammatory cells and cytokines were demonstrated histologically. Nuclei per high powered field peaked at day 7 and neodermal thickness increased daily to day 14. A novel porcine model for donor site wound healing that interrelates re-epithelilaizationand perfusion with molecular and cellular indices has been demonstrated. PMID:23511287

  19. Benzene Probes in Molecular Dynamics Simulations Reveal Novel Binding Sites for Ligand Design.

    PubMed

    Tan, Yaw Sing; Reeks, Judith; Brown, Christopher J; Thean, Dawn; Ferrer Gago, Fernando Jose; Yuen, Tsz Ying; Goh, Eunice Tze Leng; Lee, Xue Er Cheryl; Jennings, Claire E; Joseph, Thomas L; Lakshminarayanan, Rajamani; Lane, David P; Noble, Martin E M; Verma, Chandra S

    2016-09-01

    Protein flexibility poses a major challenge in binding site identification. Several computational pocket detection methods that utilize small-molecule probes in molecular dynamics (MD) simulations have been developed to address this issue. Although they have proven hugely successful at reproducing experimental structural data, their ability to predict new binding sites that are yet to be identified and characterized has not been demonstrated. Here, we report the use of benzenes as probe molecules in ligand-mapping MD (LMMD) simulations to predict the existence of two novel binding sites on the surface of the oncoprotein MDM2. One of them was serendipitously confirmed by biophysical assays and X-ray crystallography to be important for the binding of a new family of hydrocarbon stapled peptides that were specifically designed to target the other putative site. These results highlight the predictive power of LMMD and suggest that predictions derived from LMMD simulations can serve as a reliable basis for the identification of novel ligand binding sites in structure-based drug design. PMID:27532490

  20. Promoting the Adsorption of Metal Ions on Kaolinite by Defect Sites: A Molecular Dynamics Study

    PubMed Central

    Li, Xiong; Li, Hang; Yang, Gang

    2015-01-01

    Defect sites exist abundantly in minerals and play a crucial role for a variety of important processes. Here molecular dynamics simulations are used to comprehensively investigate the adsorption behaviors, stabilities and mechanisms of metal ions on defective minerals, considering different ionic concentrations, defect sizes and contents. Outer-sphere adsorbed Pb2+ ions predominate for all models (regular and defective), while inner-sphere Na+ ions, which exist sporadically only at concentrated solutions for regular models, govern the adsorption for all defective models. Adsorption quantities and stabilities of metal ions on kaolinite are fundamentally promoted by defect sites, thus explaining the experimental observations. Defect sites improve the stabilities of both inner- and outer-sphere adsorption, and (quasi) inner-sphere Pb2+ ions emerge only at defect sites that reinforce the interactions. Adsorption configurations are greatly altered by defect sites but respond weakly by changing defect sizes or contents. Both adsorption quantities and stabilities are enhanced by increasing defect sizes or contents, while ionic concentrations mainly affect adsorption quantities. We also find that adsorption of metal ions and anions can be promoted by each other and proceeds in a collaborative mechanism. Results thus obtained are beneficial to comprehend related processes for all types of minerals. PMID:26403873

  1. Thiolactomycin inhibits D-aspartate oxidase: a novel approach to probing the active site environment.

    PubMed

    Katane, Masumi; Saitoh, Yasuaki; Hanai, Toshihiko; Sekine, Masae; Furuchi, Takemitsu; Koyama, Nobuhiro; Nakagome, Izumi; Tomoda, Hiroshi; Hirono, Shuichi; Homma, Hiroshi

    2010-10-01

    D-Aspartate oxidase (DDO) and D-amino acid oxidase (DAO) are flavin adenine dinucleotide (FAD)-containing flavoproteins that catalyze the oxidative deamination of D-amino acids. While several functionally and structurally important amino acid residues have been identified in the DAO protein, little is known about the structure-function relationships of DDO. In the search for a potent DDO inhibitor as a novel tool for investigating its structure-function relationships, a large number of biologically active compounds of microbial origin were screened for their ability to inhibit the enzymatic activity of mouse DDO. We discovered several compounds that inhibited the activity of mouse DDO, and one of the compounds identified, thiolactomycin (TLM), was then characterized and evaluated as a novel DDO inhibitor. TLM reversibly inhibited the activity of mouse DDO with a mixed type of inhibition more efficiently than meso-tartrate and malonate, known competitive inhibitors of mammalian DDOs. The selectivity of TLM was investigated using various DDOs and DAOs, and it was found that TLM inhibits not only DDO, but also DAO. Further experiments with apoenzymes of DDO and DAO revealed that TLM is most likely to inhibit the activities of DDO and DAO by competition with both the substrate and the coenzyme, FAD. Structural models of mouse DDO/TLM complexes supported this finding. The binding mode of TLM to DDO was validated further by site-directed mutagenesis of an active site residue, Arg-237. Collectively, our findings show that TLM is a novel, active site-directed DDO inhibitor that will be useful for elucidating the molecular details of the active site environment of DDO. PMID:20603179

  2. Potential sites of CFTR activation by tyrosine kinases.

    PubMed

    Billet, Arnaud; Jia, Yanlin; Jensen, Timothy J; Hou, Yue-Xian; Chang, Xiu-Bao; Riordan, John R; Hanrahan, John W

    2016-05-01

    The CFTR chloride channel is tightly regulated by phosphorylation at multiple serine residues. Recently it has been proposed that its activity is also regulated by tyrosine kinases, however the tyrosine phosphorylation sites remain to be identified. In this study we examined 2 candidate tyrosine residues near the boundary between the first nucleotide binding domain and the R domain, a region which is important for channel function but devoid of PKA consensus sequences. Mutating tyrosines at positions 625 and 627 dramatically reduced responses to Src or Pyk2 without altering the activation by PKA, suggesting they may contribute to CFTR regulation. PMID:26645934

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

    SciTech Connect

    Nekrasov, Vyacheslav M.; Yurkin, Maxim A.; Chernyshev, Andrei V.; Polshchitsin, Alexey A.; Yakovleva, Galina E.; Maltsev, Valeri P.

    2014-08-14

    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.

  4. The activation strain model and molecular orbital theory

    PubMed Central

    Wolters, Lando P; Bickelhaupt, F Matthias

    2015-01-01

    The activation strain model is a powerful tool for understanding reactivity, or inertness, of molecular species. This is done by relating the relative energy of a molecular complex along the reaction energy profile to the structural rigidity of the reactants and the strength of their mutual interactions: ΔE(ζ) = ΔEstrain(ζ) + ΔEint(ζ). We provide a detailed discussion of the model, and elaborate on its strong connection with molecular orbital theory. Using these approaches, a causal relationship is revealed between the properties of the reactants and their reactivity, e.g., reaction barriers and plausible reaction mechanisms. This methodology may reveal intriguing parallels between completely different types of chemical transformations. Thus, the activation strain model constitutes a unifying framework that furthers the development of cross-disciplinary concepts throughout various fields of chemistry. We illustrate the activation strain model in action with selected examples from literature. These examples demonstrate how the methodology is applied to different research questions, how results are interpreted, and how insights into one chemical phenomenon can lead to an improved understanding of another, seemingly completely different chemical process. WIREs Comput Mol Sci 2015, 5:324–343. doi: 10.1002/wcms.1221 PMID:26753009

  5. Molecular Dynamics of Rab7::REP1::GGTase-II Ternary Complex and Identification of Their Putative Drug Binding Sites.

    PubMed

    Sindhu, Meenakshi; Saini, Vandana; Piplani, Sakshi; Kumar, A

    2013-01-01

    The structure-function correlation of membrane proteins have been a difficult task, particularly in context to transient protein complexes. The molecular simulation of ternary complex of Rab7::REP1::GGTase-II was carried out to understand the basic structural events occurring during the prenylation event of Rab proteins, using the software YASARA. The study suggested that the C-terminus of Rab7 has to be in completely extended conformation during prenylation to reach the active site of RabGGTase-II. Also, attempt was made to find putative drug binding sites on the ternary complex of Rab7::REP1::GGTase-II using Q-SiteFinder programme. The comprehensive consensus probe generated by the program revealed a total of 10 major pockets as putative drug binding sites on Rab7::REP:: GGTase-II ternary complex. These pockets were found on REP protein and GGTase protein subunits. The Rab7 was found to be devoid of any putative drug binding sites in the ternary complex. The phylogenetic analysis of 60 Rab proteins of human was carried out using PHYLIP and study indicated the close phylogenetic relationship between Rab7 and Rab9 proteins of human and hence with further in silico study, the present observations can be extrapolated to Rab9 proteins. The study paves a good platform for further experimental verifications of the findings and other in silico studies like identifying the potential drug targets by searching the putative drug binding sites, generating pharmacophoric pattern, searching or constructing suitable ligand and docking studies. PMID:23901157

  6. Site-specific labeling of proteins via sortase: protocols for the molecular biologist.

    PubMed

    Popp, Maximilian Wei-Lin

    2015-01-01

    Creation of site-specifically labeled protein bioconjugates is an important tool for the molecular biologist and cell biologist. Chemical labeling methods, while versatile with respect to the types of moieties that can be attached, suffer from lack of specificity, often targeting multiple positions within a protein. Here we describe protocols for the chemoenzymatic labeling of proteins at the C-terminus using the bacterial transpeptidase, sortase A. We detail a protocol for the purification of an improved pentamutant variant of the Staphylococcus aureus enzyme (SrtA 5(o)) that exhibits vastly improved kinetics relative to the wild-type enzyme. Importantly, a protocol for the construction of peptide probes compatible with sortase labeling using techniques that can be adapted to any cellular/molecular biology lab with no existing infrastructure for synthetic chemistry is described. Finally, we provide an example of how to optimize the labeling reaction using the improved SrtA 5(o) variant. PMID:25560076

  7. Mapping Topoisomerase IV Binding and Activity Sites on the E. coli Genome.

    PubMed

    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

  8. Mapping Topoisomerase IV Binding and Activity Sites on the E. coli Genome

    PubMed Central

    Lebailly, Elise; Pages, Carine; Cornet, Francois; Cosentino Lagomarsino, Marco

    2016-01-01

    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

  9. High Sulfation and a High Molecular Weight Are Important for Anti-hepcidin Activity of Heparin

    PubMed Central

    Asperti, Michela; Naggi, Annamaria; Esposito, Emiliano; Ruzzenenti, Paola; Di Somma, Margherita; Gryzik, Magdalena; Arosio, Paolo; Poli, Maura

    2016-01-01

    Heparins are efficient inhibitors of hepcidin expression even in vivo, where they induce an increase of systemic iron availability. Heparins seem to act by interfering with BMP6 signaling pathways that control the expression of liver hepcidin, causing the suppression of SMAD1/5/8 phosphorylation. The anti-hepcidin activity persists also when the heparin anticoagulant property is abolished or reduced by chemical reactions of oxidation/reduction (glycol-split, Gs-Heparins) or by high sulfation (SS-Heparins), but the structural characteristics needed to optimize this inhibitory activity have not been studied in detail. To this aim we analyzed three different heparins (Mucosal Heparin, the Glycol split RO-82, the partially desulfated glycol-split RO-68 and the oversulfated SSLMWH) and separated them in fractions of molecular weight in the range 4–16 kD. Since the distribution of the negative charges in heparins contributes to the activity, we produced 2-O- and 6-O-desulfated heparins. These derivatives were analyzed for the capacity to inhibit hepcidin expression in hepatic HepG2 cells and in mice. The two approaches produced consistent results and showed that the anti-hepcidin activity strongly decreases with molecular weight below 7 kD, with high N-acetylation and after 2-O and 6-O desulfation. The high sulfation and high molecular weight properties for efficient anti-hepcidin activity suggest that heparin is involved in multiple binding sites. PMID:26955355

  10. High Sulfation and a High Molecular Weight Are Important for Anti-hepcidin Activity of Heparin.

    PubMed

    Asperti, Michela; Naggi, Annamaria; Esposito, Emiliano; Ruzzenenti, Paola; Di Somma, Margherita; Gryzik, Magdalena; Arosio, Paolo; Poli, Maura

    2015-01-01

    Heparins are efficient inhibitors of hepcidin expression even in vivo, where they induce an increase of systemic iron availability. Heparins seem to act by interfering with BMP6 signaling pathways that control the expression of liver hepcidin, causing the suppression of SMAD1/5/8 phosphorylation. The anti-hepcidin activity persists also when the heparin anticoagulant property is abolished or reduced by chemical reactions of oxidation/reduction (glycol-split, Gs-Heparins) or by high sulfation (SS-Heparins), but the structural characteristics needed to optimize this inhibitory activity have not been studied in detail. To this aim we analyzed three different heparins (Mucosal Heparin, the Glycol split RO-82, the partially desulfated glycol-split RO-68 and the oversulfated SSLMWH) and separated them in fractions of molecular weight in the range 4-16 kD. Since the distribution of the negative charges in heparins contributes to the activity, we produced 2-O- and 6-O-desulfated heparins. These derivatives were analyzed for the capacity to inhibit hepcidin expression in hepatic HepG2 cells and in mice. The two approaches produced consistent results and showed that the anti-hepcidin activity strongly decreases with molecular weight below 7 kD, with high N-acetylation and after 2-O and 6-O desulfation. The high sulfation and high molecular weight properties for efficient anti-hepcidin activity suggest that heparin is involved in multiple binding sites. PMID:26955355

  11. Molecular field extrema as descriptors of biological activity: definition and validation.

    PubMed

    Cheeseright, Tim; Mackey, Mark; Rose, Sally; Vinter, Andy

    2006-01-01

    The paper describes the generation of four types of three-dimensional molecular field descriptors or 'field points' as extrema of electrostatic, steric, and hydrophobic fields. These field points are used to define the properties necessary for a molecule to bind in a characteristic way into a specified active site. The hypothesis is that compounds showing a similar field point pattern are likely to bind at the same target site regardless of structure. The methodology to test this idea is illustrated using HIV NNRTI and thrombin ligands and validated across seven other targets. From the in silico comparisons of field point overlays, the experimentally observed binding poses of these ligands in their respective sites can be reproduced from pairwise comparisons. PMID:16562997

  12. Triplex molecular beacons for sensitive recognition of melamine based on abasic-site-containing DNA and fluorescent silver nanoclusters.

    PubMed

    Wang, Ya; Sun, Qianqian; Zhu, Linling; Zhang, Junying; Wang, Fengyang; Lu, Linlin; Yu, Haijun; Xu, Zhiai; Zhang, Wen

    2015-05-01

    A melamine aptamer derived from an abasic-site-containing triplex molecular beacon (tMB) was designed and developed for sensitive recognition of melamine by integrating tMBs and fluorescent silver nanoclusters (Ag NCs). PMID:25865656

  13. Molecular dynamics study of liquid methanol with a flexible three-site model

    SciTech Connect

    Palinkas, G.; Hawlicka, E.; Heinzinger, K.

    1987-07-30

    A new potential is presented which describes the methanol-methanol interactions on the basis of a flexible three-site model. The intramolecular part of the potential has been derived from spectroscopic data. A molecular dynamics study has been performed with this potential at 286 K. The structural properties of liquid methanol calculated from the simulations are in good agreement with X-ray measurements. The average geometrical arrangement of nearest neighbors and their hydrogen bonding are discussed. The potential describes correctly the gas-liquid frequency shifts of the intramolecular vibrations. Several thermodynamic properties calculated from the simulation compare favorably with experimental results.

  14. Noninvasive Molecular Imaging of Disease Activity in Atherosclerosis.

    PubMed

    Dweck, Marc R; Aikawa, Elena; Newby, David E; Tarkin, Jason M; Rudd, James H F; Narula, Jagat; Fayad, Zahi A

    2016-07-01

    Major focus has been placed on the identification of vulnerable plaques as a means of improving the prediction of myocardial infarction. However, this strategy has recently been questioned on the basis that the majority of these individual coronary lesions do not in fact go on to cause clinical events. Attention is, therefore, shifting to alternative imaging modalities that might provide a more complete pan-coronary assessment of the atherosclerotic disease process. These include markers of disease activity with the potential to discriminate between patients with stable burnt-out disease that is no longer metabolically active and those with active atheroma, faster disease progression, and increased risk of infarction. This review will examine how novel molecular imaging approaches can provide such assessments, focusing on inflammation and microcalcification activity, the importance of these processes to coronary atherosclerosis, and the advantages and challenges posed by these techniques. PMID:27390335

  15. Molecular profiling of activated neurons by phosphorylated ribosome capture.

    PubMed

    Knight, Zachary A; Tan, Keith; Birsoy, Kivanc; Schmidt, Sarah; Garrison, Jennifer L; Wysocki, Robert W; Emiliano, Ana; Ekstrand, Mats I; Friedman, Jeffrey M

    2012-11-21

    The mammalian brain is composed of thousands of interacting neural cell types. Systematic approaches to establish the molecular identity of functional populations of neurons would advance our understanding of neural mechanisms controlling behavior. Here, we show that ribosomal protein S6, a structural component of the ribosome, becomes phosphorylated in neurons activated by a wide range of stimuli. We show that these phosphorylated ribosomes can be captured from mouse brain homogenates, thereby enriching directly for the mRNAs expressed in discrete subpopulations of activated cells. We use this approach to identify neurons in the hypothalamus regulated by changes in salt balance or food availability. We show that galanin neurons are activated by fasting and that prodynorphin neurons restrain food intake during scheduled feeding. These studies identify elements of the neural circuit that controls food intake and illustrate how the activity-dependent capture of cell-type-specific transcripts can elucidate the functional organization of a complex tissue. PMID:23178128

  16. First molecular evidence of intrauterine and surgical-site infections caused by Streptococcus dysgalactiae subsp. equisimilis.

    PubMed

    Lu, Binghuai; Diao, Baowei; Fang, Yujie; Shi, Yanli; Zhu, Fengxia; Li, Dong; Zhang, Shuchen; Cui, Yanchao; Wang, Duochun

    2016-01-01

    S. dysgalactiae subsp. equisimilis (SDSE) is infrequently associated with maternal infections during delivery in pregnant women. A rare case is presented of a woman with intrauterine infection and surgical-site infection due to SDSE after cesarean section, which had colonized her genital tract and, via the ascending pathway, reached her intact fetal membrane. All isolates were identified as Streptococcus Lancefield group G, and their emm genes that coded M protein belonged to stG6.1. The isolates tested negative for a series of streptococcal superantigen virulence genes but positive for nonsuperantigenic virulence genes. In particular, molecular typing using pulsed-field gel electrophoresis analysis disclosed that the three isolates from the different infection sites had identical profiles. Furthermore, multilocus sequence typing indicated that the three isolates belonged to a new sequence typing. Our results indicated that SDSE is potentially pathogenic for pregnant women and newborns if colonized. PMID:27367018

  17. Molecular characterization of novel splice site mutation causing protein C deficiency.

    PubMed

    Al-Hamed, Mohamed H; AlBatniji, Fatma; AlDakheel, Ghadah A; El-Faraidi, Huda; Al-Zahrani, Azzah; Al-Abbass, Fahed; Imtiaz, Faiqa

    2016-07-01

    Congenital protein C deficiency is an inherited coagulation disorder associated with an elevated risk of venous thromboembolism. A Saudi Arabian male from a consanguineous family was admitted to neonatal intensive care unit in his first days of life because of transient tachypnea and hematuria. Laboratory investigations determined low platelet and protein C deficiency. Direct sequencing of PROC gene and RNA analysis were performed. Analysis of factor V Leiden (G1691A) and factor II (G20210A) mutations was also done. Novel homozygous splice site mutation c.796+3A>T was detected in the index case and segregation was confirmed in the family. RNA analysis revealed the pathogenicity of the mutation by skipping exon 8 of PROC gene and changing the donor splice site of the exon. Detection of the molecular cause of protein C deficiency reduces life threatening and facilitates inductive carrier testing, prenatal and preimplantation genetic diagnosis for families. PMID:26656900

  18. Dynamics of the Active Sites of Dimeric Seryl tRNA Synthetase from Methanopyrus kandleri.

    PubMed

    Dutta, Saheb; Nandi, Nilashis

    2015-08-27

    Aminoacyl tRNA synthetases (aaRSs) carry out the first step of protein biosynthesis. Several aaRSs are multimeric, and coordination between the dynamics of active sites present in each monomer is a prerequisite for the fast and accurate aminoacylation. However, important lacunae of understanding exist concerning the conformational dynamics of multimeric aaRSs. Questions remained unanswered pertaining to the dynamics of the active site. Little is known concerning the conformational dynamics of the active sites in response to the substrate binding, reorganization of the catalytic residues around reactants, time-dependent changes at the reaction center, which are essential for facilitating the nucleophilic attack, and interactions at the interface of neighboring monomers. In the present work, we carried out all-atom molecular dynamics simulation of dimeric (mk)SerRS from Methanopyrus kandleri bound with tRNA using an explicit solvent system. Two dimeric states of seryl tRNA synthetase (open, substrate bound, and adenylate bound) and two monomeric states (open and substrate bound) are simulated with bound tRNA. The aim is to understand the conformational dynamics of (mk)SerRS during its reaction cycle. While the present results provide a clear dynamical perspective of the active sites of (mk)SerRS, they corroborate with the results from the time-averaged experimental data such as crystallographic and mutation analysis of methanogenic SerRS from M. kandleri and M. barkeri. It is observed from the present simulation that the motif 2 loop gates the active site and its Glu351 and Arg360 stabilizes ATP in a bent state favorable for nucleophilic attack. The flexibility of the walls of the active site gradually reduces near reaction center, which is a more organized region compared to the lid region. The motif 2 loop anchors Ser and ATP using Arg349 in a hydrogen bonded geometry crucial for nucleophilic attack and favorably influences the electrostatic potential at the

  19. Probing the Electrostatics of Active Site Microenvironments along the Catalytic Cycle for Escherichia coli Dihydrofolate Reductase

    PubMed Central

    2015-01-01

    Electrostatic interactions play an important role in enzyme catalysis by guiding ligand binding and facilitating chemical reactions. These electrostatic interactions are modulated by conformational changes occurring over the catalytic cycle. Herein, the changes in active site electrostatic microenvironments are examined for all enzyme complexes along the catalytic cycle of Escherichia coli dihydrofolate reductase (ecDHFR) by incorporation of thiocyanate probes at two site-specific locations in the active site. The electrostatics and degree of hydration of the microenvironments surrounding the probes are investigated with spectroscopic techniques and mixed quantum mechanical/molecular mechanical (QM/MM) calculations. Changes in the electrostatic microenvironments along the catalytic environment lead to different nitrile (CN) vibrational stretching frequencies and 13C NMR chemical shifts. These environmental changes arise from protein conformational rearrangements during catalysis. The QM/MM calculations reproduce the experimentally measured vibrational frequency shifts of the thiocyanate probes across the catalyzed hydride transfer step, which spans the closed and occluded conformations of the enzyme. Analysis of the molecular dynamics trajectories provides insight into the conformational changes occurring between these two states and the resulting changes in classical electrostatics and specific hydrogen-bonding interactions. The electric fields along the CN axes of the probes are decomposed into contributions from specific residues, ligands, and solvent molecules that make up the microenvironments around the probes. Moreover, calculation of the electric field along the hydride donor–acceptor axis, along with decomposition of this field into specific contributions, indicates that the cofactor and substrate, as well as the enzyme, impose a substantial electric field that facilitates hydride transfer. Overall, experimental and theoretical data provide evidence for

  20. The Histamine N-Methyltransferase T105I Polymorphism Affects Active Site Structure and Dynamics†

    PubMed Central

    Rutherford, Karen; Parson, William W.; Daggett, Valerie

    2010-01-01

    Histamine N-methyltransferase (HNMT) is the sole enzyme responsible for inactivating histamine in the mammalian brain. The human HNMT gene contains a common threonine-isoleucine polymorphism at residue 105, distal from the active site. The 105I variant has decreased activity and lower protein levels relative to the 105T protein. Crystal structures of both variants have been solved, but reveal little regarding how the T105I polymorphism affects activity. We performed molecular dynamics simulations of both 105T and 105I at 37°C to explore the structural and dynamic consequences of the polymorphism. The simulations indicate that replacing Thr with the larger Ile residue leads to greater burial of residue 105 and heightened packing interactions between residue105 and residues within helix α3 and strand β3. This altered packing is directly translated to the active site resulting in the reorientation of several co-substrate-binding residues. The simulations also show that the hydrophobic histamine-binding domain in both proteins undergoes a large-scale breathing motion that exposes key catalytic residues and lessens the hydrophobicity of the substrate-binding site. PMID:18154359

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

    SciTech Connect

    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.

  2. Chemistry in the Molecular Disks of Active Galactic Nuclei

    NASA Astrophysics Data System (ADS)

    Harada, Nanase; Herbst, Eric

    2010-06-01

    Active galactic nuclei (AGNs) are the centers of galaxies with supermassive blackholes whose accretion of mass causes very high luminosities of L˜1044-46erg s-1. An accretion disk has a molecular component that extends to hundreds of pc from the central AGN core. The question of how much central illumination affects the disk and how much star formation is present near the core have been astrophysical interests. Rotational lines from these disks at a sub-kpc scale have been observed for molecules such as CO, HCO+, HCN, and HNC. When ALMA becomes fully operational, it will be able to resolve these disks at much higher resolution than currently. Molecular observations at higher resolution may give some hints on the physics in the molecular disk. We modeled the chemical composition of a molecular disk in an AGN on a scale of tens of pc. To do this, we extended our standard gas-phase OSU network to include important processes at much higher temperatures, approaching 1000 K. We used the density model of Thompson et al., and determined the temperature by the blackbody approximation from the luminosity of the AGN core. The ionization by X-rays from the AGN core, by cosmic-rays from the AGN core, supernovae and stellar winds, and by UV-photons from OB stars are considered. We will briefly mention the effects from other factors that may change the molecular abundances such as shock waves and inhomogeneity of the density of the disk. T. Thompson, E. Quataert, and N. Murray, Astrophysical J. 630, 167 (2005)

  3. Molecular candidates for cardiac stretch-activated ion channels

    PubMed Central

    Reed, Alistair; Kohl, Peter; Peyronnet, Rémi

    2014-01-01

    The heart is a mechanically-active organ that dynamically senses its own mechanical environment. This environment is constantly changing, on a beat-by-beat basis, with additional modulation by respiratory activity and changes in posture or physical activity, and further overlaid with more slowly occurring physiological (e.g. pregnancy, endurance training) or pathological challenges (e.g. pressure or volume overload). Far from being a simple pump, the heart detects changes in mechanical demand and adjusts its performance accordingly, both via heart rate and stroke volume alteration. Many of the underlying regulatory processes are encoded intracardially, and are thus maintained even in heart transplant recipients. Over the last three decades, molecular substrates of cardiac mechanosensitivity have gained increasing recognition in the scientific and clinical communities. Nonetheless, the processes underlying this phenomenon are still poorly understood. Stretch-activated ion channels (SAC) have been identified as one contributor to mechanosensitive autoregulation of the heartbeat. They also appear to play important roles in the development of cardiac pathologies – most notably stretch-induced arrhythmias. As recently discovered, some established cardiac drugs act, in part at least, via mechanotransduction pathways suggesting SAC as potential therapeutic targets. Clearly, identification of the molecular substrate of cardiac SAC is of clinical importance and a number of candidate proteins have been identified. At the same time, experimental studies have revealed variable–and at times contrasting–results regarding their function. Further complication arises from the fact that many ion channels that are not classically defined as SAC, including voltage and ligand-gated ion channels, can respond to mechanical stimulation. Here, we summarise what is known about the molecular substrate of the main candidates for cardiac SAC, before identifying potential further

  4. Systematic Discovery of Molecular Probes Targeting Multiple Non-orthosteric and Spatially Distinct Sites in the Botulinum Neurotoxin Subtype A (BoNT/A)

    PubMed Central

    Dadgar, Saedeh; Floriano, Wely B.

    2015-01-01

    The development of molecular probes targeting proteins has traditionally relied on labeling compounds already known to bind to the protein of interest. These known ligands bind to orthosteric or allosteric sites in their target protein as a way to control their activity. Binding pockets other than known orthosteric or allosteric sites may exist that are large enough to accommodate a ligand without significantly disrupting protein activity. Such sites may provide opportunities to discriminate between subtypes or other closely related proteins, since they are under less evolutionary pressure to be conserved. The Protein Scanning with Virtual Ligand Screening (PSVLS) approach was previously used to identify a novel inhibitor and a fluorescent probe against the catalytic site of the botulinum neurotoxin subtype A (BoNT/A). PSVLS screens compound databases against multiple sites within a target protein, and the results for all the sites probed against BoNT/A, not only the catalytic site, are available online. Here, we analyze the PSVLS data for multiple sites in order to identify molecular probes with affinity for binding pockets other than the catalytic site of BoNT/A. BoNT/A is a large protein with a light (LC) and a heavy (HC) chain that can be assayed separately. We used scintillation proximity assay (SPA) to test experimentally 5 probe candidates predicted computationally to have affinity for different non-orthosteric binding regions within the HC and LC, and one compound predicted not to have affinity for either domain. The binding profiles obtained experimentally confirmed the targeting of multiple and spatially distinct pockets within BoNT/A. Moreover, inhibition assay results indicate that some of these probes do not significantly interfere with the catalytic activity of BoNT/A. PMID:25745992

  5. Site-selective Characterization of Src Homology 3 Domain Molecular Recognition with Cyanophenylalanine Infrared Probes

    PubMed Central

    Horness, Rachel E.; Basom, Edward J.; Thielges, Megan C.

    2015-01-01

    Local heterogeneity of microenvironments in proteins is important in biological function, but difficult to characterize experimentally. One approach is the combination of infrared (IR) spectroscopy and site-selective incorporation of probe moieties with spectrally resolved IR absorptions that enable characterization within inherently congested protein IR spectra. We employed this method to study molecular recognition of a Src homology 3 (SH3) domain from the yeast protein Sho1 for a peptide containing the proline-rich recognition sequence of its physiological binding partner Pbs2. Nitrile IR probes were introduced at four distinct sites in the protein by selective incorporation of p-cyanophenylalanine via the amber codon suppressor method and characterized by IR spectroscopy. Variation among the IR absorption bands reports on heterogeneity in local residue environments dictated by the protein structure, as well as on residue-dependent changes upon peptide binding. The study informs on the molecular recognition of SH3Sho1 and illustrates the speed and simplicity of this approach for characterization of select microenvironments within proteins. PMID:26491469

  6. Kinetic model of ethopropazine interaction with horse serum butyrylcholinesterase and its docking into the active site.

    PubMed

    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

  7. The active sites in the heterogeneous Baeyer-Villiger oxidation of cyclopentanone by hydrotalcite catalysts

    NASA Astrophysics Data System (ADS)

    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.

  8. Impact of single-site axonal GABAergic synaptic events on cerebellar interneuron activity

    PubMed Central

    Zorrilla de San Martin, Javier; Jalil, Abdelali

    2015-01-01

    Axonal ionotropic receptors are present in a variety of neuronal types, and their function has largely been associated with the modulation of axonal activity and synaptic release. It is usually assumed that activation of axonal GABAARs comes from spillover, but in cerebellar molecular layer interneurons (MLIs) the GABA source is different: in these cells, GABA release activates presynaptic GABAA autoreceptors (autoRs) together with postsynaptic targets, producing an autoR-mediated synaptic event. The frequency of presynaptic, autoR-mediated miniature currents is twice that of their somatodendritic counterparts, suggesting that autoR-mediated responses have an important effect on interneuron activity. Here, we used local Ca2+ photolysis in MLI axons of juvenile rats to evoke GABA release from individual varicosities to study the activation of axonal autoRs in single release sites. Our data show that single-site autoR conductances are similar to postsynaptic dendritic conductances. In conditions of high [Cl−]i, autoR-mediated conductances range from 1 to 5 nS; this corresponds to ∼30–150 GABAA channels per presynaptic varicosity, a value close to the number of channels in postsynaptic densities. Voltage responses produced by the activation of autoRs in single varicosities are amplified by a Nav-dependent mechanism and propagate along the axon with a length constant of 91 µm. Immunolabeling determination of synapse location shows that on average, one third of the synapses produce autoR-mediated signals that are large enough to reach the axon initial segment. Finally, we show that single-site activation of presynaptic GABAA autoRs leads to an increase in MLI excitability and thus conveys a strong feedback signal that contributes to spiking activity. PMID:26621773

  9. Linking neural activity and molecular oscillations in the SCN

    PubMed Central

    Colwell, Christopher S.

    2015-01-01

    Neurons in the suprachiasmatic nucleus (SCN) function as part of a central timing circuit that drives daily changes in our behaviour and underlying physiology. A hallmark feature of SCN neuronal populations is that they are mostly electrically silent during the night, start to fire action potentials near dawn and then continue to generate action potentials with a slow and steady pace all day long. Sets of currents are responsible for this daily rhythm, with the strongest evidence for persistent Na+ currents, L-type Ca2+ currents, hyperpolarization-activated currents (IH), large-conductance Ca2+ activated K+ (BK) currents and fast delayed rectifier (FDR) K+ currents. These rhythms in electrical activity are crucial for the function of the circadian timing system, including the expression of clock genes, and decline with ageing and disease. This article reviews our current understanding of the ionic and molecular mechanisms that drive the rhythmic firing patterns in the SCN. PMID:21886186

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

    PubMed Central

    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. 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.

    PubMed

    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

  12. Dynamic formation of single-atom catalytic active sites on ceria-supported gold nanoparticles

    PubMed Central

    Wang, Yang-Gang; Mei, Donghai; Glezakou, Vassiliki-Alexandra; Li, Jun; Rousseau, Roger

    2015-01-01

    Catalysis by gold supported on reducible oxides has been extensively studied, yet issues such as the nature of the catalytic site and the role of the reducible support remain fiercely debated topics. Here we present ab initio molecular dynamics simulations of an unprecedented dynamic single-atom catalytic mechanism for the oxidation of carbon monoxide by ceria-supported gold clusters. The reported dynamic single-atom catalytic mechanism results from the ability of the gold cation to strongly couple with the redox properties of the ceria in a synergistic manner, thereby lowering the energy of redox reactions. The gold cation can break away from the gold nanoparticle to catalyse carbon monoxide oxidation, adjacent to the metal/oxide interface and subsequently reintegrate back into the nanoparticle after the reaction is completed. Our study highlights the importance of the dynamic creation of active sites under reaction conditions and their essential role in catalysis. PMID:25735407

  13. Site-Specifically Labeled Immunoconjugates for Molecular Imaging--Part 1: Cysteine Residues and Glycans.

    PubMed

    Adumeau, Pierre; Sharma, Sai Kiran; Brent, Colleen; Zeglis, Brian M

    2016-02-01

    that this review fosters interest and enthusiasm for site-specific immunoconjugates within the nuclear medicine and molecular imaging communities. PMID:26754790

  14. Molecular Features of an Alcohol Binding Site in a Neuronal Potassium Channel†

    PubMed Central

    Shahidullah, Mohammad; Harris, Thanawath; Germann, Markus W.; Covarrubias, Manuel

    2008-01-01

    Aliphatic alcohols (1-alkanols) selectively inhibit the neuronal Shaw2 K+ channel at an internal binding site. This inhibition is conferred by a sequence of 13 residues that constitutes the S4–S5 loop in the pore-forming subunit. Here, we combined functional and structural approaches to gain insights into the molecular basis of this interaction. To infer the forces that are involved, we employed a fast concentration-clamp method (10–90% exchange time = 800 μs) to examine the kinetics of the interaction of three members of the homologous series of 1-alkanols (ethanol, 1-butanol, and 1-hexanol) with Shaw2 K+ channels in Xenopus oocyte inside-out patches. As expected for a second-order mechanism involving a receptor site, only the observed association rate constants were linearly dependent on the 1-alkanol concentration. While the alkyl chain length modestly influenced the dissociation rate constants (decreasing only ∼2-fold between ethanol and 1-hexanol), the second-order association rate constants increased e-fold per carbon atom. Thus, hydrophobic interactions govern the probability of productive collisions at the 1-alkanol binding site, and short-range polar interactions help to stabilize the complex. We also examined the relationship between the energetics of 1-alkanol binding and the structural properties of the S4–S5 loop. Circular dichroism spectroscopy applied to peptides corresponding to the S4–S5 loop of various K+ channels revealed a correlation between the apparent binding affinity of the 1-alkanol binding site and the α-helical propensity of the S4–S5 loop. The data suggest that amphiphilic interactions at the Shaw2 1-alkanol binding site depend on specific structural constraints in the pore-forming subunit of the channel. PMID:14503874

  15. The copper active site of CBM33 polysaccharide oxygenases.

    PubMed

    Hemsworth, Glyn R; Taylor, Edward J; Kim, Robbert Q; Gregory, Rebecca C; Lewis, Sally J; Turkenburg, Johan P; Parkin, Alison; Davies, Gideon J; Walton, Paul H

    2013-04-24

    The capacity of metal-dependent fungal and bacterial polysaccharide oxygenases, termed GH61 and CBM33, respectively, to potentiate the enzymatic degradation of cellulose opens new possibilities for the conversion of recalcitrant biomass to biofuels. GH61s have already been shown to be unique metalloenzymes containing an active site with a mononuclear copper ion coordinated by two histidines, one of which is an unusual τ-N-methylated N-terminal histidine. We now report the structural and spectroscopic characterization of the corresponding copper CBM33 enzymes. CBM33 binds copper with high affinity at a mononuclear site, significantly stabilizing the enzyme. X-band EPR spectroscopy of Cu(II)-CBM33 shows a mononuclear type 2 copper site with the copper ion in a distorted axial coordination sphere, into which azide will coordinate as evidenced by the concomitant formation of a new absorption band in the UV/vis spectrum at 390 nm. The enzyme's three-dimensional structure contains copper, which has been photoreduced to Cu(I) by the incident X-rays, confirmed by X-ray absorption/fluorescence studies of both aqueous solution and intact crystals of Cu-CBM33. The single copper(I) ion is ligated in a T-shaped configuration by three nitrogen atoms from two histidine side chains and the amino terminus, similar to the endogenous copper coordination geometry found in fungal GH61. PMID:23540833

  16. The Copper Active Site of CBM33 Polysaccharide Oxygenases

    PubMed Central

    2013-01-01

    The capacity of metal-dependent fungal and bacterial polysaccharide oxygenases, termed GH61 and CBM33, respectively, to potentiate the enzymatic degradation of cellulose opens new possibilities for the conversion of recalcitrant biomass to biofuels. GH61s have already been shown to be unique metalloenzymes containing an active site with a mononuclear copper ion coordinated by two histidines, one of which is an unusual τ-N-methylated N-terminal histidine. We now report the structural and spectroscopic characterization of the corresponding copper CBM33 enzymes. CBM33 binds copper with high affinity at a mononuclear site, significantly stabilizing the enzyme. X-band EPR spectroscopy of Cu(II)-CBM33 shows a mononuclear type 2 copper site with the copper ion in a distorted axial coordination sphere, into which azide will coordinate as evidenced by the concomitant formation of a new absorption band in the UV/vis spectrum at 390 nm. The enzyme’s three-dimensional structure contains copper, which has been photoreduced to Cu(I) by the incident X-rays, confirmed by X-ray absorption/fluorescence studies of both aqueous solution and intact crystals of Cu-CBM33. The single copper(I) ion is ligated in a T-shaped configuration by three nitrogen atoms from two histidine side chains and the amino terminus, similar to the endogenous copper coordination geometry found in fungal GH61. PMID:23540833

  17. Molecular characterization of Capra hircus lysosomal α-mannosidase and potential mutant site for the therapy of locoweed poisoning.

    PubMed

    Xiangya, Kong; Jiangye, Zhang; Ying, Wu; Jianfei, Li; Qinfan, Li

    2014-01-01

    Lysosomal α-Mannosidase (LAM) belongs to the glycoside hydrolyzing enzymes family 38 and is involved in the biosynthesis and turnover of N-linked glycoproteins process. Locoweeds, which contain swainsonine (SW) that inhibits LAM, are the main poisoning plants in many regions of the world, and thereby resulting in animal poisoning or even death. Based on regions of protein sequence conservation between LAM from Bos taurus and Homo sapiens, we cloned cDNA encoding Capra hircus LAM (chLAM). Expression of cDNA in Pichia pastoris resulted in the secretion of aLAM activity into the culture medium. The recombinant chLAM was activated 1.6 and 1.2-fold with Zn(2+) and Ca(2+), respectively. By homology modeling, molecular docking and mutant analysis, we obtained the probable binding modes of SW at the allosteric sites of chLAM, and the potential mutant sites for the resistance to SW. Prediction of SW sensitivity to A28 W/G, D58 Y/G mutant chLAM is lower than wild type chLAM. The obtained results lead to a better understanding of not only interactions between substrate/SW and chLAM, but also of a potential strategy for a novel therapy for locoweed poisoning. PMID:24660168

  18. Nanoplasmonic molecular ruler for nuclease activity and DNA footprinting

    DOEpatents

    Chen, Fanqing Frank; Liu, Gang L; Lee, Luke P

    2013-10-29

    This invention provides a nanoplasmonic molecular ruler, which can perform label-free and real-time monitoring of nucleic acid (e.g., DNA) length changes and perform nucleic acid footprinting. In various embodiments the ruler comprises a nucleic acid attached to a nanoparticle, such that changes in the nucleic acid length are detectable using surface plasmon resonance. The nanoplasmonic ruler provides a fast and convenient platform for mapping nucleic acid-protein interactions, for nuclease activity monitoring, and for other footprinting related methods.

  19. Molecular mechanisms for the conversion of zymogens to active proteolytic enzymes.

    PubMed Central

    Khan, A. R.; James, M. N.

    1998-01-01

    Proteolytic enzymes are synthesized as inactive precursors, or "zymogens," to prevent unwanted protein degradation, and to enable spatial and temporal regulation of proteolytic activity. Upon sorting or appropriate compartmentalization, zymogen conversion to the active enzyme typically involves limited proteolysis and removal of an "activation segment." The sizes of activation segments range from dipeptide units to independently folding domains comprising more than 100 residues. A common form of the activation segment is an N-terminal extension of the mature enzyme, or "prosegment," that sterically blocks the active site, and thereby prevents binding of substrates. In addition to their inhibitory role, prosegments are frequently important for the folding, stability, and/or intracellular sorting of the zymogen. The mechanisms of conversion to active enzymes are diverse in nature, ranging from enzymatic or nonenzymatic cofactors that trigger activation, to a simple change in pH that results in conversion by an autocatalytic mechanism. Recent X-ray crystallographic studies of zymogens and comparisons with their active counterparts have identified the structural changes that accompany conversion. This review will focus upon the structural basis for inhibition by activation segments, as well as the molecular events that lead to the conversion of zymogens to active enzymes. PMID:9568890

  20. Charge transmission through a molecular wire: the role of terminal sites for the current-voltage behavior.

    PubMed

    Petrov, E G; Zelinskyy, Ya R; May, V; Hänggi, P

    2007-08-28

    The current-voltage and the conductance-voltage characteristics are analyzed for a particular type of molecular wire embedded between two electrodes. The wire is characterized by internal molecular units where the lowest occupied molecular orbital (LUMO) levels are positioned much above the Fermi energy of the electrodes, as well as above the LUMO levels of the terminal wire units. The latter act as specific intermediate donor and acceptor sites which in turn control the current formation via the superexchange and sequential electron transfer mechanisms. According to the chosen wire structure, intramolecular multiphonon processes may block the superexchange component of the interelectrode current, resulting in a negative differential resistance of the molecular wire. A pronounced current rectification appears if (i) the superexchange component dominates the electron transfer between the terminal sites and if (ii) the multiphonon suppression of distant superexchange charge hopping events between those sites is nonsymmetric. PMID:17764286

  1. Differential Assembly of Catalytic Interactions within the Conserved Active Sites of Two Ribozymes

    PubMed Central

    Herschlag, Daniel

    2016-01-01

    Molecular recognition is central to biology and a critical aspect of RNA function. Yet structured RNAs typically lack the preorganization needed for strong binding and precise positioning. A striking example is the group I ribozyme from Tetrahymena, which binds its guanosine substrate (G) orders of magnitude slower than diffusion. Binding of G is also thermodynamically coupled to binding of the oligonucleotide substrate (S) and further work has shown that the transition from E•G to E•S•G accompanies a conformational change that allows G to make the active site interactions required for catalysis. The group I ribozyme from Azoarcus has a similarly slow association rate but lacks the coupled binding observed for the Tetrahymena ribozyme. Here we test, using G analogs and metal ion rescue experiments, whether this absence of coupling arises from a higher degree of preorganization within the Azoarcus active site. Our results suggest that the Azoarcus ribozyme forms cognate catalytic metal ion interactions with G in the E•G complex, interactions that are absent in the Tetrahymena E•G complex. Thus, RNAs that share highly similar active site architectures and catalyze the same reactions can differ in the assembly of transition state interactions. More generally, an ability to readily access distinct local conformational states may have facilitated the evolutionary exploration needed to attain RNA machines that carry out complex, multi-step processes. PMID:27501145

  2. Free energy simulations of active-site mutants of dihydrofolate reductase.

    PubMed

    Doron, Dvir; Stojković, Vanja; Gakhar, Lokesh; Vardi-Kilshtain, Alexandra; Kohen, Amnon; Major, Dan Thomas

    2015-01-22

    This study employs hybrid quantum mechanics-molecular mechanics (QM/MM) simulations to investigate the effect of mutations of the active-site residue I14 of E. coli dihydrofolate reductase (DHFR) on the hydride transfer. Recent kinetic measurements of the I14X mutants (X = V, A, and G) indicated slower hydride transfer rates and increasingly temperature-dependent kinetic isotope effects (KIEs) with systematic reduction of the I14 side chain. The QM/MM simulations show that when the original isoleucine residue is substituted in silico by valine, alanine, or glycine (I14V, I14A, and I14G DHFR, respectively), the free energy barrier height of the hydride transfer reaction increases relative to the wild-type enzyme. These trends are in line with the single-turnover rate measurements reported for these systems. In addition, extended dynamics simulations of the reactive Michaelis complex reveal enhanced flexibility in the mutants, and in particular for the I14G mutant, including considerable fluctuations of the donor-acceptor distance (DAD) and the active-site hydrogen bonding network compared with those detected in the native enzyme. These observations suggest that the perturbations induced by the mutations partly impair the active-site environment in the reactant state. On the other hand, the average DADs at the transition state of all DHFR variants are similar. Crystal structures of I14 mutants (V, A, and G) confirmed the trend of increased flexibility of the M20 and other loops. PMID:25382260

  3. An Active Site Water Network in the Plasminogen Activator Pla from Yersinia pestis

    SciTech Connect

    Eren, Elif; Murphy, Megan; Goguen, Jon; van den Berg, Bert

    2010-08-13

    The plasminogen activator Pla from Yersinia pestis is an outer membrane protease (omptin) that is important for the virulence of plague. Here, we present the high-resolution crystal structure of wild-type, enzymatically active Pla at 1.9 {angstrom}. The structure shows a water molecule located between active site residues D84 and H208, which likely corresponds to the nucleophilic water. A number of other water molecules are present in the active site, linking residues important for enzymatic activity. The R211 sidechain in loop L4 is close to the nucleophilic water and possibly involved in the stabilization of the oxyanion intermediate. Subtle conformational changes of H208 result from the binding of lipopolysaccharide to the outside of the barrel, explaining the unusual dependence of omptins on lipopolysaccharide for activity. The Pla structure suggests a model for the interaction with plasminogen substrate and provides a more detailed understanding of the catalytic mechanism of omptin proteases.

  4. Design, synthesis, molecular docking studies and anti-HBV activity of phenylpropanoid derivatives.

    PubMed

    Liu, Sheng; Li, Yubin; Wei, Wanxing; Wang, Kuiwu; Wang, Lisheng; Wang, Jianyi

    2016-05-01

    In this work, a series of phenylpropanoid derivatives were synthesized, and their anti-hepatitis B virus (HBV) activity was evaluated. Most of the synthesized derivatives showed effective anti-HBV activity. And compound 4d-3 showed the most effective anti-HBV activity, performing strong potent inhibitory not only on the secretion of HBsAg (IC50 = 58.28 μM, SI = 23.26) and HBeAg (IC50 = 97.21 μM, SI = 13.95), but also on the HBV DNA replication (IC50 = 42.28 μM, SI = 32.06). The structure-activity relationships (SARs) of the derivatives had been discussed, which were useful for developing phenylpropanoid derivatives as novel anti-HBV agents. Moreover, the docking study of all synthesized compounds inside the HLA-A protein (PDB ID: 3OX8) active site was carried out to explore the molecular interactions and a molecular target for activity and a modified assay method measuring the interaction between our derivatives and HBcAg was investigated, indicating that the HBV core protein might be their potential target for anti-HBV. This study identified a new class of potent non-nucleoside anti-HBV agents. PMID:26980103

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

    PubMed

    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

  6. Target-classification approach applied to active UXO sites

    NASA Astrophysics Data System (ADS)

    Shubitidze, F.; Fernández, J. P.; Shamatava, Irma; Barrowes, B. E.; O'Neill, K.

    2013-06-01

    This study is designed to illustrate the discrimination performance at two UXO active sites (Oklahoma's Fort Sill and the Massachusetts Military Reservation) of a set of advanced electromagnetic induction (EMI) inversion/discrimination models which include the orthonormalized volume magnetic source (ONVMS), joint diagonalization (JD), and differential evolution (DE) approaches and whose power and flexibility greatly exceed those of the simple dipole model. The Fort Sill site is highly contaminated by a mix of the following types of munitions: 37-mm target practice tracers, 60-mm illumination mortars, 75-mm and 4.5'' projectiles, 3.5'', 2.36'', and LAAW rockets, antitank mine fuzes with and without hex nuts, practice MK2 and M67 grenades, 2.5'' ballistic windshields, M2A1-mines with/without bases, M19-14 time fuzes, and 40-mm practice grenades with/without cartridges. The site at the MMR site contains targets of yet different sizes. In this work we apply our models to EMI data collected using the MetalMapper (MM) and 2 × 2 TEMTADS sensors. The data for each anomaly are inverted to extract estimates of the extrinsic and intrinsic parameters associated with each buried target. (The latter include the total volume magnetic source or NVMS, which relates to size, shape, and material properties; the former includes location, depth, and orientation). The estimated intrinsic parameters are then used for classification performed via library matching and the use of statistical classification algorithms; this process yielded prioritized dig-lists that were submitted to the Institute for Defense Analyses (IDA) for independent scoring. The models' classification performance is illustrated and assessed based on these independent evaluations.

  7. Differential Active Site Loop Conformations Mediate Promiscuous Activities in the Lactonase SsoPox

    PubMed Central

    Elias, Mikael; Chabriere, Eric

    2013-01-01

    Enzymes are proficient catalysts that enable fast rates of Michaelis-complex formation, the chemical step and products release. These different steps may require different conformational states of the active site that have distinct binding properties. Moreover, the conformational flexibility of the active site mediates alternative, promiscuous functions. Here we focused on the lactonase SsoPox from Sulfolobus solfataricus. SsoPox is a native lactonase endowed with promiscuous phosphotriesterase activity. We identified a position in the active site loop (W263) that governs its flexibility, and thereby affects the substrate specificity of the enzyme. We isolated two different sets of substitutions at position 263 that induce two distinct conformational sampling of the active loop and characterized the structural and kinetic effects of these substitutions. These sets of mutations selectively and distinctly mediate the improvement of the promiscuous phosphotriesterase and oxo-lactonase activities of SsoPox by increasing active-site loop flexibility. These observations corroborate the idea that conformational diversity governs enzymatic promiscuity and is a key feature of protein evolvability. PMID:24086491

  8. Molecular evidence for dual pyrethroid-receptor sites on a mosquito sodium channel

    PubMed Central

    Nomura, Yoshiko; Satar, Gul; Hu, Zhaonong; Nauen, Ralf; He, Sheng Yang; Zhorov, Boris S.; Dong, Ke

    2013-01-01

    Pyrethroid insecticides are widely used as one of the most effective control measures in the global fight against agricultural arthropod pests and mosquito-borne diseases, including malaria and dengue. They exert toxic effects by altering the function of voltage-gated sodium channels, which are essential for proper electrical signaling in the nervous system. A major threat to the sustained use of pyrethroids for vector control is the emergence of mosquito resistance to pyrethroids worldwide. Here, we report the successful expression of a sodium channel, AaNav1–1, from Aedes aegypti in Xenopus oocytes, and the functional examination of nine sodium channel mutations that are associated with pyrethroid resistance in various Ae. aegypti and Anopheles gambiae populations around the world. Our analysis shows that five of the nine mutations reduce AaNav1–1 sensitivity to pyrethroids. Computer modeling and further mutational analysis revealed a surprising finding: Although two of the five confirmed mutations map to a previously proposed pyrethroid-receptor site in the house fly sodium channel, the other three mutations are mapped to a second receptor site. Discovery of this second putative receptor site provides a dual-receptor paradigm that could explain much of the molecular mechanisms of pyrethroid action and resistance as well as the high selectivity of pyrethroids on insect vs. mammalian sodium channels. Results from this study could impact future prediction and monitoring of pyrethroid resistance in mosquitoes and other arthropod pests and disease vectors. PMID:23821746

  9. Spectroscopic Definition of the Ferroxidase Site in M Ferritin: Comparison of Binuclear Substrate vs. Cofactor Active Sites

    PubMed Central

    Schwartz, Jennifer K.; Liu, Xiaofeng S.; Tosha, Takehiko; Theil, Elizabeth C.; Solomon, Edward I.

    2008-01-01

    Maxi ferritins, 24 subunit protein nanocages, are essential in humans, plants, bacteria, and other animals for the concentration and storage of iron as hydrated ferric oxide, while minimizing free radical generation or use by pathogens. Formation of the precursors to these ferric oxides is catalyzed at a non-heme biferrous substrate site, which has some parallels with the cofactor sites in other biferrous enzymes. A combination of circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature, variable-field MCD (VTVH MCD) has been used to probe Fe(II) binding to the substrate active site in frog M ferritin. These data determined that the active site within each subunit consists of two inequivalent five-coordinate (5C) ferrous centers that are weakly anti-ferromagnetically coupled, consistent with a μ-1,3 carboxylate bridge. The active site ligand set is unusual and likely includes a terminal water bound to each Fe(II) center. The Fe(II) ions bind to the active sites in a concerted manner, and cooperativity among the sites in each subunit is observed, potentially providing a mechanism for the control of ferritin iron loading. Differences in geometric and electronic structure – including a weak ligand field, availability of two water ligands at the biferrous substrate site, and the single carboxylate bridge in ferritin – coincide with the divergent reaction pathways observed between this substrate site and the previously studied cofactor active sites. PMID:18576633

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

    SciTech Connect

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

    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.

  11. Evidence for segmental mobility in the active site of pepsin

    SciTech Connect

    Pohl, J.; Strop, P.; Senn, H.; Foundling, S.; Kostka, V.

    1986-05-01

    The low hydrolytic activity (k/sub cat/ < 0.001 s/sup -1/) of chicken pepsin (CP) towards tri- and tetrapeptides is enhanced at least 100 times by modification of its single sulfhydryl group of Cys-115, with little effect on K/sub m/-values. Modification thus simulates the effect of secondary substrate binding on pepsin catalysis. The rate of Cys-115 modification is substantially decreased in the presence of some competitive inhibitors, suggesting its active site location. Experiments with CP alkylated at Cys-115 with Acrylodan as a fluorescent probe or with N-iodoacetyl-(4-fluoro)-aniline as a /sup 19/F-nmr probe suggest conformation change around Cys-115 to occur on substrate or substrate analog binding. The difference /sup 1/H-nmr spectra (500 MHz) of unmodified free and inhibitor-complexed CP reveal chemical shifts almost exclusively in the aromatic region. The effects of Cu/sup + +/ on /sup 19/F- and /sup 1/H-nmr spectra have been studied. Examination of a computer graphics model of CP based on E. parasitica pepsin-inhibitor complex X-ray coordinates suggests that Cys-115 is located near the S/sub 3//S/sub 5/ binding site. The results are interpreted in favor of segmental mobility of this region important for pepsin substrate binding and catalysis.

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

    PubMed

    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

  13. Eel calcitonin binding site distribution and antinociceptive activity in rats

    SciTech Connect

    Guidobono, F.; Netti, C.; Sibilia, V.; Villa, I.; Zamboni, A.; Pecile, A.

    1986-03-01

    The distribution of binding site for (/sup 125/I)-eel-calcitonin (ECT) to rat central nervous system, studied by an autoradiographic technique, showed concentrations of binding in the diencephalon, the brain stem and the spinal cord. Large accumulations of grains were seen in the hypothalamus, the amygdala, in the fasciculus medialis prosencephali, in the fasciculus longitudinalis medialis, in the ventrolateral part of the periventricular gray matter, in the lemniscus medialis and in the raphe nuclei. The density of grains in the reticular formation and in the nucleus tractus spinalis nervi trigemini was more moderate. In the spinal cord, grains were scattered throughout the dorsal horns. Binding of the ligand was displaced equally by cold ECT and by salmon CT(sCT), indicating that both peptides bind to the same receptors. Human CT was much weaker than sCT in displacing (/sup 125/I)-ECT binding. The administration of ECT into the brain ventricles of rats dose-dependently induced a significant and long-lasting enhancement of hot-plate latencies comparable with that obtained with sCT. The antinociceptive activity induced by ECT is compatible with the topographical distribution of binding sites for the peptide and is a further indication that fish CTs are active in the mammalian brain.

  14. Molecular Abundances in the Disk of AN Active Galactic Nucleus

    NASA Astrophysics Data System (ADS)

    Harada, N.; Thompson, T. A.; Herbst, E.

    2011-06-01

    There are galactic nuclei that emit high luminosities L˜1044-46 erg S-1 including luminosity produced by X-rays from high mass accretion onto the central black holes. These nuclei are called active galactic nuclei (AGNs), and they are accompanied by molecular disks. Observations show high abundances of CN and HCN in the disks; the molecules are proposed to be probes of X-ray dominated regions (XDRs) created by the X-rays from AGNs. We have constructed a spatially-dependent chemical-abundance model of the molecular disk in NGC 1068, a typical AGN-dominated galaxy. Recently, new observations of CN and HCN have been made at much higher spatial resolution, and there are also detections of polyatomic molecules such as HC3N, c-C3H2, and C2H. We discuss how these observations and our simulations can help us to better understand the physical conditions, the disk structure, and conditions for star formation within molecular disks, which are still uncertain. We also include a comparison with other types of galaxies such as (ultra-) luminous infrared galaxies. Usero et al.Astronomy and Astrophysics. 419 (897), 2004. Initial results were presented at the International Symposium on Molecular Spectroscopy 2010, RF05 Garcia-Burillo et al. Astronomy and Astrophysics. 519 (2), 2010. Garcia-Burillo et al. Journal of Physics Conference Series, 131 (12031), 2008. Costagliola et al. ArXiv e-print arXiv:1101.2122, 2011. Nakajima et al. Astrophysical Journal Letters 728 (L38), 2008.

  15. Site-targeted acoustic contrast agent detects molecular expression of tissue factor after balloon angioplasty

    NASA Astrophysics Data System (ADS)

    Hall, Christopher S.; Abendschein, Dana R.; Scherrer, David E.; Scott, Michael J.; Marsh, Jon N.; Wickline, Samuel A.; Lanza, Gregory M.

    2000-04-01

    Complex molecular signaling heralds the early stages of pathologies such as angiogenesis, inflammation, and cellular responses to mechanically damaged coronary arteries after balloon angioplasty. In previous studies, we have demonstrated acoustic enhancement of blood clot morphology with the use of a nongaseous, ligand-targeted acoustic nanoparticle emulsion delivered to areas of thrombosis both in vitro and in vivo. In this paper, we characterize the early expression of tissue factor which contributes to subsequent arterial restenosis. Tissue factor is a 42kd glycoprotein responsible for blood coagulation but also plays a well-described role in cancer metastasis, angiogenesis, and vascular restenosis. This study was designed to determine whether the targeted contrast agent could localize tissue factor expressed within the wall of balloon-injured arteries. Both carotid arteries of five pigs (20 kg) were injured using an 8 X 20 mm angioplasty balloon. The carotids were treated in situ with a perfluorocarbon nanoparticle emulsion covalently complexed to either specific anti-tissue factor polyclonal F(ab) fragments (treatment) or non-specific IgG F(ab) fragments (control). Intravascular ultrasound (30 MHz) images of the arteries were obtained before and after exposure to the emulsions. Tissue- factor targeted ultrasonic contrast agent acoustically enhanced the subintima and media at the site of balloon- induced injury compared with control contrast arteries (p less than 0.05). Immunohistochemical staining confirmed the presence of increased tissue factor at the sites of acoustic enhancement. Binding of the targeted agents was demonstrated in vitro by scanning electron microscope images of cultured smooth muscle cells that constitutively express tissue factor. This study demonstrates the concept of molecular imaging and localization of carotid arteries' tissue factor in vivo using a new, nanoparticulate emulsion. Enhancement of the visualization of the molecular

  16. Active Sites Environmental Monitoring Program: Program plan. Revision 1

    SciTech Connect

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

    1992-02-01

    The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of transuranic (TRU) waste and active low-level waste (LLW) facilities at Oak Ridge National Laboratory (ORNL) in accordance with US Department of Energy (DOE) Order 5820.2A. Active LLW facilities in Solid Waste Storage Area (SWSA) 6 include Tumulus I and Tumulus II, the Interim Waste Management Facility (IWMF), LLW silos, high-range wells, asbestos silos, and fissile wells. The tumulus pads and IWMF are aboveground, high-strength concrete pads on which concrete vaults containing metal boxes of LLW are placed; the void space between the boxes and vaults is filled with grout. Eventually, these pads and vaults will be covered by an engineered multilayered cap. All other LLW facilities in SWSA 6 are below ground. In addition, this plan includes monitoring of the Hillcut Disposal Test Facility (HDTF) in SWSA 6, even though this facility was completed prior to the data of the DOE order. In SWSA 5 North, the TRU facilities include below-grade engineered caves, high-range wells, and unlined trenches. All samples from SWSA 6 are screened for alpha and beta activity, counted for gamma-emitting isotopes, and analyzed for tritium. In addition to these analytes, samples from SWSA 5 North are analyzed for specific transuranic elements.

  17. Identification of the molecular attributes required for aminoglycoside activity against Leishmania

    PubMed Central

    Shalev, Moran; Kondo, Jiro; Kopelyanskiy, Dmitry; Jaffe, Charles L.; Adir, Noam; Baasov, Timor

    2013-01-01

    Leishmaniasis, a parasitic disease caused by protozoa of the genus Leishmania, affects millions of people worldwide. Aminoglycosides are mostly known as highly potent, broad-spectrum antibiotics that exert their antibacterial activity by selectively targeting the decoding A site of the bacterial ribosome, leading to aberrant protein synthesis. Recently, some aminoglycosides have been clinically approved and are currently used worldwide for the treatment of leishmaniasis; however the molecular details by which aminoglycosides induce their deleterious effect on Leishmaina is still rather obscure. Based on high conservation of the decoding site among all kingdoms, it is assumed that the putative binding site of these agents in Leishmania is the ribosomal A site. However, although recent X-ray crystal structures of the bacterial ribosome in complex with aminoglycosides shed light on the mechanism of aminoglycosides action as antibiotics, no such data are presently available regarding their binding site in Leishmania. We present crystal structures of two different aminoglycoside molecules bound to a model of the Leishmania ribosomal A site: Geneticin (G418), a potent aminoglycoside for the treatment of leishmaniasis at a 2.65-Å resolution, and Apramycin, shown to be a strong binder to the leishmanial ribosome lacking an antileishmanial activity at 1.4-Å resolution. The structural data, coupled with in vitro inhibition measurements on two strains of Leishmania, provide insight as to the source of the difference in inhibitory activity of different Aminoglycosides. The combined structural and physiological data sets the ground for rational design of new, and more specific, aminoglycoside derivatives as potential therapeutic agents against leishmaniasis. PMID:23898171

  18. Localization of the active site of an enzyme, bacterial luciferase, using two-quantum affinity modification

    NASA Astrophysics Data System (ADS)

    Benimetskaya, L. Z.; Gitelzon, I. I.; Kozionov, Andrew L.; Novozhilov, S. Y.; Petushkov, V. N.; Rodionova, N. S.; Stockman, Mark I.

    1991-11-01

    For the first time the method of two-quantum affinity modification has been employed to probe the structure of an enzyme, bacterial luciferase. Position of the flavin-binding site of this enzyme, which was previously unknown, has been established. The obtained data indicate that the flavin site is positioned on the (alpha) -subunit. The closest contact of the protein chain of the enzyme with the chromophoric group of the flavin takes place near 80 +/- 10 and 120 +/- 10 amino acid residues; the regions 50 +/- 10 and 215 +/- 10 are also close to the flavin. The established localization does not contradict suggestions on positions of the flavin and phosphate sites of the bacterial luciferase, which had earlier been made from the data on evolutionary stability of various luciferases. The present method can, in principle, be applied to a great number of enzymes, including all flavin-dependent enzymes. Enzymatic catalysis has high speed and specificity. Creation of a method of determination of the elements of the primary structure of a protein, making up the active site (in which substratum conversion occurs), could be a significant advance in clearing up mechanisms of enzymatic catalysis. It was proposed to localize active sites of the enzymes, whose substrata are chromophores, using this method of two-quantum affinity modification. An enzyme- substratum complex is irradiated with laser light of sufficiently long wavelength ((lambda) 300 nm) which is not directly absorbed by the enzyme. Two-quantum quasiresonant excitation of the substratum activates it to the state with energy 5-7 eV, which is then radiativelessly transferred to neighboring protein groups. This energy exceeds the energy of activation of peptide bond breakage. Therefore, the enzyme will be disrupted in the vicinity of its active site. In the present paper the above approach has been implemented for the first time. Information has been obtained about the position of the flavin-binding site of bacterial

  19. Liquid acetone and chloroform: a comparison between Monte Carlo simulation, molecular Ornstein-Zernike theory, and site-site Ornstein-Zernike theory

    NASA Astrophysics Data System (ADS)

    Richardi, J.; Fries, P. H.; Fischer, R.; Rast, S.; Krienke, H.

    Site-site distribution functions, internal energies, compressibilities, and dielectric constants of liquid acetone and chloroform are computed and compared. The hypernetted chain approximation is used for the molecular Ornstein-Zernike (MOZ) and site-site Ornstein-Zernike (SSOZ) theories. Both solvent molecules are described as sets of interaction sites, which are the centres of Lennard-Jones potentials and which carry partial electric charges. How the approximations inherent in both OZ theories affect the calculated liquid properties is discussed. In contrast to SSOZ theory, MOZ theory yields a correct description of the liquid structure as defined by the site-site distribution functions of simulations. The MOZ results for the dielectric constants are in good agreement with simulation, but both OZ theories slightly underestimate the internal excess energies. In contrast to the SSOZ formalism and to the simulations, the molecular pair distribution of MOZ theory can be computed readily for all two-molecule configurations, allowing a detailed study of the complex relief of this distribution. A search was performed for configurations that are highly probable in the liquid. They can be assigned to a small number of peaks of the molecular distribution function. These peaks have major contributions to the Kirkwood factors, which measure the orientational order in the liquid, and to the electrostatic part of the excess energies. The partitioning of the whole two-molecule configuration space makes it possible to study the details of the site-site distribution functions. For instance, a sharp peak in the oxygen-oxygen distribution in acetone results from many superimposed contributions of acetone-acetone arrangements of low and medium statistical weights, and not from the highly probable arrangements.

  20. Active Site and Laminarin Binding in Glycoside Hydrolase Family 55*

    PubMed Central

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

    2015-01-01

    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

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

    PubMed

    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

  2. Active Site Loop Conformation Regulates Promiscuous Activity in a Lactonase from Geobacillus kaustophilus HTA426

    PubMed Central

    Zhang, Yu; An, Jiao; Yang, Guang-Yu; Bai, Aixi; Zheng, Baisong; Lou, Zhiyong; Wu, Geng; Ye, Wei; Chen, Hai-Feng; Feng, Yan; Manco, Giuseppe

    2015-01-01

    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. PMID:25706379

  3. Non-specific binding sites help to explain mixed inhibition in mushroom tyrosinase activities.

    PubMed

    Hassani, Sorour; Haghbeen, Kamahldin; Fazli, Mostafa

    2016-10-21

    Inhibition and activation studies of tyrosinase could prove beneficial to agricultural, food, cosmetic, and pharmaceutical industries. Although non-competitive and mixed-inhibition are frequent modes observed in kinetics studies on mushroom tyrosinase (MT) activities, the phenomena are left unexplained. In this study, dual effects of phthalic acid (PA) and cinnamic acid (CA) on MT during mono-phenolase activity were demonstrated. PA activated and inhibited MT at concentrations lower and higher than 150 μM, respectively. In contrast, CA inhibited and activated MT at concentrations lower and higher than 5 μM. The mode of inhibition for both effectors was mixed-type. Complex kinetics of MT in the presence of a modulator could partly be ascribed to its mixed-cooperativity. However, to explain mixed-inhibition mode, it is necessary to demonstrate how the ternary complex of substrate/enzyme/effector is formed. Therefore, we looked for possible non-specific binding sites using MT tropolone-bound PDB (2Y9X) in the computational studies. When tropolone was in MTPa (active site), PA and CA occupied different pockets (named MTPb and MTPc, respectively). The close Moldock scores of PA binding posed in MTPb and MTPa suggested that MTPb could be a secondary binding site for PA. Similar results were obtained for CA. Ensuing results from 10 ns molecular dynamics simulations for 2Y9X-effector complexes indicated that the structures were gradually stabilized during simulation. Tunnel analysis by using CAVER Analyst and CHEXVIS resulted in identifying two distinct channels that assumingly participate in exchanging the effectors when the direct channel to MTPa is not accessible. PMID:27344491

  4. Migfilin, a Molecular Switch in Regulation of Integrin Activation*S⃞

    PubMed Central

    Ithychanda, Sujay Subbayya; Das, Mitali; Ma, Yan-Qing; Ding, Keyang; Wang, Xiaoxia; Gupta, Sudhiranjan; Wu, Chuanyue; Plow, Edward F.; Qin, Jun

    2009-01-01

    The linkage of heterodimeric (α/β) integrin receptors with their extracellular matrix ligands and intracellular actin cytoskeleton is a fundamental step for controlling cell adhesion and migration. Binding of the actin-linking protein, talin, to integrin β cytoplasmic tails (CTs) induces high affinity ligand binding (integrin activation), whereas binding of another actin-linking protein, filamin, to the integrin β CTs negatively regulates this process by blocking the talin-integrin interaction. Here we show structurally that migfilin, a novel cytoskeletal adaptor highly enriched in the integrin adhesion sites, strongly interacts with the same region in filamin where integrin β CTs bind. We further demonstrate that the migfilin interaction dissociates filamin from integrin and promotes the talin/integrin binding and integrin activation. Migfilin thus acts as a molecular switch to disconnect filamin from integrin for regulating integrin activation and dynamics of extracellular matrix-actin linkage. PMID:19074766

  5. Molecular electrostatic potential at the atomic sites in the effective core potential approximation.

    PubMed

    Lesiuk, Michał; Zachara, Janusz

    2013-02-21

    Considering calculations of the molecular electrostatic potential at the atomic sites (MEP@AS) in the presence of effective core potentials (ECP), we found that the consequent use of the definition of MEP@AS based on the energy derivative with respect to nuclear charge leads to a formula that differs by one term from the result of simple application of Coulomb's law. We have developed a general method to analytically treat derivatives of ECP with respect to nuclear charge. Benchmarking calculations performed on a set of simple molecules show that our formula leads to a systematic decrease in the error connected with the introduction of ECP when compared to all-electron results. Because of a straightforward implementation and relatively low costs of the developed procedure we suggest to use it by default. PMID:23444997

  6. Molecular electrostatic potential at the atomic sites in the effective core potential approximation

    NASA Astrophysics Data System (ADS)

    Lesiuk, Michał; Zachara, Janusz

    2013-02-01

    Considering calculations of the molecular electrostatic potential at the atomic sites (MEP@AS) in the presence of effective core potentials (ECP), we found that the consequent use of the definition of MEP@AS based on the energy derivative with respect to nuclear charge leads to a formula that differs by one term from the result of simple application of Coulomb's law. We have developed a general method to analytically treat derivatives of ECP with respect to nuclear charge. Benchmarking calculations performed on a set of simple molecules show that our formula leads to a systematic decrease in the error connected with the introduction of ECP when compared to all-electron results. Because of a straightforward implementation and relatively low costs of the developed procedure we suggest to use it by default.

  7. Theory of exciton annihilation in complexes of a finite number of molecular sites.

    PubMed

    Gülen, D

    1990-11-01

    A theory of the kinematics of singlet exciton annihilation in complexes of a finite number of molecular sites is developed. The theory is based on a specific scheme suggested earlier by Gülen, Wittmershaus, and Knox [Biophys J. 49:469-477 (1986)]. It is adequate to address the excitation kinetics and dynamics in such systems, especially under high excitation intensities. A Pauli master equation is formulated and is solved to give explicit expressions for observables such as quantum yield and fluorescence intensity. The excitation intensity dependence of the observables is taken into account by introducing Poisson statistics. Details relevant to its application to the annihilation of excitons in photosynthetic systems and its connection to earlier theories are presented. PMID:2134489

  8. Molecular docking sites designed for the generation of highly crystalline covalent organic frameworks

    NASA Astrophysics Data System (ADS)

    Ascherl, Laura; Sick, Torben; Margraf, Johannes T.; Lapidus, Saul H.; Calik, Mona; Hettstedt, Christina; Karaghiosoff, Konstantin; Döblinger, Markus; Clark, Timothy; Chapman, Karena W.; Auras, Florian; Bein, Thomas

    2016-04-01

    Covalent organic frameworks (COFs) formed by connecting multidentate organic building blocks through covalent bonds provide a platform for designing multifunctional porous materials with atomic precision. As they are promising materials for applications in optoelectronics, they would benefit from a maximum degree of long-range order within the framework, which has remained a major challenge. We have developed a synthetic concept to allow consecutive COF sheets to lock in position during crystal growth, and thus minimize the occurrence of stacking faults and dislocations. Hereby, the three-dimensional conformation of propeller-shaped molecular building units was used to generate well-defined periodic docking sites, which guided the attachment of successive building blocks that, in turn, promoted long-range order during COF formation. This approach enables us to achieve a very high crystallinity for a series of COFs that comprise tri- and tetradentate central building blocks. We expect this strategy to be transferable to a broad range of customized COFs.

  9. Proteolytic regulation of epithelial sodium channels by urokinase plasminogen activator: cutting edge and cleavage sites.

    PubMed

    Ji, Hong-Long; Zhao, Runzhen; Komissarov, Andrey A; Chang, Yongchang; Liu, Yongfeng; Matthay, Michael A

    2015-02-27

    Plasminogen activator inhibitor 1 (PAI-1) level is extremely elevated in the edematous fluid of acutely injured lungs and pleurae. Elevated PAI-1 specifically inactivates pulmonary urokinase-type (uPA) and tissue-type plasminogen activators (tPA). We hypothesized that plasminogen activation and fibrinolysis may alter epithelial sodium channel (ENaC) activity, a key player in clearing edematous fluid. Two-chain urokinase (tcuPA) has been found to strongly stimulate heterologous human αβγ ENaC activity in a dose- and time-dependent manner. This activity of tcuPA was completely ablated by PAI-1. Furthermore, a mutation (S195A) of the active site of the enzyme also prevented ENaC activation. By comparison, three truncation mutants of the amino-terminal fragment of tcuPA still activated ENaC. uPA enzymatic activity was positively correlated with ENaC current amplitude prior to reaching the maximal level. In sharp contrast to uPA, neither single-chain tPA nor derivatives, including two-chain tPA and tenecteplase, affected ENaC activity. Furthermore, γ but not α subunit of ENaC was proteolytically cleaved at ((177)GR↓KR(180)) by tcuPA. In summary, the underlying mechanisms of urokinase-mediated activation of ENaC include release of self-inhibition, proteolysis of γ ENaC, incremental increase in opening rate, and activation of closed (electrically "silent") channels. This study for the first time demonstrates multifaceted mechanisms for uPA-mediated up-regulation of ENaC, which form the cellular and molecular rationale for the beneficial effects of urokinase in mitigating mortal pulmonary edema and pleural effusions. PMID:25555911

  10. Reverse evolution leads to genotypic incompatibility despite functional and active site convergence.

    PubMed

    Kaltenbach, Miriam; Jackson, Colin J; Campbell, Eleanor C; Hollfelder, Florian; Tokuriki, Nobuhiko

    2015-01-01

    Understanding the extent to which enzyme evolution is reversible can shed light on the fundamental relationship between protein sequence, structure, and function. Here, we perform an experimental test of evolutionary reversibility using directed evolution from a phosphotriesterase to an arylesterase, and back, and examine the underlying molecular basis. We find that wild-type phosphotriesterase function could be restored (>10(4)-fold activity increase), but via an alternative set of mutations. The enzyme active site converged towards its original state, indicating evolutionary constraints imposed by catalytic requirements. We reveal that extensive epistasis prevents reversions and necessitates fixation of new mutations, leading to a functionally identical sequence. Many amino acid exchanges between the new and original enzyme are not tolerated, implying sequence incompatibility. Therefore, the evolution was phenotypically reversible but genotypically irreversible. Our study illustrates that the enzyme's adaptive landscape is highly rugged, and different functional sequences may constitute separate fitness peaks. PMID:26274563

  11. Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site.

    PubMed

    Sunden, Fanny; Peck, Ariana; Salzman, Julia; Ressl, Susanne; Herschlag, Daniel

    2015-01-01

    Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called 'catalytic residues' are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modes of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes. PMID:25902402

  12. Key Role of Active-Site Water Molecules in Bacteriorhodopsin Proton-Transfer Reactions

    SciTech Connect

    Bondar, A.N.; Baudry, Jerome Y; Suhai, Sandor; Fischer, S.; Smith, Jeremy C

    2008-10-01

    The functional mechanism of the light-driven proton pump protein bacteriorhodopsin depends on the location of water molecules in the active site at various stages of the photocycle and on their roles in the proton-transfer steps. Here, free energy computations indicate that electrostatic interactions favor the presence of a cytoplasmic-side water molecule hydrogen bonding to the retinal Schiff base in the state preceding proton transfer from the retinal Schiff base to Asp85. However, the nonequilibrium nature of the pumping process means that the probability of occupancy of a water molecule in a given site depends both on the free energies of insertion of the water molecule in this and other sites during the preceding photocycle steps and on the kinetic accessibility of these sites on the time scale of the reaction steps. The presence of the cytoplasmic-side water molecule has a dramatic effect on the mechanism of proton transfer: the proton is channeled on the Thr89 side of the retinal, whereas the transfer on the Asp212 side is hindered. Reaction-path simulations and molecular dynamics simulations indicate that the presence of the cytoplasmic-side water molecule permits a low-energy bacteriorhodopsin conformer in which the water molecule bridges the twisted retinal Schiff base and the proton acceptor Asp85. From this low-energy conformer, proton transfer occurs via a concerted mechanism in which the water molecule participates as an intermediate proton carrier.

  13. Single-site Lennard-Jones models via polynomial chaos surrogates of Monte Carlo molecular simulation.

    PubMed

    Kadoura, Ahmad; Siripatana, Adil; Sun, Shuyu; Knio, Omar; Hoteit, Ibrahim

    2016-06-01

    In this work, two Polynomial Chaos (PC) surrogates were generated to reproduce Monte Carlo (MC) molecular simulation results of the canonical (single-phase) and the NVT-Gibbs (two-phase) ensembles for a system of normalized structureless Lennard-Jones (LJ) particles. The main advantage of such surrogates, once generated, is the capability of accurately computing the needed thermodynamic quantities in a few seconds, thus efficiently replacing the computationally expensive MC molecular simulations. Benefiting from the tremendous computational time reduction, the PC surrogates were used to conduct large-scale optimization in order to propose single-site LJ models for several simple molecules. Experimental data, a set of supercritical isotherms, and part of the two-phase envelope, of several pure components were used for tuning the LJ parameters (ε, σ). Based on the conducted optimization, excellent fit was obtained for different noble gases (Ar, Kr, and Xe) and other small molecules (CH4, N2, and CO). On the other hand, due to the simplicity of the LJ model used, dramatic deviations between simulation and experimental data were observed, especially in the two-phase region, for more complex molecules such as CO2 and C2 H6. PMID:27276951

  14. Single-site Lennard-Jones models via polynomial chaos surrogates of Monte Carlo molecular simulation

    NASA Astrophysics Data System (ADS)

    Kadoura, Ahmad; Siripatana, Adil; Sun, Shuyu; Knio, Omar; Hoteit, Ibrahim

    2016-06-01

    In this work, two Polynomial Chaos (PC) surrogates were generated to reproduce Monte Carlo (MC) molecular simulation results of the canonical (single-phase) and the NVT-Gibbs (two-phase) ensembles for a system of normalized structureless Lennard-Jones (LJ) particles. The main advantage of such surrogates, once generated, is the capability of accurately computing the needed thermodynamic quantities in a few seconds, thus efficiently replacing the computationally expensive MC molecular simulations. Benefiting from the tremendous computational time reduction, the PC surrogates were used to conduct large-scale optimization in order to propose single-site LJ models for several simple molecules. Experimental data, a set of supercritical isotherms, and part of the two-phase envelope, of several pure components were used for tuning the LJ parameters (ɛ, σ). Based on the conducted optimization, excellent fit was obtained for different noble gases (Ar, Kr, and Xe) and other small molecules (CH4, N2, and CO). On the other hand, due to the simplicity of the LJ model used, dramatic deviations between simulation and experimental data were observed, especially in the two-phase region, for more complex molecules such as CO2 and C2 H6.

  15. PEST Control of Molecular Stripping of NFκB from DNA Transcription Sites.

    PubMed

    Potoyan, Davit A; Zheng, Weihua; Ferreiro, Diego U; Wolynes, Peter G; Komives, Elizabeth A

    2016-08-25

    We recently proposed a model for IκBα-mediated molecular stripping of NFκB from transcription sites. IκBα was shown experimentally to form a transient ternary complex with DNA-bound NFκB, but the mechanism by which the IκBα accelerates dissociation of the NFκB from the DNA was unknown. In this paper we construct and compute free energy profiles for the wild-type IκBα-mediated molecular stripping reaction of NFκB from DNA and compare with that for a mutant of IκBα bearing a charge-neutralized PEST. The differences in the free energy profile for stripping originate from the frustrated electrostatic interactions between the negatively charged PEST and the DNA. The PEST occupies two different conformations in the NFκB-IκBα binary complex, one of which occupies the DNA-binding cavity. Specific interactions with positively charged residues in the N-terminal domains of both p50 and p65 apparently draw the domains closer together hindering reassociation of DNA. Comparison with the charge-neutralized mutant reveals that all of these functional consequences result from the negative charges in the PEST sequence of IκBα. PMID:27098223

  16. Structure–function studies on the active site of the coelenterazine-dependent luciferase from Renilla

    PubMed Central

    Woo, Jongchan; Howell, Matthew H.; von Arnim, Albrecht G.

    2008-01-01

    Renilla luciferase (RLUC) is a versatile tool for gene expression assays and in vivo biosensor applications, but its catalytic mechanism remains to be elucidated. RLUC is evolutionarily related to the α/β hydrolase family. Its closest known homologs are bacterial dehalogenases, raising the question of how a protein with a hydrolase fold can function as a decarboxylating oxygenase. Molecular docking simulations with the coelenterazine substrate against an RLUC homology model as well as a recently determined RLUC crystal structure were used to build hypotheses to identify functionally important residues, which were subsequently tested by site-directed mutagenesis, heterologous expression, and bioluminescence emission spectroscopy. The data highlighted two triads of residues that are critical for catalysis. The putative catalytic triad residues D120, E144, and H285 bear only limited resemblance to those found in the active site of aequorin, a coelenterazine-utilizing photoprotein, suggesting that the reaction scheme employed by RLUC differs substantially from the one established for aequorin. The role of H285 in catalysis was further supported by inhibition using diethylpyrocarbonate. Multiple substitutions of N53, W121, and P220—three other residues implicated in product binding in the homologous dehalogenase Sphingomonas LinB—also supported their involvement in catalysis. Together with luminescence spectra, our data lead us to propose that the conserved catalytic triad of RLUC is directly involved in the decarboxylation reaction of coelenterazine to produce bioluminescence, while the other active-site residues are used for binding of the substrate. PMID:18359861

  17. The Mechanism by which 146-N-Glycan Affects the Active Site of Neuraminidase.

    PubMed

    Liu, Pi; Wang, Zhonghua; Zhang, Lijie; Li, Dongmei; Lin, Jianping

    2015-01-01

    One of the most conserved glycosylation sites of neuraminidase (NA) is 146-N-glycan. This site is adjacent to the 150-cavity of NA, which is found within the active site and thought to be a target for rational drug development against the antiviral resistance of influenza. Here, through a total of 2.4 μs molecular dynamics (MD) simulations, we demonstrated that 146-N-glycan can stabilize the conformation of the 150-loop that controls the volume of the 150-cavity. Moreover, with 146-N-glycan, our simulation result was more consistent with crystal structures of NAs than simulations conducted without glycans. Cluster analysis of the MD trajectories showed that 146-N-glycan adopted three distinct conformations: monomer-bridged, dimer-bridged and standing. Of these conformations, the dimer-bridged 146-N-glycan was the most stable one and contributed to stabilization of the 150-loop conformation. Furthermore, our simulation revealed that various standing conformations of 146-N-glycan could block the entrance of the binding pocket. This result was consistent with experimental data and explained the relatively low activity of inhibitors with flexible substituents toward the 150-cavity. Together, our results lead us to hypothesize that rigid and hydrophobic substituents could serve as better inhibitors targeting the 150-cavity. PMID:26267136

  18. A Unique Chitinase with Dual Active Sites and Triple Substrate Binding Sites from the Hyperthermophilic Archaeon Pyrococcus kodakaraensis KOD1

    PubMed Central

    Tanaka, Takeshi; Fujiwara, Shinsuke; Nishikori, Shingo; Fukui, Toshiaki; Takagi, Masahiro; Imanaka, Tadayuki

    1999-01-01

    We have found that the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 produces an extracellular chitinase. The gene encoding the chitinase (chiA) was cloned and sequenced. The chiA gene was found to be composed of 3,645 nucleotides, encoding a protein (1,215 amino acids) with a molecular mass of 134,259 Da, which is the largest among known chitinases. Sequence analysis indicates that ChiA is divided into two distinct regions with respective active sites. The N-terminal and C-terminal regions show sequence similarity with chitinase A1 from Bacillus circulans WL-12 and chitinase from Streptomyces erythraeus (ATCC 11635), respectively. Furthermore, ChiA possesses unique chitin binding domains (CBDs) (CBD1, CBD2, and CBD3) which show sequence similarity with cellulose binding domains of various cellulases. CBD1 was classified into the group of family V type cellulose binding domains. In contrast, CBD2 and CBD3 were classified into that of the family II type. chiA was expressed in Escherichia coli cells, and the recombinant protein was purified to homogeneity. The optimal temperature and pH for chitinase activity were found to be 85°C and 5.0, respectively. Results of thin-layer chromatography analysis and activity measurements with fluorescent substrates suggest that the enzyme is an endo-type enzyme which produces a chitobiose as a major end product. Various deletion mutants were constructed, and analyses of their enzyme characteristics revealed that both the N-terminal and C-terminal halves are independently functional as chitinases and that CBDs play an important role in insoluble chitin binding and hydrolysis. Deletion mutants which contain the C-terminal half showed higher thermostability than did N-terminal-half mutants and wild-type ChiA. PMID:10583986

  19. The molecular regulation of Janus kinase (JAK) activation.

    PubMed

    Babon, Jeffrey J; Lucet, Isabelle S; Murphy, James M; Nicola, Nicos A; Varghese, Leila N

    2014-08-15

    The JAK (Janus kinase) family members serve essential roles as the intracellular signalling effectors of cytokine receptors. This family, comprising JAK1, JAK2, JAK3 and TYK2 (tyrosine kinase 2), was first described more than 20 years ago, but the complexities underlying their activation, regulation and pleiotropic signalling functions are still being explored. Here, we review the current knowledge of their physiological functions and the causative role of activating and inactivating JAK mutations in human diseases, including haemopoietic malignancies, immunodeficiency and inflammatory diseases. At the molecular level, recent studies have greatly advanced our knowledge of the structures and organization of the component FERM (4.1/ezrin/radixin/moesin)-SH2 (Src homology 2), pseudokinase and kinase domains within the JAKs, the mechanism of JAK activation and, in particular, the role of the pseudokinase domain as a suppressor of the adjacent tyrosine kinase domain's catalytic activity. We also review recent advances in our understanding of the mechanisms of negative regulation exerted by the SH2 domain-containing proteins, SOCS (suppressors of cytokine signalling) proteins and LNK. These recent studies highlight the diversity of regulatory mechanisms utilized by the JAK family to maintain signalling fidelity, and suggest alternative therapeutic strategies to complement existing ATP-competitive kinase inhibitors. PMID:25057888

  20. The molecular regulation of Janus kinase (JAK) activation

    PubMed Central

    2014-01-01

    The Janus Kinase (JAK) family members serve essential roles as the intracellular signalling effectors of cytokine receptors. This family, comprising JAK1, JAK2, JAK3 and TYK2, was first described more than 20 years ago, but the complexities underlying their activation, regulation and pleiotropic signalling functions are still being explored. Here, we review the current knowledge of their physiological functions and the causative role of activating and inactivating JAK mutations in human diseases, including haematopoietic malignancies, immunodeficiency and inflammatory diseases. At the molecular level, recent studies have greatly advanced our knowledge of the structures and organisation of the component FERM-SH2, pseudokinase and kinase domains within the JAKs, the mechanism of JAK activation and, in particular, the role of the pseudokinase domain as a suppressor of the adjacent tyrosine kinase domain's catalytic activity. We also review recent advances in our understanding of the mechanisms of negative regulation exerted by the SH2 domain containing proteins, SOCS (Suppressors of Cytokine Signalling) proteins and Lnk. These recent advances highlight the diversity of regulatory mechanisms utilised by the JAK family to maintain signalling fidelity, and suggest alternative therapeutic strategies to complement existing ATP-competitive kinase inhibitors. PMID:25057888

  1. In Vivo Molecular Dissection of the Effects of HIV-1 in Active Tuberculosis

    PubMed Central

    Bell, Lucy C. K.; Pollara, Gabriele; Pascoe, Mellissa; Tomlinson, Gillian S.; Lehloenya, Rannakoe J.; Roe, Jennifer; Meldau, Richard; Miller, Robert F.; Ramsay, Alan; Chain, Benjamin M.; Dheda, Keertan; Noursadeghi, Mahdad

    2016-01-01

    Increased risk of tuberculosis (TB) associated with HIV-1 infection is primarily attributed to deficient T helper (Th)1 immune responses, but most people with active TB have robust Th1 responses, indicating that these are not sufficient to protect against disease. Recent findings suggest that favourable outcomes following Mycobacterium tuberculosis infection arise from finely balanced inflammatory and regulatory pathways, achieving pathogen control without immunopathology. We hypothesised that HIV-1 and antiretroviral therapy (ART) exert widespread changes to cell mediated immunity, which may compromise the optimal host protective response to TB and provide novel insights into the correlates of immune protection and pathogenesis. We sought to define these effects in patients with active TB by transcriptional profiling of tuberculin skin tests (TST) to make comprehensive molecular level assessments of in vivo human immune responses at the site of a standardised mycobacterial challenge. We showed that the TST transcriptome accurately reflects the molecular pathology at the site of human pulmonary TB, and used this approach to investigate immune dysregulation in HIV-1/TB co-infected patients with distinct clinical phenotypes associated with TST reactivity or anergy and unmasking TB immune reconstitution inflammatory syndrome (IRIS) after initiation of ART. HIV-1 infected patients with positive TSTs exhibited preserved Th1 responses but deficient immunoregulatory IL10-inducible responses. Those with clinically negative TSTs revealed profound anergy of innate as well as adaptive immune responses, except for preservation of type 1 interferon activity, implicated in impaired anti-mycobacterial immunity. Patients with unmasking TB IRIS showed recovery of Th1 immunity to normal levels, but exaggerated Th2-associated responses specifically. These mechanisms of immune dysregulation were localised to the tissue microenvironment and not evident in peripheral blood. TST

  2. Molecular photoacoustic imaging of breast cancer using an actively targeted conjugated polymer

    PubMed Central

    Balasundaram, Ghayathri; Ho, Chris Jun Hui; Li, Kai; Driessen, Wouter; Dinish, US; Wong, Chi Lok; Ntziachristos, Vasilis; Liu, Bin; Olivo, Malini

    2015-01-01

    Conjugated polymers (CPs) are upcoming optical contrast agents in view of their unique optical properties and versatile synthetic chemistry. Biofunctionalization of these polymer-based nanoparticles enables molecular imaging of biological processes. In this work, we propose the concept of using a biofunctionalized CP for noninvasive photoacoustic (PA) molecular imaging of breast cancer. In particular, after verifying the PA activity of a CP nanoparticle (CP dots) in phantoms and the targeting efficacy of a folate-functionalized version of the same (folate-CP dots) in vitro, we systemically administered the probe into a folate receptor-positive (FR+ve) MCF-7 breast cancer xenograft model to demonstrate the possible application of folate-CP dots for imaging FR+ve breast cancers in comparison to CP dots with no folate moieties. We observed a strong PA signal at the tumor site of folate-CP dots-administered mice as early as 1 hour after administration as a result of the active targeting of the folate-CP dots to the FR+ve tumor cells but a weak PA signal at the tumor site of CP-dots-administered mice as a result of the passive accumulation of the probe by enhanced permeability and retention effect. We also observed that folate-CP dots produced ~4-fold enhancement in the PA signal in the tumor, when compared to CP dots. These observations demonstrate the great potential of this active-targeting CP to be used as a contrast agent for molecular PA diagnostic imaging in various biomedical applications. PMID:25609951

  3. In Vivo Molecular Dissection of the Effects of HIV-1 in Active Tuberculosis.

    PubMed

    Bell, Lucy C K; Pollara, Gabriele; Pascoe, Mellissa; Tomlinson, Gillian S; Lehloenya, Rannakoe J; Roe, Jennifer; Meldau, Richard; Miller, Robert F; Ramsay, Alan; Chain, Benjamin M; Dheda, Keertan; Noursadeghi, Mahdad

    2016-03-01

    Increased risk of tuberculosis (TB) associated with HIV-1 infection is primarily attributed to deficient T helper (Th)1 immune responses, but most people with active TB have robust Th1 responses, indicating that these are not sufficient to protect against disease. Recent findings suggest that favourable outcomes following Mycobacterium tuberculosis infection arise from finely balanced inflammatory and regulatory pathways, achieving pathogen control without immunopathology. We hypothesised that HIV-1 and antiretroviral therapy (ART) exert widespread changes to cell mediated immunity, which may compromise the optimal host protective response to TB and provide novel insights into the correlates of immune protection and pathogenesis. We sought to define these effects in patients with active TB by transcriptional profiling of tuberculin skin tests (TST) to make comprehensive molecular level assessments of in vivo human immune responses at the site of a standardised mycobacterial challenge. We showed that the TST transcriptome accurately reflects the molecular pathology at the site of human pulmonary TB, and used this approach to investigate immune dysregulation in HIV-1/TB co-infected patients with distinct clinical phenotypes associated with TST reactivity or anergy and unmasking TB immune reconstitution inflammatory syndrome (IRIS) after initiation of ART. HIV-1 infected patients with positive TSTs exhibited preserved Th1 responses but deficient immunoregulatory IL10-inducible responses. Those with clinically negative TSTs revealed profound anergy of innate as well as adaptive immune responses, except for preservation of type 1 interferon activity, implicated in impaired anti-mycobacterial immunity. Patients with unmasking TB IRIS showed recovery of Th1 immunity to normal levels, but exaggerated Th2-associated responses specifically. These mechanisms of immune dysregulation were localised to the tissue microenvironment and not evident in peripheral blood. TST

  4. Preparation, characterization and molecular modeling of PEGylated human growth hormone with agonist activity.

    PubMed

    Khameneh, Bahman; Jaafari, Mahmoud Reza; Hassanzadeh-Khayyat, Mohammad; Varasteh, AbdolReza; Chamani, JamshidKhan; Iranshahi, Mehrdad; Mohammadpanah, Hamid; Abnous, Khalil; Saberi, Mohammad Reza

    2015-09-01

    In this study, site-specific PEGylated human growth hormone (hGH) was prepared by microbial transglutaminase, modeled and characterized. To this end, the effects of different reaction parameters including reaction media, PEG:protein ratios, reaction time and pH value were investigated. PEG-hGH was purified by size exclusion chromatography method and analyzed by SDS-PAGE, BCA, peptide mapping, ESI and MALDI-TOF-TOF mass spectroscopy methods. Biophysical and biological properties of PEG-hGH were evaluated. Molecular simulation was utilized to provide molecular insight into the protein-receptor interaction. The optimum conditions that were obtained for PEGylation were phosphate buffer with pH of 7.4, 48 h of stirring and PEG:protein ratio of 40:1. By this method, mono-PEG-hGH with high reaction yield was obtained and PEGylation site was at Gln-40 residue. The circular dichroism and fluorescence spectrum indicated that PEGylation did not change the secondary structure while tertiary structure was altered. Upon enzymatic PEGylation, agonistic activity of hGH was preserved; however, Somavert(®), which is prepared by chemical PEGylation, is an antagonist form of protein. These data were confirmed by the total energy of affinity obtained by computational protein-receptor interaction. In conclusion, PEGylation of hGH was led to prepare a novel form of hormone with an agonist activity which merits further investigations. PMID:26116386

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

    PubMed

    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. Peptidylprolyl cis/trans isomerase activity and molecular evolution of vertebrate Cyclophilin A.

    PubMed

    Liqian, Ren; Wei, Liu; Wenbo, Li; Wenjun, Liu; Lei, Sun

    2016-08-01

    Peptidylprolyl isomerases (PPIase) cyclophilin A (CypA, encoded by PPIA) is a typical member of the Cyclophilin family and is involved in protein folding/translocation, signal transduction, inflammation, immune system regulation, apoptosis and virus replication. In the present study, we investigated the PPIase activity and genetic variation of vertebrate CypA. According to the GenBank reference sequences, vertebrate PPIA genes were cloned, among which the bat (Myotis davidi) and duck (Anas platyrhynchos) PPIA genes were reported for the first time. Then PPIA genes were sub-cloned into the expression vector pGEX-6p-1 and expressed in Escherichia coli. Recombinant CypA proteins were purified by using sepharose 4B affinity chromatography and the GST tag was cleaved, followed by gel filtration. The PPIase activity assay indicated that there was no significant difference in the catalytic activity of prolyl peptide bond isomerization among 12 different vertebrate CypA proteins. In addition, the genetic variation and molecular evolution analysis showed that these vertebrate CypA proteins had the same CsA binding site and the PPIase active sites. Furthermore, the predicted structure and gene localization were remarkable conserved. Our data suggested that the important residues of CypA were highly conserved, which is crucial for its PPIase activity and cellular functions. PMID:27531612

  7. Molecular pathogenesis of H5 highly pathogenic avian influenza: the role of the haemagglutinin cleavage site motif.

    PubMed

    Luczo, Jasmina M; Stambas, John; Durr, Peter A; Michalski, Wojtek P; Bingham, John

    2015-11-01

    The emergence of H5N1 highly pathogenic avian influenza has caused a heavy socio-economic burden through culling of poultry to minimise human and livestock infection. Although human infections with H5N1 have to date been limited, concerns for the pandemic potential of this zoonotic virus have been greatly intensified following experimental evidence of aerosol transmission of H5N1 viruses in a mammalian infection model. In this review, we discuss the dominance of the haemagglutinin cleavage site motif as a pathogenicity determinant, the host-pathogen molecular interactions driving cleavage activation, reverse genetics manipulations and identification of residues key to haemagglutinin cleavage site functionality and the mechanisms of cell and tissue damage during H5N1 infection. We specifically focus on the disease in chickens, as it is in this species that high pathogenicity frequently evolves and from which transmission to the human population occurs. With >75% of emerging infectious diseases being of zoonotic origin, it is necessary to understand pathogenesis in the primary host to explain spillover events into the human population. PMID:26467906

  8. Conformational Disorganization within the Active Site of a Recently Evolved Organophosphate Hydrolase Limits Its Catalytic Efficiency.

    PubMed

    Mabbitt, Peter D; Correy, Galen J; Meirelles, Tamara; Fraser, Nicholas J; Coote, Michelle L; Jackson, Colin J

    2016-03-01

    The evolution of new enzymatic activity is rarely observed outside of the laboratory. In the agricultural pest Lucilia cuprina, a naturally occurring mutation (Gly137Asp) in α-esterase 7 (LcαE7) results in acquisition of organophosphate hydrolase activity and confers resistance to organophosphate insecticides. Here, we present an X-ray crystal structure of LcαE7:Gly137Asp that, along with kinetic data, suggests that Asp137 acts as a general base in the new catalytic mechanism. Unexpectedly, the conformation of Asp137 observed in the crystal structure obstructs the active site and is not catalytically productive. Molecular dynamics simulations reveal that alternative, catalytically competent conformers of Asp137 are sampled on the nanosecond time scale, although these states are less populated. Thus, although the mutation introduces the new reactive group responsible for organophosphate detoxification, the catalytic efficiency appears to be limited by conformational disorganization: the frequent sampling of low-energy nonproductive states. This result is consistent with a model of molecular evolution in which initial function-changing mutations can result in enzymes that display only a fraction of their catalytic potential due to conformational disorganization. PMID:26881849

  9. Adapting SAFT-γ perturbation theory to site-based molecular dynamics simulation. I. Homogeneous fluids

    SciTech Connect

    Ghobadi, Ahmadreza F.; Elliott, J. Richard

    2013-12-21

    In this work, we aim to develop a version of the Statistical Associating Fluid Theory (SAFT)-γ equation of state (EOS) that is compatible with united-atom force fields, rather than experimental data. We rely on the accuracy of the force fields to provide the relation to experimental data. Although, our objective is a transferable theory of interfacial properties for soft and fused heteronuclear chains, we first clarify the details of the SAFT-γ approach in terms of site-based simulations for homogeneous fluids. We show that a direct comparison of Helmholtz free energy to molecular simulation, in the framework of a third order Weeks-Chandler-Andersen perturbation theory, leads to an EOS that takes force field parameters as input and reproduces simulation results for Vapor-Liquid Equilibria (VLE) calculations. For example, saturated liquid density and vapor pressure of n-alkanes ranging from methane to dodecane deviate from those of the Transferable Potential for Phase Equilibria (TraPPE) force field by about 0.8% and 4%, respectively. Similar agreement between simulation and theory is obtained for critical properties and second virial coefficient. The EOS also reproduces simulation data of mixtures with about 5% deviation in bubble point pressure. Extension to inhomogeneous systems and united-atom site types beyond those used in description of n-alkanes will be addressed in succeeding papers.

  10. Molecular oxygen migration through the xenon docking sites of human hemoglobin in the R-state.

    PubMed

    Lepeshkevich, Sergei V; Gilevich, Syargey N; Parkhats, Marina V; Dzhagarov, Boris M

    2016-09-01

    A nanosecond laser flash-photolysis technique was used to study bimolecular and geminate molecular oxygen (O2) rebinding to tetrameric human hemoglobin and its isolated α and β chains in buffer solutions equilibrated with 1atm of air and up to 25atm of xenon. Xenon binding to the isolated α chains and to the α subunits within tetrameric hemoglobin was found to cause a decrease in the efficiency of O2 escape by a factor of ~1.30 and 3.3, respectively. A kinetic model for O2 dissociation, rebinding, and migration through two alternative pathways in the hemoglobin subunits was introduced and discussed. It was shown that, in the isolated α chains and α subunits within tetrameric hemoglobin, nearly one- and two-third escaping molecules of O2 leave the protein via xenon docking sites, respectively. The present experimental data support the idea that O2 molecule escapes from the β subunits mainly through the His(E7) gate, and show unambiguously that, in the α subunits, in addition to the direct E7 channel, there is at least one alternative escape route leading to the exterior via the xenon docking sites. PMID:27288155

  11. Preferential sites for InAsP/InP quantum wire nucleation using molecular dynamics

    NASA Astrophysics Data System (ADS)

    Nuñez-Moraleda, Bernardo; Pizarro, Joaquin; Guerrero, Elisa; Guerrero-Lebrero, Maria P.; Yáñez, Andres; Molina, Sergio Ignacio; Galindo, Pedro Luis

    2014-11-01

    In this paper, stress fields at the surface of the capping layer of self-assembled InAsP quantum wires grown on an InP (001) substrate have been determined from atomistic models using molecular dynamics and Stillinger-Weber potentials. To carry out these calculations, the quantum wire compositional distribution was extracted from previous works, where the As and P distributions were determined by electron energy loss spectroscopy and high-resolution aberration-corrected Z-contrast imaging. Preferential sites for the nucleation of wires on the surface of the capping layer were studied and compared with (i) previous simulations using finite element analysis to solve anisotropic elastic theory equations and (ii) experimentally measured locations of stacked wires. Preferential nucleation sites of stacked wires were determined by the maximum stress location at the MD model surface in good agreement with experimental results and those derived from finite element analysis. This indicates that MD simulations based on empirical potentials provide a suitable and flexible tool to study strain dependent atom processes.

  12. Molecularly Imprinted Polymer Microspheres Containing Photoswitchable Spiropyran-Based Binding Sites

    PubMed Central

    2013-01-01

    A versatile approach for the preparation of photoswitchable molecularly imprinted polymers (MIPs) is proposed where the selective recognition and the photoresponsive function are assumed by two different monomers. As a proof of concept, MIP microspheres were synthesized by precipitation polymerization for recognizing terbutylazine, a triazine-type herbicide. Formation of the selective binding sites was based upon H-bonding interactions between the template and the functional monomer methacrylic acid, whereas a polymerizable spiropyran unit was incorporated into the polymer matrix to provide light-controllable characteristics. A trifunctional monomer, trimethylolpropane trimethacrylate, was used as a cross-linker. The imprinted particles exhibited considerable morphological differences compared to their nonimprinted counterparts as observed by scanning electron microscopy. The imprinting effect was confirmed by equilibrium rebinding studies. The photoresponsiveness of the polymer particles was visualized by fluorescence microscopy and further characterized by spectroscopy. The template binding behavior could be regulated by alternating UV and visible light illumination when analyte release and uptake was observed, respectively. Binding isotherms fitted by the Freundlich model revealed the photomodulation of the number of binding sites and their average affinity. This facile synthetic approach may give an attractive starting point to endow currently existing highly selective MIPs with photoswitchable properties, thereby extending the scope of spiropyran-based photoresponsive smart materials. PMID:23961698

  13. Direct activation and anti-repression functions of GAL4-VP16 use distinct molecular mechanisms.

    PubMed Central

    Lyons, J G; Chambon, P

    1995-01-01

    In order to determine whether the molecular mechanisms used for direct activation by GAL4-VP16 are the same as those used for anti-repression, we have employed monoclonal antibodies specific for the VP16 activation domain. In the absence of added repressors, GAL4-VP16 was able to stimulate transcription from a template containing GAL4-binding sites, and the antibodies raised against the VP16 activation domain failed to inhibit this direct activation. GAL4-VP16 also was able to prevent histone H1-mediated repression by a mechanism that was strongly dependent on the presence of specific GAL4-binding elements in the promoter. However, in contrast to the assays conducted in the absence of repressors, the antibodies were strong inhibitors of GAL4-VP16-activated transcription in the presence of histone H1. Thus the binding of the antibodies distinguished between the direct activation and anti-repression functions of GAL4-VP16, indicating that these functions operate through distinct molecular mechanisms. The anti-repression-specific mechanism that is inhibitable by the antibodies acted at an early stage of preinitiation complex formation. Deletions of individual subdomains of the VP16 activation domain demonstrated that there was not a discrete subdomain responsible for the anti-repression function of GAL4-VP16. Thus, the inhibitory effect of the antibodies appeared to be due to the location of the epitope within the activator protein rather than to some inherent biochemical property of that region of the protein that is required specifically for anti-repression. The inhibitory effect of the antibodies also ruled out the possibility that steric exclusion of repressor proteins from the promoter was the sole means of anti-repression by the transcriptional activator. Images Figure 1 Figure 2 PMID:8554536

  14. Rational targeting of active-site tyrosine residues using sulfonyl fluoride probes.

    PubMed

    Hett, Erik C; Xu, Hua; Geoghegan, Kieran F; Gopalsamy, Ariamala; Kyne, Robert E; Menard, Carol A; Narayanan, Arjun; Parikh, Mihir D; Liu, Shenping; Roberts, Lee; Robinson, Ralph P; Tones, Michael A; Jones, Lyn H

    2015-04-17

    This work describes the first rational targeting of tyrosine residues in a protein binding site by small-molecule covalent probes. Specific tyrosine residues in the active site of the mRNA-decapping scavenger enzyme DcpS were modified using reactive sulfonyl fluoride covalent inhibitors. Structure-based molecular design was used to create an alkyne-tagged probe bearing the sulfonyl fluoride warhead, thus enabling the efficient capture of the protein from a complex proteome. Use of the probe in competition experiments with a diaminoquinazoline DcpS inhibitor permitted the quantification of intracellular target occupancy. As a result, diaminoquinazoline upregulators of survival motor neuron protein that are used for the treatment of spinal muscular atrophy were confirmed as inhibitors of DcpS in human primary cells. This work illustrates the utility of sulfonyl fluoride probes designed to react with specific tyrosine residues of a protein and augments the chemical biology toolkit by these probes uses in target validation and molecular pharmacology. PMID:25571984

  15. Structural and Molecular Mechanism for Autoprocessing of MARTX Toxin of Vibrio cholerae at Multiple Sites

    SciTech Connect

    Prochazkova, Katerina; Shuvalova, Ludmilla A.; Minasov, George; Voburka, Zdeněk; Anderson, Wayne F.; Satchell, Karla J.F.

    2009-10-05

    The multifunctional autoprocessing repeats-in-toxin (MARTX) toxin of Vibrio cholerae causes destruction of the actin cytoskeleton by covalent cross-linking of actin and inactivation of Rho GTPases. The effector domains responsible for these activities are here shown to be independent proteins released from the large toxin by autoproteolysis catalyzed by an embedded cysteine protease domain (CPD). The CPD is activated upon binding inositol hexakisphosphate (InsP{sub 6}). In this study, we demonstrated that InsP{sub 6} is not simply an allosteric cofactor, but rather binding of InsP{sub 6} stabilized the CPD structure, facilitating formation of the enzyme-substrate complex. The 1.95-{angstrom} crystal structure of this InsP{sub 6}-bound unprocessed form of CPD was determined and revealed the scissile bond Leu{sup 3428}-Ala{sup 3429} captured in the catalytic site. Upon processing at this site, CPD was converted to a form with 500-fold reduced affinity for InsP{sub 6}, but was reactivated for high affinity binding of InsP{sub 6} by cooperative binding of both a new substrate and InsP{sub 6}. Reactivation of CPD allowed cleavage of the MARTX toxin at other sites, specifically at leucine residues between the effector domains. Processed CPD also cleaved other proteins in trans, including the leucine-rich protein YopM, demonstrating that it is a promiscuous leucine-specific protease.

  16. Anaesthetic potencies of primary alkanols: implications for the molecular dimensions of the anaesthetic site.

    PubMed Central

    Alifimoff, J. K.; Firestone, L. L.; Miller, K. W.

    1989-01-01

    1. We have redetermined the anaesthetic potencies (EC50S) for a series of primary alkanols, to resolve uncertainties about the molecular dimensions of the anaesthetic site resulting from the use of data from different laboratories. 2. For each alkanol, concentration-response relationships for loss of righting reflex (LRR) were plotted for over one hundred tadpoles, and the median effective concentrations determined. Aqueous concentrations present during potency assays were determined independently, and for alkanols with chain length greater than nonanol, correction was made for depletion from the aqueous phase. 3. The EC50S were found to decrease logarithmically with increasing number of carbon atoms in the hydrocarbon chain of the alkanol (CN), such that, on average, each additional methylene group was associated with an approximately four fold increase in potency. 4. The relationship between log EC50 and CN was best described by the quadratic equation, log EC50 = 0.022 (+/- 0.0038) CN2 + 0.76 (+/- 0.051) CN + 3.7 (+/- 0.14) (r2 = 0.9951). 5. A previously described correlation between the apparent changes in the free energy of binding of an additional methylene group both to luciferase and to the sites for LRR in tadpoles was not confirmed. 6. A cut-off in potency beyond dodecanol was established in experiments where tadpoles were maintained in supersaturated solutions of tridecanol for 20 h without demonstrable LRR. 7. These findings indicate that the soluble enzyme firefly luciferase does not adequately model the anaesthetic site. Specifically, there are discrepancies in the position of cut-off, and the apparent changes in the free energy of binding, per methylene group, of an alkanol to luciferase do not parallel that for tadpoles. PMID:2784337

  17. Molecular mechanisms of COUP-TF-mediated transcriptional repression: evidence for transrepression and active repression.

    PubMed Central

    Leng, X; Cooney, A J; Tsai, S Y; Tsai, M J

    1996-01-01

    COUP-TF, an orphan member of the nuclear receptor superfamily, has been proposed to play a key role in regulating organogenesis, neurogenesis, and cellular differentiation during embryonic development. Since heterodimierization is a common theme within the nuclear receptor superfamily and has been demonstrated to modulate transcriptional properties of heterodimeric partners via allosteric interactions, we have devised a strategy to examine the silencing function of COUP-TF in a heterodimeric context. We find that the intrinsic active repression function of COUP-TF is not affected by heterodimerization. Moreover, COUP-TF can transrepress the ligand-dependent activation of its heterodimeric partners without its own DNA binding site. Using receptor deletion mutants in transfection assays, we show that the region necessary for COUP-TF silencing function is not sufficient for its transrepression activity. Furthermore, our studies indicate that in addition to its active repression function, COUP-TF can repress several different types of activator-dependent transactivation. However, this active repression function of COUP-TF may be differentially regulated by some other activator(s). These studies provide new insights into the molecular mechanism(s) of COUP-TF-mediated repression. PMID:8628300

  18. Molecular mechanism of L-DNase II activation and function as a molecular switch in apoptosis.

    PubMed

    Torriglia, Alicia; Leprêtre, Chloé; Padrón-Barthe, Laura; Chahory, Sabine; Martin, Elisabeth

    2008-12-01

    The discovery of caspase activation counts as one of the most important finds in the biochemistry of apoptosis. However, targeted disruption of caspases does not impair every type of apoptosis. Other proteases can replace caspases and several so called "caspase independent" pathways are now described. Here we review our current knowledge on one of these pathways, the LEI/L-DNase II. It is a serine protease-dependent pathway and its key event is the transformation of LEI (leukocyte elastase inhibitor, a serine protease inhibitor) into L-DNase II (an endonuclease). The molecular events leading to this change of enzymatic function as well as the cross-talk and interactions of this molecule with other apoptotic pathway, including caspases, are discussed. PMID:18761000

  19. Antituberculosis Activity of the Molecular Libraries Screening Center Network Library

    PubMed Central

    MADDRY, JOSEPH A.; ANANTHAN, SUBRAMANIAM; GOLDMAN, ROBERT C.; HOBRATH, JUDITH V.; KWONG, CECIL D.; MADDOX, CLINTON; RASMUSSEN, LYNN; REYNOLDS, ROBERT C.; SECRIST, JOHN A.; SOSA, MELINDA I.; WHITE, E. LUCILE; ZHANG, WEI

    2009-01-01

    SUMMARY There is an urgent need for the discovery and development of new antitubercular agents that target novel biochemical pathways and treat drug-resistant forms of the disease. One approach to addressing this need is through high-throughput screening of drug-like small molecule libraries against the whole bacterium in order to identify a variety of new, active scaffolds that will stimulate additional biological research and drug discovery. Through the Molecular Libraries Screening Center Network, the NIAID Tuberculosis Antimicrobial Acquisition and Coordinating Facility tested a 215,110-compound library against M. tuberculosis strain H37Rv. A medicinal chemistry survey of the results from the screening campaign is reported herein. PMID:19783214

  20. Hybrid [FeFe]-hydrogenases with modified active sites show remarkable residual enzymatic activity.

    PubMed

    Siebel, Judith F; Adamska-Venkatesh, Agnieszka; Weber, Katharina; Rumpel, Sigrun; Reijerse, Edward; Lubitz, Wolfgang

    2015-02-24

    [FeFe]-hydrogenases are to date the only enzymes for which it has been demonstrated that the native inorganic binuclear cofactor of the active site Fe2(adt)(CO)3(CN)2 (adt = azadithiolate = [S-CH2-NH-CH2-S](2-)) can be synthesized on the laboratory bench and subsequently inserted into the unmaturated enzyme to yield fully functional holo-enzyme (Berggren, G. et al. (2013) Nature 499, 66-70; Esselborn, J. et al. (2013) Nat. Chem. Biol. 9, 607-610). In the current study, we exploit this procedure to introduce non-native cofactors into the enzyme. Mimics of the binuclear subcluster with a modified bridging dithiolate ligand (thiodithiolate, N-methylazadithiolate, dimethyl-azadithiolate) and three variants containing only one CN(-) ligand were inserted into the active site of the enzyme. We investigated the activity of these variants for hydrogen oxidation as well as proton reduction and their structural accommodation within the active site was analyzed using Fourier transform infrared spectroscopy. Interestingly, the monocyanide variant with the azadithiolate bridge showed ∼50% of the native enzyme activity. This would suggest that the CN(-) ligands are not essential for catalytic activity, but rather serve to anchor the binuclear subsite inside the protein pocket through hydrogen bonding. The inserted artificial cofactors with a propanedithiolate and an N-methylazadithiolate bridge as well as their monocyanide variants also showed residual activity. However, these activities were less than 1% of the native enzyme. Our findings indicate that even small changes in the dithiolate bridge of the binuclear subsite lead to a rather strong decrease of the catalytic activity. We conclude that both the Brønsted base function and the conformational flexibility of the native azadithiolate amine moiety are essential for the high catalytic activity of the native enzyme. PMID:25633077

  1. MEK1/2 Inhibitors: Molecular Activity and Resistance Mechanisms.

    PubMed

    Wu, Pui-Kei; Park, Jong-In

    2015-12-01

    Aberrant activation of the three-layered protein kinase cascade, Raf/MEK/ERK, is often detected in human cancer, which is mainly attributed to the oncogenic alterations of RAF, or its upstream activators RAS or cell surface receptor tyrosine kinases. Deregulated activity of the Raf/MEK/ERK pathway drives uncontrolled tumor cell proliferation and survival, thus providing a rational therapeutic target for the treatment of many cancers. While Raf, MEK1/2, and ERK1/2 are equally important targets for the design of therapeutic small molecular weight inhibitors, the effort to develop MEK1/2-specific inhibitors has been greatly successful. Particularly, MEK1/2 have been relatively advantageous for the design of highly selective adenosine triphosphate (ATP)-noncompetitive inhibitors. Indeed, a plethora of highly selective and potent MEK1/2 inhibitors are now available and many of those inhibitors have been evaluated for their therapeutic potential. Herein, we review different MEK1/2 inhibitors that have been studied for their inhibitory mechanisms and therapeutic potential in cancer. Some of the key structural features of MEK1/2 that are important for the efficacy of these inhibitors are also discussed. In addition, we discuss current challenges and future prospective in using these advanced MEK1/2 inhibitors for cancer therapy. PMID:26615130

  2. Restricted dynamics of molecular hydrogen confined in activated carbon nanopores

    SciTech Connect

    Contescu, Cristian I; Saha, Dipendu; Gallego, Nidia C; Mamontov, Eugene; Kolesnikov, Alexander I; Bhat, Vinay V

    2012-01-01

    Quasi-elastic neutron scattering was used for characterization of dynamics of molecular hydrogen confined in narrow nanopores of two activated carbon materials: PFAC (derived from polyfurfuryl alcohol) and UMC (ultramicroporous carbon). Fast, but incomplete ortho-para conversion was observed at 10 K, suggesting that scattering originates from the fraction of unconverted ortho isomer which is rotation-hindered because of confinement in nanopores. Hydrogen molecules entrapped in narrow nanopores (<7 ) were immobile below 22-25 K. Mobility increased rapidly with temperature above this threshold, which is 8 K higher than the melting point of bulk hydrogen. Diffusion obeyed fixed-jump length mechanism, indistinguishable between 2D and 3D processes. Thermal activation of diffusion was characterized between ~22 and 37 K, and structure-dependent differences were found between the two carbons. Activation energy of diffusion was higher than that of bulk solid hydrogen. Classical notions of liquid and solid do not longer apply for H2 confined in narrow nanopores.

  3. An undergraduate laboratory activity on molecular dynamics simulations.

    PubMed

    Spitznagel, Benjamin; Pritchett, Paige R; Messina, Troy C; Goadrich, Mark; Rodriguez, Juan

    2016-01-01

    Vision and Change [AAAS, 2011] outlines a blueprint for modernizing biology education by addressing conceptual understanding of key concepts, such as the relationship between structure and function. The document also highlights skills necessary for student success in 21st century Biology, such as the use of modeling and simulation. Here we describe a laboratory activity that allows students to investigate the dynamic nature of protein structure and function through the use of a modeling technique known as molecular dynamics (MD). The activity takes place over two lab periods that are 3 hr each. The first lab period unpacks the basic approach behind MD simulations, beginning with the kinematic equations that all bioscience students learn in an introductory physics course. During this period students are taught rudimentary programming skills in Python while guided through simple modeling exercises that lead up to the simulation of the motion of a single atom. In the second lab period students extend concepts learned in the first period to develop skills in the use of expert MD software. Here students simulate and analyze changes in protein conformation resulting from temperature change, solvation, and phosphorylation. The article will describe how these activities can be carried out using free software packages, including Abalone and VMD/NAMD. PMID:26751047

  4. Crystallographic characterization of the ribosomal binding site and molecular mechanism of action of Hygromycin A

    PubMed Central

    Kaminishi, Tatsuya; Schedlbauer, Andreas; Fabbretti, Attilio; Brandi, Letizia; Ochoa-Lizarralde, Borja; He, Cheng-Guang; Milón, Pohl; Connell, Sean R.; Gualerzi, Claudio O.; Fucini, Paola

    2015-01-01

    Hygromycin A (HygA) binds to the large ribosomal subunit and inhibits its peptidyl transferase (PT) activity. The presented structural and biochemical data indicate that HygA does not interfere with the initial binding of aminoacyl-tRNA to the A site, but prevents its subsequent adjustment such that it fails to act as a substrate in the PT reaction. Structurally we demonstrate that HygA binds within the peptidyl transferase center (PTC) and induces a unique conformation. Specifically in its ribosomal binding site HygA would overlap and clash with aminoacyl-A76 ribose moiety and, therefore, its primary mode of action involves sterically restricting access of the incoming aminoacyl-tRNA to the PTC. PMID:26464437

  5. Site-specific PEGylation of lidamycin and its antitumor activity.

    PubMed

    Li, Liang; Shang, Boyang; Hu, Lei; Shao, Rongguang; Zhen, Yongsu

    2015-05-01

    In this study, N-terminal site-specific mono-PEGylation of the recombinant lidamycin apoprotein (rLDP) of lidamycin (LDM) was prepared using a polyethyleneglycol (PEG) derivative (M w 20 kDa) through a reactive terminal aldehyde group under weak acidic conditions (pH 5.5). The biochemical properties of mPEG-rLDP-AE, an enediyne-integrated conjugate, were analyzed by SDS-PAGE, RP-HPLC, SEC-HPLC and MALDI-TOF. Meanwhile, in vitro and in vivo antitumor activity of mPEG-rLDP-AE was evaluated by MTT assays and in xenograft model. The results indicated that mPEG-rLDP-AE showed significant antitumor activity both in vitro and in vivo. After PEGylation, mPEG-rLDP still retained the binding capability to the enediyne AE and presented the physicochemical characteristics similar to that of native LDP. It is of interest that the PEGylation did not diminish the antitumor efficacy of LDM, implying the possibility that this derivative may function as a payload to deliver novel tumor-targeted drugs. PMID:26579455

  6. Chemical and thermal modulation of molecular motor activities

    NASA Astrophysics Data System (ADS)

    Hong, Weili

    Molecular motors of kinesin and dynein families are responsible for various intracellular activities, from long distance movement of organelles, vesicles, protein complexes, and mRNAs to powering mitotic processes. They can take nanometer steps using chemical energy from the hydrolysis of ATP (adenosine triphosphate), and their dysfunction is involved in many neurodegenerative diseases that require long distance transport of cargos. Here I report on the study of the properties of molecular motors at a single-molecule level using optical trappings. I first studied the inhibition properties of kinesin motors by marine natural compound adociasulfates. I showed that adociasulfates compete with microtubules for binding to kinesins and thus inhibit kinesins' activity. Although adociasulfates are a strong inhibitor for all kinesin members, they show a much higher inhibition effect for conventional kinesins than for mitotic kinesins. Thus adociasulfates can be used to specifically inhibit conventional kinesins. By comparing the inhibition of kinesins by two structurally similar adociasulfates, one can see that the negatively charged sulfate residue of adociasulfates can be replaced by other negative residues and thus make it possible for adociasulfate-derived compounds to be more cell permeable. Kinesins and dyneins move cargos towards opposite directions along a microtubule. Cargos with both kinesins and dyneins attached often move bidirectionally due to undergoing a tug-of-war between the oppositely moving kinesin and dynein motors. Here I studied the effect of temperature on microtubule-based kinesin and dynein motor transport. While kinesins' and dyneins' velocities are closely matched above 15 °C, below this temperature the dyneins' velocity decreases much faster than the kinesins'. The kinesins' and dyneins' forces do not measurably change with temperature. The results suggest that temperature has significant effects on bidirectional transport and can be used to

  7. Investigating the Conformational Structure and Potential Site Interactions of SOD Inhibitors on Ec-SOD in Marine Mud Crab Scylla serrata: A Molecular Modeling Approach.

    PubMed

    Paital, Biswaranjan; Sablok, Gaurav; Kumar, Sunil; Singh, Sanjeev Kumar; Chainy, G B N

    2016-09-01

    Superoxide dismutases (SODs) act as a first line of the enzymatic antioxidant defense system to control cellular superoxide anion toxicity. Previously, several inhibitors have been widely identified and catalogued for inhibition of SOD activity; however, still the information about the mechanism of interaction and points toward the inhibitor interactions in structures of SODs in general and in extracellular (Ec)-SOD in particular is still in naive. In the present research, we present an insight to elucidate the molecular basis of interactions of SOD inhibitors with Ec-SOD in mud crab Scylla serrata using molecular modeling and docking approaches. Different inhibitors of SOD such as hydrogen peroxide [Formula: see text], potassium cyanide, sodium dodecyl sulfate (SDS), [Formula: see text]-mercaptoethanol and dithiocarbamate were screened to understand the potential sites that may act as sites for cleavage or blocking in the protein. SOD-SDS and [Formula: see text] complex interactions indicate residues Pro72 and Asp102 of the predicted crab Ec-SOD as common targets. The GOLD result indicates that Pro72, Asp102 and Thr103 are commonly acting as the site of interaction in Ec-SOD of S. serrata with SOD inhibitors. For the first time, the results of this study provide an insight into the structural properties of Ec-SOD of S. serrata and define the possible involvements between the amino acids present in its active sites, i.e., in the regions from 70 to 84 and from 101 to 103 and different inhibitors. PMID:26286009

  8. Understanding the Cellular and Molecular Mechanisms of Physical Activity-Induced Health Benefits.

    PubMed

    Neufer, P Darrell; Bamman, Marcas M; Muoio, Deborah M; Bouchard, Claude; Cooper, Dan M; Goodpaster, Bret H; Booth, Frank W; Kohrt, Wendy M; Gerszten, Robert E; Mattson, Mark P; Hepple, Russell T; Kraus, William E; Reid, Michael B; Bodine, Sue C; Jakicic, John M; Fleg, Jerome L; Williams, John P; Joseph, Lyndon; Evans, Mary; Maruvada, Padma; Rodgers, Mary; Roary, Mary; Boyce, Amanda T; Drugan, Jonelle K; Koenig, James I; Ingraham, Richard H; Krotoski, Danuta; Garcia-Cazarin, Mary; McGowan, Joan A; Laughlin, Maren R

    2015-07-01

    The beneficial effects of physical activity (PA) are well documented, yet the mechanisms by which PA prevents disease and improves health outcomes are poorly understood. To identify major gaps in knowledge and potential strategies for catalyzing progress in the field, the NIH convened a workshop in late October 2014 entitled "Understanding the Cellular and Molecular Mechanisms of Physical Activity-Induced Health Benefits." Presentations and discussions emphasized the challenges imposed by the integrative and intermittent nature of PA, the tremendous discovery potential of applying "-omics" technologies to understand interorgan crosstalk and biological networking systems during PA, and the need to establish an infrastructure of clinical trial sites with sufficient expertise to incorporate mechanistic outcome measures into adequately sized human PA trials. Identification of the mechanisms that underlie the link between PA and improved health holds extraordinary promise for discovery of novel therapeutic targets and development of personalized exercise medicine. PMID:26073496

  9. Synthetic Molecular Machines for Active Self-Assembly: Prototype Algorithms, Designs, and Experimental Study

    NASA Astrophysics Data System (ADS)

    Dabby, Nadine L.

    behaviors. This class of behaviors includes any behavior where a passive physical system simply does not have enough physical energy to perform the specified tasks in the requisite amount of time. As we will demonstrate and prove, a sufficiently expressive implementation of an "active" molecular self-assembly approach can achieve these behaviors. Using an external source of fuel solves part of the problem, so the system is not "energetically incomplete." But the programmable system also needs to have sufficient expressive power to achieve the specified behaviors. Perhaps surprisingly, some of these systems do not even require Turing completeness to be sufficiently expressive. Building on a large variety of work by other scientists in the fields of DNA nanotechnology, chemistry and reconfigurable robotics, this thesis introduces several research contributions in the context of active self-assembly. We show that simple primitives such as insertion and deletion are able to generate complex and interesting results such as the growth of a linear polymer in logarithmic time and the ability of a linear polymer to treadmill. To this end we developed a formal model for active-self assembly that is directly implementable with DNA molecules. We show that this model is computationally equivalent to a machine capable of producing strings that are stronger than regular languages and, at most, as strong as context-free grammars. This is a great advance in the theory of active self-assembly as prior models were either entirely theoretical or only implementable in the context of macro-scale robotics. We developed a chain reaction method for the autonomous exponential growth of a linear DNA polymer. Our method is based on the insertion of molecules into the assembly, which generates two new insertion sites for every initial one employed. The building of a line in logarithmic time is a first step toward building a shape in logarithmic time. We demonstrate the first construction of a synthetic

  10. Molecular Biological Detection of Anaerobic Gut Fungi (Neocallimastigales) from Landfill Sites

    PubMed Central

    Lockhart, Robert J.; Van Dyke, Michele I.; Beadle, Ian R.; Humphreys, Paul; McCarthy, Alan J.

    2006-01-01

    Oligonucleotide primers were designed for the 18S rRNA genes of members of the Neocallimastigales and used in a nested PCR protocol to amplify 787-bp fragments of DNA from landfill site samples. The specificities of the primers were confirmed by phylogenetic analysis of the environmental clone sequences, and this method can therefore now be used to investigate the ecology of the obligately anaerobic fungi. To our knowledge, this is the first demonstration of the occurrence of members of the Neocallimastigales outside the mammalian gut, and their distribution across the landfill samples examined here suggests that they are actively involved in cellulose degradation. PMID:16885325

  11. Fluctuation driven active molecular transport in passive channel proteins

    NASA Astrophysics Data System (ADS)

    Kosztin, Ioan

    2006-03-01

    Living cells interact with their extracellular environment through the cell membrane, which acts as a protective permeability barrier for preserving the internal integrity of the cell. However, cell metabolism requires controlled molecular transport across the cell membrane, a function that is fulfilled by a wide variety of transmembrane proteins, acting as either passive or active transporters. In this talk it is argued that, contrary to the general belief, in active cell membranes passive and spatially asymmetric channel proteins can act as active transporters by consuming energy from nonequilibrium fluctuations fueled by cell metabolism. This assertion is demonstrated in the case of the E. coli aquaglyceroporin GlpF channel protein, whose high resolution crystal structure is manifestly asymmetric. By calculating the glycerol flux through GlpF within the framework of a stochastic model, it is found that, as a result of channel asymmetry, glycerol uptake driven by a concentration gradient is enhanced significantly in the presence of non-equilibrium fluctuations. Furthermore, the enhancement caused by a ratchet-like mechanism is larger for the outward, i.e., from the cytoplasm to the periplasm, flux than for the inward one, suggesting that the same non-equilibrium fluctuations also play an important role in protecting the interior of the cell against poisoning by excess uptake of glycerol. Preliminary data on water and sugar transport through aquaporin and maltoporin channels, respectively, are indicative of the universality of the proposed nonequilibrium-fluctuation-driven active transport mechanism. This work was supported by grants from the Univ. of Missouri Research Board, the Institute for Theoretical Sciences and the Department of Energy (DOE Contract W-7405-ENG-36), and the National Science Foundation (FIBR-0526854).

  12. Organic aerosol molecular composition and gas-particle partitioning coefficients at a Mediterranean site (Corsica).

    PubMed

    Rossignol, Stéphanie; Couvidat, Florian; Rio, Caroline; Fable, Sébastien; Grignion, Guillaume; Savelli; Pailly, Olivier; Leoz-Garziandia, Eva; Doussin, Jean-Francois; Chiappini, Laura

    2016-02-01

    Molecular speciation of atmospheric organic matter was investigated during a short summer field campaign performed in a citrus fruit field in northern Corsica (June 2011). Aimed at assessing the performance on the field of newly developed analytical protocols, this work focuses on the molecular composition of both gas and particulate phases and provides an insight into partitioning behavior of the semi-volatile oxygenated fraction. Limonene ozonolysis tracers were specifically searched for, according to gas chromatography-mass spectrometry (GC-MS) data previously recorded for smog chamber experiments. A screening of other oxygenated species present in the field atmosphere was also performed. About sixty polar molecules were positively or tentatively identified in gas and/or particle phases. These molecules comprise a wide range of branched and linear, mono and di-carbonyls (C3-C7), mono and di-carboxylic acids (C3-C18), and compounds bearing up to three functionalities. Among these compounds, some can be specifically attributed to limonene oxidation and others can be related to α- or β-pinene oxidation. This provides an original snapshot of the organic matter composition at a Mediterranean site in summer. Furthermore, for compounds identified and quantified in both gaseous and particulate phases, an experimental gas/particle partitioning coefficient was determined. Several volatile products, which are not expected in the particulate phase assuming thermodynamic equilibrium, were nonetheless present in significant concentrations. Hypotheses are proposed to explain these observations, such as the possible aerosol viscosity that could hinder the theoretical equilibrium to be rapidly reached. PMID:26969549

  13. Active site hydrophobicity is critical to the bioluminescence activity of Vibrio harveyi luciferase.

    PubMed

    Li, Chi-Hui; Tu, Shiao-Chun

    2005-10-01

    Vibrio harveyi luciferase is an alphabeta heterodimer containing a single active site, proposed earlier to be at a cleft in the alpha subunit. In this work, six conserved phenylalanine residues at this proposed active site were subjected to site-directed mutations to investigate their possible functional roles and to delineate the makeup of luciferase active site. After initial screening of Phe --> Ala mutants, alphaF46, alphaF49, alphaF114, and alphaF117 were chosen for additional mutations to Asp, Ser, and Tyr. Comparisons of the general kinetic properties of wild-type and mutated luciferases indicated that the hydrophobic nature of alphaF46, alphaF49, alphaF114, and alphaF117 was important to luciferase V(max) and V(max)/K(m), which were reduced by 3-5 orders of magnitude for the Phe --> Asp mutants. Both alphaF46 and alphaF117 also appeared to be involved in the binding of reduced flavin substrate. Additional studies on the stability and yield of the 4a-hydroperoxyflavin intermediate II and measurements of decanal substrate oxidation by alphaF46D, alphaF49D, alphaF114D, and alphaF117D revealed that their marked reductions in the overall quantum yield (phi( degrees )) were a consequence of diminished yields of luciferase intermediates and, with the exception of alphaF114D, emission quantum yield of the excited emitter due to the replacement of the hydrophobic Phe by the anionic Asp. The locations of these four critical Phe residues in relation to other essential and/or hydrophobic residues are depicted in a refined map of the active site. Functional implications of these residues are discussed. PMID:16185065

  14. Molecular environment of the phencyclidine binding site in the nicotinic acetylcholine receptor membrane

    SciTech Connect

    Palma, A.L.; Wang, H.H. )

    1991-06-01

    Phencyclidine is a highly specific noncompetitive inhibitor of the nicotinic acetylcholine receptor. In a novel approach to study this site, a spin-labeled analogue of phencyclidine, 4-phenyl-4-(1-piperidinyl)-2,2,6,6-tetramethylpiperidinoxyl (PPT) was synthesized. The binding of PPT inhibits 86Rb flux (IC50 = 6.6 microM), and (3H)phencyclidine binding to both resting and desensitized acetylcholine receptor (IC50 = 17 microM and 0.22 microM, respectively). From an indirect Hill plot of the inhibition of (3H)phencyclidine binding by PPT, a Hill coefficient of approximately one was obtained in the presence of carbamylcholine and 0.8 in alpha-bungarotoxin-treated preparations. Taken together, these results indicate that PPT mimics phencyclidine in its ability to bind to the noncompetitive inhibitor site and is functionally active in blocking ion flux across the acetylcholine receptor channel. Analysis of the electron spin resonance signal of the bound PPT suggests that the environment surrounding the probe within the ion channel is hydrophobic, with a hydrophobicity parameter of 1.09. A dielectric constant for the binding site was estimated to be in the range of 2-3 units.

  15. Molecular Dynamics of "Fuzzy" Transcriptional Activator-Coactivator Interactions.

    PubMed

    Scholes, Natalie S; Weinzierl, Robert O J

    2016-05-01

    Transcriptional activation domains (ADs) are generally thought to be intrinsically unstructured, but capable of adopting limited secondary structure upon interaction with a coactivator surface. The indeterminate nature of this interface made it hitherto difficult to study structure/function relationships of such contacts. Here we used atomistic accelerated molecular dynamics (aMD) simulations to study the conformational changes of the GCN4 AD and variants thereof, either free in solution, or bound to the GAL11 coactivator surface. We show that the AD-coactivator interactions are highly dynamic while obeying distinct rules. The data provide insights into the constant and variable aspects of orientation of ADs relative to the coactivator, changes in secondary structure and energetic contributions stabilizing the various conformers at different time points. We also demonstrate that a prediction of α-helical propensity correlates directly with the experimentally measured transactivation potential of a large set of mutagenized ADs. The link between α-helical propensity and the stimulatory activity of ADs has fundamental practical and theoretical implications concerning the recruitment of ADs to coactivators. PMID:27175900

  16. Molecular Dynamics of "Fuzzy" Transcriptional Activator-Coactivator Interactions

    PubMed Central

    Scholes, Natalie S.; Weinzierl, Robert O. J.

    2016-01-01

    Transcriptional activation domains (ADs) are generally thought to be intrinsically unstructured, but capable of adopting limited secondary structure upon interaction with a coactivator surface. The indeterminate nature of this interface made it hitherto difficult to study structure/function relationships of such contacts. Here we used atomistic accelerated molecular dynamics (aMD) simulations to study the conformational changes of the GCN4 AD and variants thereof, either free in solution, or bound to the GAL11 coactivator surface. We show that the AD-coactivator interactions are highly dynamic while obeying distinct rules. The data provide insights into the constant and variable aspects of orientation of ADs relative to the coactivator, changes in secondary structure and energetic contributions stabilizing the various conformers at different time points. We also demonstrate that a prediction of α-helical propensity correlates directly with the experimentally measured transactivation potential of a large set of mutagenized ADs. The link between α-helical propensity and the stimulatory activity of ADs has fundamental practical and theoretical implications concerning the recruitment of ADs to coactivators. PMID:27175900

  17. Nanoplasmonic molecular ruler for nuclease activity and DNAfootprinting

    SciTech Connect

    Chen, Fanqing Frank; Liu, Gang L.; Yin, Yadong; Gerion, Daniele; Kunchakarra, Siri; Mukherjee, Bipasha; Jett, Stephen D.; Bear, David G.; Alivisatos, Paul; Lee, Luke P.

    2006-08-15

    We have constructed a nanoplasmonic molecular ruler, which can perform label-free and real-time monitoring of DNA length changes and perform DNA footprinting. The ruler was created by tethering double-stranded DNA to single Au nanoparticles. The scattering spectra of Au-DNA nanoconjugates showed red-shifted peak plasmon resonance wavelength dependent on DNA length, which can be measured with sub-nanometer axial resolution, averaging {approx}1.24 nm peak wavelength shift per DNA base pair. The spectra of individual Au-DNA nanoconjugates in the presence of nuclease showed a time-resolved dependence on the reaction dynamics, allowing quantitative, kinetic and real-time measurement of nuclease activity. The ruler was further developed into a new DNA footprinting platform. We showed the specific binding of a protein to DNA and the accurate mapping of its footprint. This work promises a very fast and convenient platform for mapping DNA-protein interactions, for nuclease activity monitoring, and for other DNA size-based methods.

  18. Correlation of active site metal content in human diamine oxidase with trihydroxyphenylalanine quinone cofactor biogenesis .

    PubMed

    McGrath, Aaron P; Caradoc-Davies, Tom; Collyer, Charles A; Guss, J Mitchell

    2010-09-28

    Copper-containing amine oxidases (CAOs) require a protein-derived topaquinone cofactor (TPQ) for activity. TPQ biogenesis is a self-processing reaction requiring the presence of copper and molecular oxygen. Recombinant human diamine oxidase (hDAO) was heterologously expressed in Drosophila S2 cells, and analysis indicates that the purified hDAO contains substoichiometric amounts of copper and TPQ. The crystal structure of a complex of an inhibitor, aminoguanidine, and hDAO at 2.05 Å resolution shows that the aminoguanidine forms a covalent adduct with the TPQ and that the site is ∼75% occupied. Aminoguanidine is a potent inhibitor of hDAO with an IC(50) of 153 ± 9 nM. The structure indicates that the catalytic metal site, normally occupied by copper, is fully occupied. X-ray diffraction data recorded below the copper edge, between the copper and zinc edges, and above the zinc edge have been used to show that the metal site is occupied approximately 75% by copper and 25% by zinc and the formation of the TPQ cofactor is correlated with copper occupancy. PMID:20722416

  19. Non-native ligands define the active site of Pennisetum glaucum (L.) R. Br dehydroascorbate reductase.

    PubMed

    Krishna Das, Bhaba; Kumar, Amit; Maindola, Priyank; Mahanty, Srikrishna; Jain, S K; Reddy, Mallireddy K; Arockiasamy, Arulandu

    2016-05-13

    Dehydroascorbate reductase (DHAR), a member of the glutathione-S-transferase (GST) family, reduces dehydroascorbate (DHA) to ascorbate (AsA; Vitamin-C) in a glutathione (GSH)-dependent manner and in doing so, replenishes the critical AsA pool of the cell. To understand the enzyme mechanism in detail, we determined the crystal structure of a plant DHAR from Pennisetum glaucum (PgDHAR) using Iodide-Single Anomalous Dispersion (SAD) and Molecular replacement methods, in two different space groups. Here, we show PgDHAR in complex with two non-native ligands, viz. an acetate bound at the G-site, which resembles the γ-carboxyl moiety of GSH, and a glycerol at the H-site, which shares the backbone of AsA. We also show that, in the absence of bound native substrates, these non-native ligands help define the critical 'hook points' in the DHAR enzyme active site. Further, our data suggest that these non-native ligands can act as the logical bootstrapping points for iterative design of inhibitors/analogs for DHARs. PMID:27067046

  20. Inhibition of Plasma Kallikrein by a Highly Specific Active Site Blocking Antibody

    PubMed Central

    Kenniston, Jon A.; Faucette, Ryan R.; Martik, Diana; Comeau, Stephen R.; Lindberg, Allison P.; Kopacz, Kris J.; Conley, Gregory P.; Chen, Jie; Viswanathan, Malini; Kastrapeli, Niksa; Cosic, Janja; Mason, Shauna; DiLeo, Mike; Abendroth, Jan; Kuzmic, Petr; Ladner, Robert C.; Edwards, Thomas E.; TenHoor, Christopher; Adelman, Burt A.; Nixon, Andrew E.; Sexton, Daniel J.

    2014-01-01

    Plasma kallikrein (pKal) proteolytically cleaves high molecular weight kininogen to generate the potent vasodilator and the pro-inflammatory peptide, bradykinin. pKal activity is tightly regulated in healthy individuals by the serpin C1-inhibitor, but individuals with hereditary angioedema (HAE) are deficient in C1-inhibitor and consequently exhibit excessive bradykinin generation that in turn causes debilitating and potentially fatal swelling attacks. To develop a potential therapeutic agent for HAE and other pKal-mediated disorders, we used phage display to discover a fully human IgG1 monoclonal antibody (DX-2930) against pKal. In vitro experiments demonstrated that DX-2930 potently inhibits active pKal (Ki = 0.120 ± 0.005 nm) but does not target either the zymogen (prekallikrein) or any other serine protease tested. These findings are supported by a 2.1-Å resolution crystal structure of pKal complexed to a DX-2930 Fab construct, which establishes that the pKal active site is fully occluded by the antibody. DX-2930 injected subcutaneously into cynomolgus monkeys exhibited a long half-life (t½ ∼12.5 days) and blocked high molecular weight kininogen proteolysis in activated plasma in a dose- and time-dependent manner. Furthermore, subcutaneous DX-2930 reduced carrageenan-induced paw edema in rats. A potent and long acting inhibitor of pKal activity could be an effective treatment option for pKal-mediated diseases, such as HAE. PMID:24970892

  1. Role of Bassoon and Piccolo in Assembly and Molecular Organization of the Active Zone

    PubMed Central

    Gundelfinger, Eckart D.; Reissner, Carsten; Garner, Craig C.

    2016-01-01

    Bassoon and Piccolo are two very large scaffolding proteins of the cytomatrix assembled at the active zone (CAZ) where neurotransmitter is released. They share regions of high sequence similarity distributed along their entire length and seem to share both overlapping and distinct functions in organizing the CAZ. Here, we survey our present knowledge on protein-protein interactions and recent progress in understanding of molecular functions of these two giant proteins. These include roles in the assembly of active zones (AZ), the localization of voltage-gated Ca2+ channels (VGCCs) in the vicinity of release sites, synaptic vesicle (SV) priming and in the case of Piccolo, a role in the dynamic assembly of the actin cytoskeleton. Piccolo and Bassoon are also important for the maintenance of presynaptic structure and function, as well as for the assembly of CAZ specializations such as synaptic ribbons. Recent findings suggest that they are also involved in the regulation activity-dependent communication between presynaptic boutons and the neuronal nucleus. Together these observations suggest that Bassoon and Piccolo use their modular structure to organize super-molecular complexes essential for various aspects of presynaptic function. PMID:26793095

  2. STUDIES OF RELATIONSHIPS BETWEEN MOLECULAR STRUCTURE AND BIOLOGICAL ACTIVITY BY PATTERN RECOGNITION METHODS

    EPA Science Inventory

    The attempt to rationalize the connections between the molecular structures of organic compounds and their biological activities comprises the field of structure-activity relations (SAR) studies. Correlations between structure and activity are important for the understanding and ...

  3. A proposed definition of the 'activity' of surface sites on lactose carriers for dry powder inhalation.

    PubMed

    Grasmeijer, Floris; Frijlink, Henderik W; de Boer, Anne H

    2014-06-01

    A new definition of the activity of surface sites on lactose carriers for dry powder inhalation is proposed which relates to drug detachment during dispersion. The new definition is expected to improve the understanding of 'carrier surface site activity', which stimulates the unambiguous communication about this subject and may aid in the rational design and interpretation of future formulation studies. In contrast to the currently prevailing view on carrier surface site activity, it follows from the newly proposed definition that carrier surface site activity depends on more variables than just the physicochemical properties of the carrier surface. Because the term 'active sites' is ambiguous, it is recommended to use the term 'highly active sites' instead to denote carrier surface sites with a relatively high activity. PMID:24613490

  4. Drug Repositioning by Kernel-Based Integration of Molecular Structure, Molecular Activity, and Phenotype Data

    PubMed Central

    Wang, Yongcui; Chen, Shilong; Deng, Naiyang; Wang, Yong

    2013-01-01

    Computational inference of novel therapeutic values for existing drugs, i.e., drug repositioning, offers the great prospect for faster and low-risk drug development. Previous researches have indicated that chemical structures, target proteins, and side-effects could provide rich information in drug similarity assessment and further disease similarity. However, each single data source is important in its own way and data integration holds the great promise to reposition drug more accurately. Here, we propose a new method for drug repositioning, PreDR (Predict Drug Repositioning), to integrate molecular structure, molecular activity, and phenotype data. Specifically, we characterize drug by profiling in chemical structure, target protein, and side-effects space, and define a kernel function to correlate drugs with diseases. Then we train a support vector machine (SVM) to computationally predict novel drug-disease interactions. PreDR is validated on a well-established drug-disease network with 1,933 interactions among 593 drugs and 313 diseases. By cross-validation, we find that chemical structure, drug target, and side-effects information are all predictive for drug-disease relationships. More experimentally observed drug-disease interactions can be revealed by integrating these three data sources. Comparison with existing methods demonstrates that PreDR is competitive both in accuracy and coverage. Follow-up database search and pathway analysis indicate that our new predictions are worthy of further experimental validation. Particularly several novel predictions are supported by clinical trials databases and this shows the significant prospects of PreDR in future drug treatment. In conclusion, our new method, PreDR, can serve as a useful tool in drug discovery to efficiently identify novel drug-disease interactions. In addition, our heterogeneous data integration framework can be applied to other problems. PMID:24244318

  5. Exploration of the ligand binding site of the human 5-HT4 receptor by site-directed mutagenesis and molecular modeling

    PubMed Central

    Mialet, Jeanne; Dahmoune, Yamina; Lezoualc'h, Frank; Berque-Bestel, Isabelle; Eftekhari, Pierre; Hoebeke, Johan; Sicsic, Sames; Langlois, Michel; Fischmeister, Rodolphe

    2000-01-01

    Among the five human 5-HT4 (h5-HT4) receptor isoforms, the h5-HT4(a) receptor was studied with a particular emphasis on the molecular interactions involved in ligand binding. For this purpose, we used site-directed mutagenesis of the transmembrane domain. Twelve mutants were constructed with a special focus on the residue P4.53 of helix IV which substitutes in h5-HT4 receptors the highly conserved S residue among the rhodopsin family receptors. The mutated receptors were transiently expressed in COS-7 cells.Ligand binding or competition studies with two h5-HT4 receptor agonists, serotonin and ML10302 and two h5-HT4 receptor antagonists, [3H]-GR113808 and ML10375 were performed on wild type and mutant receptors. Functional activity of the receptors was evaluated by measuring the ability of serotonin to stimulate adenylyl cyclase.Ligand binding experiments revealed that [3H]-GR113808 did not bind to mutants P4.53A, S5.43A, F6.51A, Y7.43A and to double mutant F6.52V/N6.55L. On the other hand mutations D3.32N, S5.43A and Y7.43A appeared to promote a dramatic decrease of h5-HT4(a) receptor functional activity. From these studies, S5.43 and Y7.43 clearly emerged as common anchoring sites to antagonist [3H]-GR113808 and to serotonin.According to these results, we propose ligand-receptor complex models with serotonin and [3H]-GR113808. For serotonin, three interaction points were selected including ionic interaction with D3.32, a stabilizing interaction of this ion pair by Y7.43 and a hydrogen bond with S5.43. [3H]-GR113808 was also docked, based on the same type of interactions with S5.43 and D3.32: the proposed model suggested a possible role of P4.53 in helix IV structure allowing the involvement of a close hydrophobic residue, W4.50, in a hydrophobic pocket for hydrophobic interactions with the indole ring of [3H]-GR113808. PMID:10821780

  6. Homology modeling and molecular docking of human pituitary adenylate cyclase-activating polypeptide I receptor

    PubMed Central

    WU, LUSHENG; GUANG, WENHUA; CHEN, XIAOJIA; HONG, AN

    2014-01-01

    Pituitary adenylate cyclase-activating peptide I receptor (PAC1R) is member of the B class of G protein-coupled seven-transmembrane receptors, with molecular functions associated with neural cell differentiation, regeneration and the inhibition of apoptosis. However, the integrity of the protein structure is difficult to be determined in vitro. In the present study, the physicochemical properties of PAC1R were analyzed, the extracellular, transmembrane and intracellular regions were constructed and a three-dimensional structure model of PAC1R was produced using extracellular loop region optimization and the energy minimization homology modeling method. Preliminary studies on the PAC1R protein and ligand interactions used a molecular docking method. The results indicated that the interaction sites of PAC1R were at Ile63, Ser100 and Gln105. These were the sites where the PAC1R combined with a hydrazide small molecule inhibitor. This study provides a theoretical basis for further studies on the model for the development of PAC1R target drugs. PMID:25069645

  7. Molecular Assessment of litter decay dynamics across old and young forest sites

    NASA Astrophysics Data System (ADS)

    Filley, T. R.; Crow, S.; Gamblin, D.; McCormick, M.; Whigham, D.; Taylor, D. L.

    2006-12-01

    The response of soil organic matter pools to changes in litter input, land cover, and ýinvertebrate activity is a research area of intensive study given the proposed impacts that ýrising CO2 and surface temperatures may have on forest productivity and distribution of ýinvasive species. In a mixed deciduous forest at the Smithsonian Environmental ýResearch Center litter amendment plots were established in old (120-150 y) and young ýý(50-70 y) forests. In May 2004, six plots were amended with locally collected ýLirodendron tulipifera wood (chipped) and leaves. At the same time, leaf and wood litter ýbag decomposition experiments on the sites were also started. Changes in the ýconcentration and composition of biopolymers, e.g. lignin and cutin/suberin, after ýapproximately four months of decay were tracked by alkaline CuO extraction. Resultant ýleaf and wood litter in the surface amendments was distinct between age groupings. ýYoung sites exhibited the greatest change in chemical character showing increased lignin ýand decreased cutin/suberin resulting in a cutin-poor residue. Minor changes to ýbiopolymer character were observed in older sites with residues exhibiting small but ýopposite trends to the young sites. In contrast, the litter bag studies exhibited little to no ývariation in chemistry with age of stand; although, generally leaf litter showed the ýgreatest age-related effect. These patterns in litter decay are consistent with both ýmicrobial activity and relative biomass of invasive earthworms; young forests exhibit ýrelatively higher activity of both phenol oxidase and B-glucosidase in the soil (0-5 cm) ýplots and greater biomass and relative abundance invasive earthworms. These results are ýimportant as they show how stand age and the presence of invertebrate species may have ýimportant controls on the impact that many global change drivers may have on forest soil ýand carbon exchange dynamics.ý

  8. Implication of crystal water molecules in inhibitor binding at ALR2 active site.

    PubMed

    Hymavati; Kumar, Vivek; Sobhia, M Elizabeth

    2012-01-01

    Water molecules play a crucial role in mediating the interaction between a ligand and a macromolecule. The solvent environment around such biomolecule controls their structure and plays important role in protein-ligand interactions. An understanding of the nature and role of these water molecules in the active site of a protein could greatly increase the efficiency of rational drug design approaches. We have performed the comparative crystal structure analysis of aldose reductase to understand the role of crystal water in protein-ligand interaction. Molecular dynamics simulation has shown the versatile nature of water molecules in bridge H bonding during interaction. Occupancy and life time of water molecules depend on the type of cocrystallized ligand present in the structure. The information may be useful in rational approach to customize the ligand, and thereby longer occupancy and life time for bridge H-bonding. PMID:22649481

  9. On the binding mode of urease active site inhibitors: A density functional study

    NASA Astrophysics Data System (ADS)

    Leopoldini, M.; Marino, T.; Russo, N.; Toscano, M.

    The way with which boric acid, a rapid reversible competitive inhibitor, binds the urease active site was explored at density functional B3LYP level of theory. The catalytic core of the enzyme was simulated by two models of different size. In both cases, amino acid residues belonging to the inner and to the outer coordination spheres of nickel ions were replaced by smaller molecular species. Contrary to the experimental indication that attributes the inhibitory ability of this acid to the lack of a nucleophilic attack by the enzyme to the boron atom, we instead found that another possibility exists based on the presence of a strong covalent sigma bond between boron and urease that we think can be hardly broken to allow any course of the reaction.

  10. Disturbance opens recruitment sites for bacterial colonization in activated sludge.

    PubMed

    Vuono, David C; Munakata-Marr, Junko; Spear, John R; Drewes, Jörg E

    2016-01-01

    Little is known about the role of immigration in shaping bacterial communities or the factors that may dictate success or failure of colonization by bacteria from regional species pools. To address these knowledge gaps, the influence of bacterial colonization into an ecosystem (activated sludge bioreactor) was measured through a disturbance gradient (successive decreases in the parameter solids retention time) relative to stable operational conditions. Through a DNA sequencing approach, we show that the most abundant bacteria within the immigrant community have a greater probability of colonizing the receiving ecosystem, but mostly as low abundance community members. Only during the disturbance do some of these bacterial populations significantly increase in abundance beyond background levels and in few cases become dominant community members post-disturbance. Two mechanisms facilitate the enhanced enrichment of immigrant populations during disturbance: (i) the availability of resources left unconsumed by established species and (ii) the increased availability of niche space for colonizers to establish and displace resident populations. Thus, as a disturbance decreases local diversity, recruitment sites become available to promote colonization. This work advances our understanding of microbial resource management and diversity maintenance in complex ecosystems. PMID:25727891

  11. Construction of DNA recognition sites active in Haemophilus transformation.

    PubMed Central

    Danner, D B; Smith, H O; Narang, S A

    1982-01-01

    Competent Haemophilus cells recognize and preferentially take up Haemophilus DNA during genetic transformation. This preferential uptake is correlated with the presence on incoming DNA of an 11-base-pair (bp) sequence, 5'-A-A-G-T-G-C-G-G-T-C-A-3'. To prove that this sequence is the recognition site that identifies Haemophilus DNA to the competent cell, we have now constructed a series of plasmids, each of which contains the 11-bp sequence. Using two different assay systems we have tested the ability of fragments from these plasmids to compete with cloned Haemophilus DNA fragments that naturally contain the 11-bp sequence. We find that the addition of the 11-bp sequence to a DNA fragment is necessary and sufficient for preferential uptake of that fragment. However, plasmid DNAs containing this sequence may vary as much as 48-fold in uptake activity, and this variation correlates with the A+T-richness of the DNA flanking the 11-mer. Images PMID:6285382

  12. Characterization of active site residues of nitroalkane oxidase.

    PubMed

    Valley, Michael P; Fenny, Nana S; Ali, Shah R; Fitzpatrick, Paul F

    2010-06-01

    The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the corresponding aldehydes and ketones plus nitrite. The structure of the enzyme shows that Ser171 forms a hydrogen bond to the flavin N5, suggesting that it plays a role in catalysis. Cys397 and Tyr398 were previously identified by chemical modification as potential active site residues. To more directly probe the roles of these residues, the S171A, S171V, S171T, C397S, and Y398F enzymes have been characterized with nitroethane as substrate. The C397S and Y398 enzymes were less stable than the wild-type enzyme, and the C397S enzyme routinely contained a substoichiometric amount of FAD. Analysis of the steady-state kinetic parameters for the mutant enzymes, including deuterium isotope effects, establishes that all of the mutations result in decreases in the rate constants for removal of the substrate proton by approximately 5-fold and decreases in the rate constant for product release of approximately 2-fold. Only the S171V and S171T mutations alter the rate constant for flavin oxidation. These results establish that these residues are not involved in catalysis, but rather are required for maintaining the protein structure. PMID:20056514

  13. Detection limit for activation measurements in ultralow background sites

    NASA Astrophysics Data System (ADS)

    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.

  14. N6-Methyldeoxyadenosine Marks Active Transcription Start Sites in Chlamydomonas

    PubMed Central

    Chen, Kai; Deng, Xin; Yu, Miao; Han, Dali; Hao, Ziyang; Liu, Jianzhao; Lu, Xingyu; Dore, Louis C; Weng, Xiaocheng; Ji, Quanjiang; Mets, Laurens; He, Chuan

    2015-01-01

    SUMMARY N6-methyldeoxyadenosine (6mA or m6A) is a DNA modification preserved in prokaryotes to eukaryotes. It is widespread in bacteria, and functions in DNA mismatch repair, chromosome segregation, and virulence regulation. In contrast, the distribution and function of 6mA in eukaryotes have been unclear. Here we present a comprehensive analysis of the 6mA landscape in the genome of Chlamydomonas using new sequencing approaches. We identified the 6mA modification in 84% of genes in Chlamydomonas. We found that 6mA mainly locates at ApT dinucleotides around transcription start sites (TSS) with a bimodal distribution, and appears to mark active genes. A periodic pattern of 6mA deposition was also observed at base resolution, which is associated with nucleosome distribution near the TSS, suggesting a possible role in nucleosome positioning. The new genome-wide mapping of 6mA and its unique distribution in the Chlamydomonas genome suggest potential regulatory roles of 6mA in gene expression in eukaryotic organisms. PMID:25936837

  15. rRNA Binding Sites and the Molecular Mechanism of Action of the Tetracyclines.

    PubMed

    Chukwudi, Chinwe U

    2016-08-01

    The tetracycline antibiotics are known to be effective in the treatment of both infectious and noninfectious disease conditions. The 16S rRNA binding mechanism currently held for the antibacterial action of the tetracyclines does not explain their activity against viruses, protozoa that lack mitochondria, and noninfectious conditions. Also, the mechanism by which the tetracyclines selectively inhibit microbial protein synthesis against host eukaryotic protein synthesis despite conservation of ribosome structure and functions is still questionable. Many studies have investigated the binding of the tetracyclines to the 16S rRNA using the small ribosomal subunit of different bacterial species, but there seems to be no agreement between various reports on the exact binding site on the 16S rRNA. The wide range of activity of the tetracyclines against a broad spectrum of bacterial pathogens, viruses, protozoa, and helminths, as well as noninfectious conditions, indicates a more generalized effect on RNA. In the light of recent evidence that the tetracyclines bind to various synthetic double-stranded RNAs (dsRNAs) of random base sequences, suggesting that the double-stranded structures may play a more important role in the binding of the tetracyclines to RNA than the specific base pairs, as earlier speculated, it is imperative to consider possible alternative binding modes or sites that could help explain the mechanisms of action of the tetracyclines against various pathogens and disease conditions. PMID:27246781

  16. Testing a structural model for viral DNA packaging motor function by optical tweezers measurements, site directed mutagenesis, and molecular dynamics calculations

    NASA Astrophysics Data System (ADS)

    Keller, Nicholas A.; Migliori, Amy D.; Arya, Gaurav; Rao, Venigalla B.; Smith, Douglas E.

    2013-09-01

    Many double-stranded DNA viruses employ a molecular motor to package DNA into preformed capsid shells. Based on structures of phage T4 motor proteins determined by X-ray crystallography and cryo-electron microscopy, Rao, Rossmann and coworkers recently proposed a structural model for motor function. They proposed that DNA is ratcheted by a large conformational change driven by electrostatic interactions between charged residues at an interface between two globular domains of the motor protein. We have conducted experiments to test this model by studying the effect on packaging under applied load of site-directed changes altering these residues. We observe significant impairment of packaging activity including reductions in packaging rate, percent time packaging, and time active under high load. We show that these measured impairments correlate well with alterations in free energies associated with the conformational change predicted by molecular dynamics simulations.

  17. Molecular insights of protein contour recognition with ligand pharmacophoric sites through combinatorial library design and MD simulation in validating HTLV-1 PR inhibitors.

    PubMed

    Selvaraj, Chandrabose; Omer, Ankur; Singh, Poonam; Singh, Sanjeev Kumar

    2015-01-01

    Retroviruses HIV-1 and HTLV-1 are chiefly considered to be the most dangerous pathogens in Homo sapiens. These two viruses have structurally unique protease (PR) enzymes, which are having common function of its replication mechanism. Though HIV PR drugs failed to inhibit HTLV-1 infections, they emphatically emphasise the need for designing new lead compounds against HTLV-1 PR. Therefore, we tried to understand the binding level interactions through the charge environment present in both ligand and protein active sites. The domino effect illustrates that libraries of purvalanol-A are attuned to fill allosteric binding site of HTLV-1 PR through molecular recognition and shows proper binding of ligand pharmacophoric features in receptor contours. Our screening evaluates seven compounds from purvalanol-A libraries, and these compounds' pharmacophore searches for an appropriate place in the binding site and it places well according to respective receptor contour surfaces. Thus our result provides a platform for the progress of more effective compounds, which are better in free energy calculation, molecular docking, ADME and molecular dynamics studies. Finally, this research provided novel chemical scaffolds for HTLV-1 drug discovery. PMID:25335799

  18. Ligand-dependent dynamics of the active-site lid in bacterial dimethylarginine dimethylaminohydrolase.

    PubMed

    Rasheed, Masooma; Richter, Christine; Chisty, Liisa T; Kirkpatrick, John; Blackledge, Martin; Webb, Martin R; Driscoll, Paul C

    2014-02-18

    The dimethylarginine dimethylaminohydrolase (DDAH) enzyme family has been the subject of substantial investigation as a potential therapeutic target for the regulation of vascular tension. DDAH enzymes catalyze the conversion of asymmetric N(η),N(η)-dimethylarginine (ADMA) to l-citrulline. Here the influence of substrate and product binding on the dynamic flexibility of DDAH from Pseudomonas aeruginosa (PaDDAH) has been assessed. A combination of heteronuclear NMR spectroscopy, static and time-resolved fluorescence measurements, and atomistic molecular dynamics simulations was employed. A monodisperse monomeric variant of the wild-type enzyme binds the reaction product l-citrulline with a low millimolar dissociation constant. A second variant, engineered to be catalytically inactive by substitution of the nucleophilic Cys249 residue with serine, can still convert the substrate ADMA to products very slowly. This PaDDAH variant also binds l-citrulline, but with a low micromolar dissociation constant. NMR and molecular dynamics simulations indicate that the active site "lid", formed by residues Gly17-Asp27, exhibits a high degree of internal motion on the picosecond-to-nanosecond time scale. This suggests that the lid is open in the apo state and allows substrate access to the active site that is otherwise buried. l-Citrulline binding to both protein variants is accompanied by an ordering of the lid. Modification of PaDDAH with a coumarin fluorescence reporter allowed measurement of the kinetic mechanism of the PaDDAH reaction. A combination of NMR and kinetic data shows that the catalytic turnover of the enzyme is not limited by release of the l-citrulline product. The potential to develop the coumarin-PaDDAH adduct as an l-citrulline sensor is discussed. PMID:24484052

  19. Multiple, Ligand-Dependent Routes from the Active Site of Cytochrome P450 2C9

    SciTech Connect

    Cojocaru, Vlad; Winn, Peter J.; Wade, Rebecca C.

    2012-02-13

    The active site of liver-specific, drug-metabolizing cytochrome P450 (CYP) monooxygenases is deeply buried in the protein and is connected to the protein surface through multiple tunnels, many of which were found open in different CYP crystal structures. It has been shown that different tunnels could serve as ligand passage routes in different CYPs. However, it is not understood whether one CYP uses multiple routes for substrate access and product release and whether these routes depend on ligand properties. From 300 ns of molecular dynamics simulations of CYP2C9, the second most abundant CYP in the human liver we found four main ligand exit routes, the occurrence of each depending on the ligand type and the conformation of the F-G loop, which is likely to be affected by the CYP-membrane interaction. A non-helical F-G loop favored exit towards the putative membrane-embedded region. Important protein features that direct ligand exit include aromatic residues that divide the active site and whose motions control access to two pathways. The ligands interacted with positively charged residues on the protein surface through hydrogen bonds that appear to select for acidic substrates. The observation of multiple, ligand-dependent routes in a CYP aids understanding of how CYP mutations affect drug metabolism and provides new possibilities for CYP inhibition.

  20. The role of active site aromatic residues in substrate degradation by the human chitotriosidase.

    PubMed

    Eide, Kristine Bistrup; Stockinger, Linn Wilhelmsen; Lewin, Anna Sofia; Tøndervik, Anne; Eijsink, Vincent G H; Sørlie, Morten

    2016-02-01

    Human chitotriosidase (HCHT) is a glycoside hydrolase family 18 chitinase synthesized and secreted in human macrophages thought be an innate part of the human immune system. It consists of a catalytic domain with the (β/α)8 TIM barrel fold having a large area of solvent-exposed aromatic amino acids in the active site and an additional family 14 carbohydrate-binding module. To gain further insight into enzyme functionality, especially the effect of the active site aromatic residues, we expressed two variants with mutations in subsites on either side of the catalytic acid, subsite -3 (W31A) and +2 (W218A), and compared their catalytic properties on chitin and high molecular weight chitosans. Exchange of Trp to Ala in subsite -3 resulted in a 12-fold reduction in extent of degradation and a 20-fold reduction in kcat(app) on chitin, while the values are 5-fold and 10-fold for subsite +2. Moreover, aromatic residue mutation resulted in a decrease of the rate of chitosan degradation contrasting previous observations for bacterial family 18 chitinases. Interestingly, the presence of product polymers of 40 sugar moieties and higher starts to disappear already at 8% degradation for HCHT50-W31A. Such behavior contrast that of the wild type and HCHT-W218A and resembles the action of endo-nonprocessive chitinases. PMID:26621384

  1. Leaving Group Ability Observably Affects Transition State Structure in a Single Enzyme Active Site.

    PubMed

    Roston, Daniel; Demapan, Darren; Cui, Qiang

    2016-06-15

    A reaction's transition state (TS) structure plays a critical role in determining reactivity and has important implications for the design of catalysts, drugs, and other applications. Here, we explore TS structure in the enzyme alkaline phosphatase using hybrid Quantum Mechanics/Molecular Mechanics simulations. We find that minor perturbations to the substrate have major effects on TS structure and the way the enzyme stabilizes the TS. Substrates with good leaving groups (LGs) have little cleavage of the phosphorus-LG bond at the TS, while substrates with poor LGs have substantial cleavage of that bond. The results predict nonlinear free energy relationships for a single rate-determining step, and substantial differences in kinetic isotope effects for different substrates; both trends were observed in previous experimental studies, although the original interpretations differed from the present model. Moreover, due to different degrees of phosphorus-LG bond cleavage at the TS for different substrates, the LG is stabilized by different interactions at the TS: while a poor LG is directly stabilized by an active site zinc ion, a good LG is mainly stabilized by active site water molecules. Our results demonstrate the considerable plasticity of TS structure and stabilization in enzymes. Furthermore, perturbations to reactivity that probe TS structure experimentally (i.e., substituent effects) may substantially perturb the TS they aim to probe, and thus classical experimental approaches such as free energy relations should be interpreted with care. PMID:27186960

  2. Similarities in the HIV-1 and ASV Integrease Active Site Upon Metal Binding

    SciTech Connect

    Lins, Roberto D.; Straatsma, TP; Briggs, J. M.

    2000-04-05

    The HIV-1 integrase, which is essential for viral replication, catalyzes the insertion of viral DNA into the host chromosome thereby recruiting host cell machinery into making viral proteins. It represents the third main HIV enzyme target for inhibitor design, the first two being the reverse transcriptase and the protease. We report here a fully hydrated 2 ns molecular dynamics simulation performed using parallel NWChem3.2.1 with the AMBER95 force field. The HIV-1 integrase catalytic domain previously determined by crystallography (1B9D) and modeling including two Mg2+ ions placed into the active site based on an alignment against an ASV integrase structure containing two divalent metals (1VSH), was used as the starting structure. The simulation reveals a high degree of flexibility in the region of residues 140-149 even in the presence of a second divalent metal ion and a dramatic conformational change of the side chain of E152 when the second metal ion is present. This study shows similarities in the behavior of the catalytic residues in the HIV-1 and ASV integrases upon metal binding. The present simulation also provides support to the hypothesis that the second metal ion is likely to be carried into the HIV-1 integrase active site by the substrate, a strand of DNA.

  3. Characterization of the Dielectric Constant in the Trichoderma reesei Cel7B Active Site.

    PubMed

    Song, Xiangfei; Wang, Yefei; Zhang, Shujun; Yan, Shihai; Li, Tong; Yao, Lishan

    2015-07-27

    An attempt is made to evaluate the dielectric constant of the Trichoderma reesei Cel7B active site. Through kinetic measurements, the pKa value of the catalytic acid E201 is determined. Mutations (away from E201) with net charge changes are introduced to perturb the E201 pKa. It is shown that the mutation with a +1 charge change (including G225R, G230R, and A335R) decreases the pKa of E201, whereas the mutation with a -1 charge change (including Q149E, A222D, G225D, and G230D) increases the pKa. This effect is consistent with the electrostatic interaction between the changed charge and the E201 side chain. The fitting of the experimental data yields an apparent dielectric constant of 25-80. Molecular dynamics simulations with explicit water molecules indicate that the high solvent accessibility of the active site contributes largely to the high dielectric constant. ONIOM calculations show that high dielectric constant benefits the catalysis through decreasing the energy of the transition state relative to that of the enzyme substrate complex. PMID:26114648

  4. Theoretical study of nitrodibenzofurans: A possible relationship between molecular properties and mutagenic activity.

    PubMed

    Stanković, B; Ostojić, B D; Popović, A; Gruden, M А; Đorđević, D S

    2016-11-15

    In this study we present a theoretical investigation of the molecular properties of nitrodibenzofurans (NDFs) and dinitrodibenzofurans (DNDFs) and their relation to mutagenic activity. Equilibrium geometries, relative energies, vertical ionization potentials (IP), vertical electron activities (EA), electronic dipole polarizabilities, and dipole moments of all NDFs and three DNDFs calculated by Density Functional Theory (DFT) methods are reported. The Ziegler/Rauk Energy Decomposition Analysis (EDA) is employed for a direct estimate of the variations of the orbital interaction and steric repulsion terms corresponding to the nitro group and the oxygen of the central ring of NDFs. The results indicate differences among NDF isomers for the cleavage of the related bonds and steric effects in the active site. The results show a good linear relationship between polarizability (<α>), anisotropy of polarizability (Δα), the summation of IR intensities (ΣIIR) and the summation of Raman activities (ΣARaman) over all 3N-6 vibrational modes and experimental mutagenic activities of NDF isomers in Salmonella typhimurium TA98 strain. The polarizability changes with respect to the νsNO+CN vibrational mode are in correlation with the mutagenic activities of NDFs and suggest that intermolecular interactions are favoured along this coordinate. PMID:27475460

  5. Molecular and biochemical characterizations of a new low-temperature active mannanase.

    PubMed

    Zhang, Rui; Zhou, Junpei; Gao, Yajie; Guan, Yaping; Li, Junjun; Tang, Xianghua; Xu, Bo; Ding, Junmei; Huang, Zunxi

    2015-11-01

    A mannanase-coding gene was cloned from Sphingobacterium sp. GN25 isolated from the feces of Grus nigricollis. The gene encodes a 371-residue polypeptide (ManAGN25) showing less than 74 % identity with a number of hypothetical proteins and putative glucanases and mannanases. Before experiment's performance, ManAGN25 was predicted to be a low-temperature active mannanase based on the molecular characterization, including (1) ManAGN25 shared the highest identity of 41.1 % with the experimentally verified low-temperature active mannanase (ManAJB13) from Sphingomonas sp. JB13; (2) compared with their mesophilic and thermophilic counterparts, ManAGN25 and ManAJB13 had increased number of amino acid residues around their catalytic sites; (3) these increased number of amino acid residues built longer loops, more α-helices, and larger total accessible surface area and packing volume. Then the experiments of biochemical characterization verified that the purified recombinant ManAGN25 is a low-temperature active mannanase: the enzyme showed apparently optimal activity at 35-40 °C and retained 78.2, 44.8, and 15.0 % of its maximum activity when assayed at 30, 20, and 10 °C, respectively; the half-life of the enzyme was approximately 60 min at 37 °C; the enzyme presented a K m of 4.2 mg/ml and a k cat of 0.4/s in McIlvaine buffer (pH 7.0) at 35 °C using locust bean gum as the substrate; and the activation energy for hydrolysis of locust bean gum by the enzyme was 36.0 kJ/mol. This study is the first to report the molecular and biochemical characterizations of a mannanase from a strain. PMID:25868895

  6. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

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

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  8. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  9. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

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

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  11. Noradrenergic Regulation of Glial Activation: Molecular Mechanisms and Therapeutic Implications

    PubMed Central

    Braun, David; Madrigal, Jose L.M; Feinstein, Douglas L

    2014-01-01

    It has been known for many years that the endogenous neurotransmitter noradrenaline (NA) exerts anti-inflammatory and neuroprotective effects both in vitro and in vivo. In many cases the site of action of NA are beta-adrenergic receptors (βARs), causing an increase in intracellular levels of cAMP which initiates a broad cascade of events including suppression of inflammatory transcription factor activities, alterations in nuclear localization of proteins, and induction of patterns of gene expression mediated through activity of the CREB transcription factor. These changes lead not only to reduced inflammatory events, but also contribute to neuroprotective actions of NA by increasing expression of neurotrophic substances including BDNF, GDNF, and NGF. These properties have prompted studies to determine if treatments with drugs to raise CNS NA levels could provide benefit in various neurological conditions and diseases having an inflammatory component. Moreover, increasing evidence shows that disruptions in endogenous NA levels occurs in several diseases and conditions including Alzheimer’s disease (AD), Parkinson’s disease (PD), Down’s syndrome, posttraumatic stress disorder (PTSD), and multiple sclerosis (MS), suggesting that damage to NA producing neurons is a common factor that contributes to the initiation or progression of neuropathology. Methods to increase NA levels, or to reduce damage to noradrenergic neurons, therefore represent potential preventative as well as therapeutic approaches to disease. PMID:25342942

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

    SciTech Connect

    Ohori, Makoto; Kinoshita, Takayoshi; Yoshimura, Seiji; Warizaya, Masaichi; Nakajima, Hidenori . E-mail: hidenori.nakajima@jp.astellas.com; Miyake, Hiroshi

    2007-02-16

    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 TGF{beta}-induced AP-1-dependent luciferase expression with respective IC{sub 50} values of 0.08 and 0.05 {mu}M. 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 {alpha},{beta}-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 S{gamma} of ERK2 Cys166, hydrogen bonds with the backbone NH of Met108, N{zeta} of Lys114, backbone C=O of Ser153, N{delta}2 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 p38MAPK{alpha}/{beta}/{gamma}/{delta} 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.

  13. Various Molecular Species of Diacylglycerol Hydroperoxide Activate Human Neutrophils via PKC Activation

    PubMed Central

    Kambayashi, Yasuhiro; Takekoshi, Susumu; Tanino, Yutaka; Watanabe, Keiichi; Nakano, Minoru; Hitomi, Yoshiaki; Takigawa, Tomoko; Ogino, Keiki; Yamamoto, Yorihiro

    2007-01-01

    We have proposed that diacylglycerol hydroperoxide-induced unregulated signal transduction causes oxidative stress-related diseases. In this study, we investigated which molecular species of diacylglycerol hydroperoxide activated human peripheral neutrophils. All diacylglycerol hydroperoxides, diacylglycerol hydroxides, and diacyglycerols tested in the present study induced superoxide production by neutrophils. The ability to activate neutrophils among molecular species containing the same fatty acid composition was as follows; diacylglycerol hydroperoxide>diacylglycerol hydroxide≥diacylglycerol. The diacylglycerol hydroperoxide composed of linoleate was a stronger activator for neutrophils than that composed of arachidonate. 1-Palmitoyl-2-linoleoylglycerol hydroperoxide (PLG-OOH) was the strongest stimulator for neutrophils. We reconfirmed that PLG-OOH activated protein kinase C (PKC) in neutrophils. PLG-OOH induced the phosphorylation of p47phox, a substrate of PKC and a cytosolic component of NADPH oxidase, in neutrophils, as did N-formyl-methionyl-leucyl-phenylalanine or 4β-phorbol-12β-myristate-13α-acetate. Moreover, the time course of p47phox phosphorylation was comparable to that of superoxide production. These results suggest that PLG-OOH activated intracellular protein kinase C. PLG-OOH, produced via an uncontrolled process, can act as a biological second messenger to cause inflammatory disease from oxidative stress. PMID:18392102

  14. Antiaging activity of low molecular weight peptide from Paphia undulate

    NASA Astrophysics Data System (ADS)

    Chen, Xin; Cai, Bingna; Chen, Hua; Pan, Jianyu; Chen, Deke; Sun, Huili

    2013-05-01

    Low molecular weight peptide (LMWP) was prepared from clam Paphia undulate and its antiaging effect on D-galactose-induced acute aging in rats, aged Kunming mice, ultraviolet-exposed rats, and thermally injured rats was investigated. P. undulate flesh was homogenized and digested using papain under optimal conditions, then subjected to Sephadex G-25 chromatography to isolate the LMWP. Administration of LMWP significantly reversed D-galactose-induced oxidative stress by increasing the activities of glutathione peroxidase (GPx) and catalase (CAT), and by decreasing the level of malondialdehyde (MDA). This process was accompanied by increased collagen synthesis. The LMWP prevented photoaging and promoted dermis recovery and remission of elastic fiber hyperplasia. Furthermore, treatment with the LMWP helped to regenerate elastic fibers and the collagen network, increased superoxide dismutase (SOD) in the serum and significantly decreased MDA. Thermal scald-induced inflammation and edema were also relieved by the LWMP, while wound healing in skin was promoted. These results suggest that the LMWP from P. undulate could serve as a new antiaging substance in cosmetics.

  15. Molecularly imprinted hydrogels as functional active packaging materials.

    PubMed

    Benito-Peña, Elena; González-Vallejo, Victoria; Rico-Yuste, Alberto; Barbosa-Pereira, Letricia; Cruz, José Manuel; Bilbao, Ainhoa; Alvarez-Lorenzo, Carmen; Moreno-Bondi, María Cruz

    2016-01-01

    This paper describes the synthesis of novel molecularly imprinted hydrogels (MIHs) for the natural antioxidant ferulic acid (FA), and their application as packaging materials to prevent lipid oxidation of butter. A library of MIHs was synthesized using a synthetic surrogate of FA, 3-(4-hydroxy-3-methoxyphenyl)propionic acid (HFA), as template molecule, ethyleneglycol dimethacrylate (EDMA) as cross-linker, and 1-allylpiperazine (1-ALPP) or 2-(dimethylamino)ethyl methacrylate (DMAEMA), in combination with 2-hydroxyethyl methacrylate (HEMA) as functional monomers, at different molar concentrations. The DMAEMA/HEMA-based MIHs showed the greatest FA loading capacity, while the 1-ALLP/HEMA-based polymers exhibited the highest imprinting effect. During cold storage, FA-loaded MIHs protected butter from oxidation and led to TBARs values that were approximately half those of butter stored without protection and 25% less than those recorded for butter covered with hydrogels without FA, potentially extending the shelf life of butter. Active packaging is a new field of application for MIHs with great potential in the food industry. PMID:26213001

  16. Active-site mutagenesis of tetanus neurotoxin implicates TYR-375 and GLU-271 in metalloproteolytic activity.

    PubMed

    Rossetto, O; Caccin, P; Rigoni, M; Tonello, F; Bortoletto, N; Stevens, R C; Montecucco, C

    2001-08-01

    Tetanus neurotoxin (TeNT) blocks neurotransmitter release by cleaving VAMP/synaptobrevin, a membrane associated protein involved in synaptic vesicle fusion. Such activity is exerted by the N-terminal 50kDa domain of TeNT which is a zinc-dependent endopeptidase (TeNT-L-chain). Based on the three-dimensional structure of botulinum neurotoxin serotype A (BoNT/A) and serotype B (BoNT/B), two proteins closely related to TeNT, and on X-ray scattering studies of TeNT, we have designed mutations at two active site residues to probe their involvement in activity. The active site of metalloproteases is composed of a primary sphere of residues co-ordinating the zinc atom, and a secondary sphere of residues that determines proteolytic specificity and activity. Glu-261 and Glu-267 directly co-ordinates the zinc atom in BoNT/A and BoNT/B respectively and the corresponding residue of TeNT was replaced by Asp or by the non conservative residue Ala. Tyr-365 is 4.3A away from zinc in BoNT/A, and the corresponding residue of TeNT was replaced by Phe or by Ala. The purified mutants had CD, fluorescence and UV spectra closely similar to those of the wild-type molecule. The proteolytic activity of TeNT-Asp-271 (E271D) is similar to that of the native molecule, whereas that of TeNT-Phe-375 (Y375F) is lower than the control. Interestingly, the two Ala mutants are completely devoid of enzymatic activity. These results demonstrate that both Glu-271 and Tyr-375 are essential for the proteolytic activity of TeNT. PMID:11306125

  17. Molecular activities, biosynthesis and evolution of triterpenoid saponins.

    PubMed

    Augustin, Jörg M; Kuzina, Vera; Andersen, Sven B; Bak, Søren

    2011-04-01

    Saponins are bioactive compounds generally considered to be produced by plants to counteract pathogens and herbivores. Besides their role in plant defense, saponins are of growing interest for drug research as they are active constituents of several folk medicines and provide valuable pharmacological properties. Accordingly, much effort has been put into unraveling the modes of action of saponins, as well as in exploration of their potential for industrial processes and pharmacology. However, the exploitation of saponins for bioengineering crop plants with improved resistances against pests as well as circumvention of laborious and uneconomical extraction procedures for industrial production from plants is hampered by the lack of knowledge and availability of genes in saponin biosynthesis. Although the ability to produce saponins is rather widespread among plants, a complete synthetic pathway has not been elucidated in any single species. Current conceptions consider saponins to be derived from intermediates of the phytosterol pathway, and predominantly enzymes belonging to the multigene families of oxidosqualene cyclases (OSCs), cytochromes P450 (P450s) and family 1 UDP-glycosyltransferases (UGTs) are thought to be involved in their biosynthesis. Formation of unique structural features involves additional biosynthetical enzymes of diverse phylogenetic background. As an example of this, a serine carboxypeptidase-like acyltransferase (SCPL) was recently found to be involved in synthesis of triterpenoid saponins in oats. However, the total number of identified genes in saponin biosynthesis remains low as the complexity and diversity of these multigene families impede gene discovery based on sequence analysis and phylogeny. This review summarizes current knowledge of triterpenoid saponin biosynthesis in plants, molecular activities, evolutionary aspects and perspectives for further gene discovery. PMID:21333312

  18. GAS HYDRATES AT TWO SITES OF AN ACTIVE CONTINENTAL MARGIN.

    USGS Publications Warehouse

    Kvenvolden, K.A.

    1985-01-01

    Sediment containing gas hydrates from two distant Deep Sea Drilling Project sites (565 and 568), located about 670 km apart on the landward flank of the Middle America Trench, was studied to determine the geochemical conditions that characterize the occurrence of gas hydrates. Site 565 was located in the Pacific Ocean offshore the Nicoya Peninsula of Costa Rica in 3,111 m of water. The depth of the hole at this site was 328 m, and gas hydrates were recovered from 285 and 319 m. Site 568 was located about 670 km to the northwest offshore Guatemala in 2,031 m of water. At this site the hole penetrated to 418 m, and gas hydrates were encountered at 404 m.

  19. Dynamically Achieved Active Site Precision in Enzyme Catalysis

    PubMed Central

    2015-01-01

    Conspectus The grand challenge in enzymology is to define and understand all of the parameters that contribute to enzymes’ enormous rate accelerations. The property of hydrogen tunneling in enzyme reactions has moved the focus of research away from an exclusive focus on transition state stabilization toward the importance of the motions of the heavy atoms of the protein, a role for reduced barrier width in catalysis, and the sampling of a protein conformational landscape to achieve a family of protein substates that optimize enzyme–substrate interactions and beyond. This Account focuses on a thermophilic alcohol dehydrogenase for which the chemical step of hydride transfer is rate determining across a wide range of experimental conditions. The properties of the chemical coordinate have been probed using kinetic isotope effects, indicating a transition in behavior below 30 °C that distinguishes nonoptimal from optimal C–H activation. Further, the introduction of single site mutants has the impact of either enhancing or eliminating the temperature dependent transition in catalysis. Biophysical probes, which include time dependent hydrogen/deuterium exchange and fluorescent lifetimes and Stokes shifts, have also been pursued. These studies allow the correlation of spatially resolved transitions in protein motions with catalysis. It is now possible to define a long-range network of protein motions in ht-ADH that extends from a dimer interface to the substrate binding domain across to the cofactor binding domain, over a distance of ca. 30 Å. The ongoing challenge to obtaining spatial and temporal resolution of catalysis-linked protein motions is discussed. PMID:25539048

  20. Influence of protonation on substrate and inhibitor interactions at the active site of human monoamine oxidase-A.

    PubMed

    Zapata-Torres, Gerald; Fierro, Angelica; Miranda-Rojas, Sebastian; Guajardo, Carlos; Saez-Briones, Patricio; Salgado, J Cristian; Celis-Barros, Cristian

    2012-05-25

    Although substrate conversion mediated by human monoaminooxidase (hMAO) has been associated with the deprotonated state of their amine moiety, data regarding the influence of protonation on substrate binding at the active site are scarce. Thus, in order to assess protonation influence, steered molecular dynamics (SMD) runs were carried out. These simulations revealed that the protonated form of the substrate serotonin (5-HT) exhibited stronger interactions at the protein surface compared to the neutral form. The latter displayed stronger interactions in the active site cavity. These observations support the possible role of the deprotonated form in substrate conversion. Multigrid docking studies carried out to rationalize the role of 5-HT protonation in other sites besides the active site indicated two energetically favored docking sites for the protonated form of 5-HT on the enzyme surface. These sites seem to be interconnected with the substrate/inhibitor cavity, as revealed by the tunnels observed by means of CAVER program. pK(a) calculations in the surface loci pointed to Glu³²⁷, Asp³²⁸, His⁴⁸⁸, and Asp¹³² as candidates for a possible in situ deprotonation step. Docking analysis of a group of inhibitors (structurally related to substrates) showed further interactions with the same two docking access sites. Interestingly, the protonated/deprotonated amine moiety of almost all compounds attained different docking poses in the active site, none of them oriented to the flavin moiety, thus producing a more variable and less productive orientations to act as substrates. Our results highlight the role of deprotonation in facilitating substrate conversion and also might reflect the necessity of inhibitor molecules to adopt specific orientations to achieve enzyme inhibition. PMID:22540832

  1. Effect of location and filling of d-states on methane activation in single site Fe-based catalysts

    NASA Astrophysics Data System (ADS)

    Sahoo, Sanjubala; Reber, Arthur C.; Khanna, Shiv N.

    2016-09-01

    Theoretical studies on the activation of the C-H bond in methane by an Iron atom bound to four different sites on a silica model support indicate that the lowest activation barrier is found for the case when the Fe is bound to three exposed silicon sites. A molecular orbital analysis reveals that the transition state is stabilized by two filled 3d orbitals that mix with the HOMO and LUMO of methane respectively, indicating how the energy and occupation of the 3d orbitals determine the reaction barrier. The studies offer a strategy for identifying candidates with optimal electronic structure for maximizing C-H bond activation using non-precious metals.

  2. Activation of molecular oxygen and the nature of the active oxygen species for CO oxidation on oxide supported Au catalysts.

    PubMed

    Widmann, D; Behm, R J

    2014-03-18

    Although highly dispersed Au catalysts with Au nanoparticles (NPs) of a few nanometers in diameter are well-known for their high catalytic activity for several oxidation and reduction reactions already at rather low temperatures for almost 30 years, central aspects of the reaction mechanism are still unresolved. While most studies focused on the active site, the active Au species, and the effect of the support material, the most crucial step during oxidation reactions, the activation of molecular oxygen and the nature of the resulting active oxygen species (Oact), received more attention just recently. This is topic of this Account, which focuses on the formation, location, and nature of the Oact species present on metal oxide supported Au catalysts under typical reaction conditions, at room temperature and above. It is mainly based on quantitative temporal analysis of products (TAP) reactor measurements, which different from most spectroscopic techniques are able to detect and quantify these species even at the extremely low concentrations present under realistic reaction conditions. Different types of pulse experiments were performed, during which the highly dispersed, realistic powder catalysts are exposed to very low amounts of reactants, CO and/or O2, in order to form and reactively remove Oact species and gain information on their formation, nature, and the active site for Oact formation. Our investigations have shown that the active oxygen species for CO oxidation on Au/TiO2 for reaction at 80 °C and higher is a highly stable atomic species, which at 80 °C is formed only at the perimeter of the Au-oxide interface and whose reactive removal by CO is activated, but not its formation. From these findings, it is concluded that surface lattice oxygen represents the Oact species for the CO oxidation. Accordingly, the CO oxidation proceeds via a Au-assisted Mars-van Krevelen mechanism, during which surface lattice oxygen close to the Au NPs is removed by reaction

  3. Site-directed mutagenesis and molecular modelling studies show the role of Asp82 and cysteines in rat acylase 1, a member of the M20 family

    SciTech Connect

    Herga, Sameh; Brutus, Alexandre; Vitale, Rosa Maria; Miche, Helene; Perrier, Josette; Puigserver, Antoine; Scaloni, Andrea; Giardina, Thierry . E-mail: thierry.giardina@univ.u-3mrs.fr

    2005-05-06

    Acylase 1 from rat kidney catalyzes the hydrolysis of acyl-amino acids. Sequence alignment has shown that this enzyme belongs to the metalloprotein family M20. Site-directed mutagenesis experiments led to the identification of one functionally important amino acid residue located near one of the zinc coordinating residues, which play a critical role in the enzymatic activity. The D82N- and D82E-substituted forms showed no significant activity and very low activity, respectively, along with a loss of zinc coordination. Molecular modelling investigations indicated a putative role of D82 in ensuring a proper protonation of catalytic histidine. In addition, none of the five cysteine residues present in the rat kidney acylase 1 sequence seemed involved in the catalytic process: the loss of activity induced by the C294A substitution was probably due to a conformational change in the 3D structure.

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

    SciTech Connect

    Teese, G.D.

    1995-09-28

    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.

  5. Characterization of the Lipid-Binding Site of Equinatoxin II by NMR and Molecular Dynamics Simulation

    PubMed Central

    Weber, Daniel K.; Yao, Shenggen; Rojko, Nejc; Anderluh, Gregor; Lybrand, Terry P.; Downton, Matthew T.; Wagner, John; Separovic, Frances

    2015-01-01

    Equinatoxin II (EqtII) is a soluble, 20 kDa pore-forming protein toxin isolated from the sea anemone Actinia equina. Although pore formation has long been known to occur in distinct stages, including monomeric attachment to phospholipid membranes followed by detachment of the N-terminal helical domain and oligomerization into the final pore assembly, atomistic-level detail of the protein-lipid interactions underlying these events remains elusive. Using high-resolution solution state NMR of uniformly-15N-labeled EqtII at the critical micelle concentration of dodecylphosphocholine, we have mapped the lipid-binding site through chemical shift perturbations. Subsequent docking of an EqtII monomer onto a dodecylphosphocholine micelle, followed by 400 ns of all-atom molecular dynamics simulation, saw several high-occupancy lipid-binding pockets stabilized by cation-π, hydrogen bonding, and hydrophobic interactions; and stabilization of the loop housing the conserved arginine-glycine-aspartate motif. Additional simulation of EqtII with an N-acetyl sphingomyelin micelle, for which high-resolution NMR data cannot be obtained due to aggregate formation, revealed that sphingomyelin specificity might occur via hydrogen bonding to the 3-OH and 2-NH groups unique to the ceramide backbone by side chains of D109 and Y113; and main chains of P81 and W112. Furthermore, a binding pocket formed by K30, K77, and P81, proximate to the hinge region of the N-terminal helix, was identified and may be implicated in triggering pore formation. PMID:25902438

  6. Proteolytic Activation of the Human Epithelial Sodium Channel by Trypsin IV and Trypsin I Involves Distinct Cleavage Sites*

    PubMed Central

    Haerteis, Silke; Krappitz, Annabel; Krappitz, Matteus; Murphy, Jane E.; Bertog, Marko; Krueger, Bettina; Nacken, Regina; Chung, Hyunjae; Hollenberg, Morley D.; Knecht, Wolfgang; Bunnett, Nigel W.; Korbmacher, Christoph

    2014-01-01

    Proteolytic activation is a unique feature of the epithelial sodium channel (ENaC). However, the underlying molecular mechanisms and the physiologically relevant proteases remain to be identified. The serine protease trypsin I can activate ENaC in vitro but is unlikely to be the physiologically relevant activating protease in ENaC-expressing tissues in vivo. Herein, we investigated whether human trypsin IV, a form of trypsin that is co-expressed in several extrapancreatic epithelial cells with ENaC, can activate human ENaC. In Xenopus laevis oocytes, we monitored proteolytic activation of ENaC currents and the appearance of γENaC cleavage products at the cell surface. We demonstrated that trypsin IV and trypsin I can stimulate ENaC heterologously expressed in oocytes. ENaC cleavage and activation by trypsin IV but not by trypsin I required a critical cleavage site (Lys-189) in the extracellular domain of the γ-subunit. In contrast, channel activation by trypsin I was prevented by mutating three putative cleavage sites (Lys-168, Lys-170, and Arg-172) in addition to mutating previously described prostasin (RKRK178), plasmin (Lys-189), and neutrophil elastase (Val-182 and Val-193) sites. Moreover, we found that trypsin IV is expressed in human renal epithelial cells and can increase ENaC-mediated sodium transport in cultured human airway epithelial cells. Thus, trypsin IV may regulate ENaC function in epithelial tissues. Our results show, for the first time, that trypsin IV can stimulate ENaC and that trypsin IV and trypsin I activate ENaC by cleavage at distinct sites. The presence of distinct cleavage sites may be important for ENaC regulation by tissue-specific proteases. PMID:24841206

  7. Improving upon Nature: Active site remodeling produces highly efficient aldolase activity towards hydrophobic electrophilic substrates

    PubMed Central

    Cheriyan, Manoj; Toone, Eric J.; Fierke, Carol A.

    2012-01-01

    Substrate specificity of enzymes is frequently narrow and constrained by multiple interactions, limiting the use of natural enzymes in biocatalytic applications. Aldolases have important synthetic applications, but the usefulness of these enzymes is hampered by their narrow reactivity profile with unnatural substrates. To explore the determinants of substrate selectivity and alter the specificity of E. coli 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, we employed structure-based mutagenesis coupled with library screening of mutant enzymes localized to the bacterial periplasm. We identified two active site mutations (T161S/S184L) that work additively to enhance the substrate specificity of this aldolase to include catalysis of retro-aldol cleavage of (4S)-2-keto-4-hydroxy-4-(2′-pyridyl)butyrate (S-KHPB). These mutations improve the value of kcat/KMS-KHPB by >450-fold, resulting in a catalytic efficiency that is comparable to that of the wild-type enzyme with the natural substrate while retaining high stereoselectivity. Moreover, the value of kcatS-KHPB for this mutant enzyme, a parameter critical for biocatalytic applications, is 3-fold higher than the maximum value achieved by the natural aldolase with any substrate. This mutant also possesses high catalytic efficiency for the retro-aldol cleavage of the natural substrate, KDPG, and a >50-fold improved activity for cleavage of 2-keto-4-hydroxy-octonoate (KHO), a non-functionalized hydrophobic analog. These data suggest a substrate binding mode that illuminates the origin of facial selectivity in aldol addition reactions catalyzed by KDPG and 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolases. Furthermore, targeting mutations to the active site provides marked improvement in substrate selectivity, demonstrating that structure-guided active site mutagenesis combined with selection techniques can efficiently identify proteins with characteristics that compare favorably to naturally occurring enzymes. PMID

  8. Atomically-thin two-dimensional sheets for understanding active sites in catalysis.

    PubMed

    Sun, Yongfu; Gao, Shan; Lei, Fengcai; Xie, Yi

    2015-02-01

    Catalysis can speed up chemical reactions and it usually occurs on the low coordinated steps, edges, terraces, kinks and corner atoms that are often called "active sites". However, the atomic level interplay between active sites and catalytic activity is still an open question, owing to the large difference between idealized models and real catalysts. This stimulates us to pursue a suitable material model for studying the active sites-catalytic activity relationship, in which the atomically-thin two-dimensional sheets could serve as an ideal model, owing to their relatively simple type of active site and the ultrahigh fraction of active sites that are comparable to the overall atoms. In this tutorial review, we focus on the recent progress in disclosing the factors that affect the activity of reactive sites, including characterization of atomic coordination number, structural defects and disorder in ultrathin two-dimensional sheets by X-ray absorption fine structure spectroscopy, positron annihilation spectroscopy, electron spin resonance and high resolution transmission electron microscopy. Also, we overview their applications in CO catalytic oxidation, photocatalytic water splitting, electrocatalytic oxygen and hydrogen evolution reactions, and hence highlight the atomic level interplay among coordination number, structural defects/disorder, active sites and catalytic activity in the two-dimensional sheets with atomic thickness. Finally, we also present the major challenges and opportunities regarding the role of active sites in catalysis. We believe that this review provides critical insights for understanding the catalysis and hence helps to develop new catalysts with high catalytic activity. PMID:25382246

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

    PubMed

    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

  10. SET7/9 Catalytic Mutants Reveal the Role of Active Site Water Molecules in Lysine Multiple Methylation

    SciTech Connect

    Del Rizzo, Paul A.; Couture, Jean-François; Dirk, Lynnette M.A.; Strunk, Bethany S.; Roiko, Marijo S.; Brunzelle, Joseph S.; Houtz, Robert L.; Trievel, Raymond C.

    2010-11-15

    SET domain lysine methyltransferases (KMTs) methylate specific lysine residues in histone and non-histone substrates. These enzymes also display product specificity by catalyzing distinct degrees of methylation of the lysine {epsilon}-amino group. To elucidate the molecular mechanism underlying this specificity, we have characterized the Y245A and Y305F mutants of the human KMT SET7/9 (also known as KMT7) that alter its product specificity from a monomethyltransferase to a di- and a trimethyltransferase, respectively. Crystal structures of these mutants in complex with peptides bearing unmodified, mono-, di-, and trimethylated lysines illustrate the roles of active site water molecules in aligning the lysine {epsilon}-amino group for methyl transfer with S-adenosylmethionine. Displacement or dissociation of these solvent molecules enlarges the diameter of the active site, accommodating the increasing size of the methylated {epsilon}-amino group during successive methyl transfer reactions. Together, these results furnish new insights into the roles of active site water molecules in modulating lysine multiple methylation by SET domain KMTs and provide the first molecular snapshots of the mono-, di-, and trimethyl transfer reactions catalyzed by these enzymes.

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

    SciTech Connect

    Shaya, D.; Hahn, Bum-Soo; Bjerkan, Tonje Marita; Kim, Wan Seok; Park, Nam Young; Sim, Joon-Soo; Kim, Yeong-Shik; Cygler, M.

    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.

  12. Extension of polyphenolics by CWPO-C peroxidase mutant containing radical-robust surface active site.

    PubMed

    Pham, L T Mai; Kim, S Jin; Ahn, U Suk; Choi, J Weon; Song, B Keun; Kim, Y Hwan

    2014-01-01

    Expressed as insoluble forms in Escherichia coli, native cationic cell wall peroxidase (CWPO-C) from the poplar tree and mutant variants were successfully reactivated via refolding experiments and used to elucidate the previously presumed existence of an electron transfer (ET) pathway in the CWPO-C structure. Their catalytic properties were fully characterized through various analyses including steady-state kinetic, direct oxidation of lignin macromolecules and their respective stabilities during the polymerization reactions. The analysis results proved that the 74th residue on the CWPO-C surface plays an important role in catalyzing the macromolecules via supposed ET mechanism. By comparing the residual activities of wild-type CWPO-C and mutant 74W CWPO-C after 3 min, mutation of tyrosine 74 residue to tryptophan increased the radical resistance of peroxidase up to ten times dramatically while maintaining its capability to oxidize lignin macromolecules. Furthermore, extension of poly(catechin) as well as lignin macromolecules with CWPO-C Y74W mutant clearly showed that this radical-resistant peroxidase mutant can increase the molecular weight of various kinds of polyphenolics by using surface-located active site. The anti-oxidation activity of the synthesized poly(catechin) was confirmed by xanthine oxidase assay. The elucidation of a uniquely catalytic mechanism in CWPO-C may improve the applicability of the peroxidase/H2O2 catalyst to green polymer chemistry. PMID:24122664

  13. Hydrogen production by the naked active site of the di-iron hydrogenases in water.

    PubMed

    Zipoli, Federico; Car, Roberto; Cohen, Morrel H; Selloni, Annabella

    2009-10-01

    We explored the reactivity of the active center of the [FeFe]-hydrogenases detached from the enzyme and immersed in acidified water by first-principles Car-Parrinello molecular-dynamics simulations. We focused on the identification of the structures that are stable and metastable in acidified water and on their activity for hydrogen production. Our calculations revealed that the naked active center could be an efficient catalyst provided that electrons are transferred to the cluster. We found that both bridging and terminal isomers are present at equilibrium and that the bridging configuration is essential for efficient hydrogen production. The formation of the hydrogen molecule occurs via sequential protonations of the distal iron and of the N-atom of the S-CH(2)-NH-CH(2)-S chelating group. H(2) desorption does not involve a significant energy barrier, making the process very efficient at room temperature. We established that the bottleneck in the reaction is the direct proton transfer from water to the vacant site of the distal iron. Moreover, we found that even if the terminal isomer is present at the equilibrium, its strong local hydrophobicity prevents poisoning of the cluster. PMID:19737003

  14. Actinobacterial diversity in limestone deposit sites in Hundung, Manipur (India) and their antimicrobial activities

    PubMed Central

    Nimaichand, Salam; Devi, Asem Mipeshwaree; Tamreihao, K.; Ningthoujam, Debananda S.; Li, Wen-Jun

    2015-01-01

    Studies on actinobacterial diversity in limestone habitats are scarce. This paper reports profiling of actinobacteria isolated from Hundung limestone samples in Manipur, India using ARDRA as the molecular tool for preliminary classification. A total of 137 actinobacteria were clustered into 31 phylotypic groups based on the ARDRA pattern generated and representative of each group was subjected to 16S rRNA gene sequencing. Generic diversity of the limestone isolates consisted of Streptomyces (15 phylotypic groups), Micromonospora (4), Amycolatopsis (3), Arthrobacter (3), Kitasatospora (2), Janibacter (1), Nocardia (1), Pseudonocardia (1) and Rhodococcus (1). Considering the antimicrobial potential of these actinobacteria, 19 showed antimicrobial activities against at least one of the bacterial and candidal test pathogens, while 45 exhibit biocontrol activities against at least one of the rice fungal pathogens. Out of the 137 actinobacterial isolates, 118 were found to have at least one of the three biosynthetic gene clusters (PKS-I, PKS-II, NRPS). The results indicate that 86% of the strains isolated from Hundung limestone deposit sites possessed biosynthetic gene clusters of which 40% exhibited antimicrobial activities. It can, therefore, be concluded that limestone habitat is a promising source for search of novel secondary metabolites. PMID:25999937

  15. Micro- and Nanostructured Materials for Active Devices and Molecular Electronics

    SciTech Connect

    Martin, Peter M.; Graff, Gordon L.; Gross, Mark E.; Burrows, Paul E.; Bennett, Wendy D.; Mast, Eric S.; Hall, Michael G.; Bonham, Charles C.; Zumhoff, Mac R.; Williford, Rick E.

    2003-10-01

    Traditional single layer barrier coatings are not adequate in preventing degradation of the performance of organic molecular electronic and other active devices. Most advanced devices used in display technology now consist of micro and nanostructured small molecule, polymer and inorganic coatings with thin high reactive group 1A metals. This includes organic electronics such as organic light emitting devices (OLED). The lifetimes of these devices rapidly degrades when they are exposed to atmospheric oxygen and water vapor. Thin film photovoltaics and batteries are also susceptible to degradation by moisture and oxygen. Using in-line coating techniques we apply a composite nanostructured inorganic/polymer thin film barrier that restricts moisture and oxygen permeation to undetectable levels using conventional permeation test equipment. We describe permeation mechanisms for this encapsulation coating and flat panel display and other device applications. Permeation through the multilayer barrier coating is defect and pore limited and can be described by Knudsen diffusion involving a long and tortuous path. Device lifetime is also enhanced by the long lag times required to reach the steady state flux regime. Permeation rates in the range of 10-6 cc,g/m2/d have been achieved and OLED device lifetimes. The structure is robust, yet flexible. The resulting device performance and lifetimes will also be described. The barrier film can be capped with a thin film of transparent conductive oxide yielding an engineered nanostructured device for next generation, rugged, lightweight or flexible displays. This enables, for the first time, thin film encapsulation of emissive organic displays.

  16. Photodynamic activation as a molecular switch to promote osteoblast cell differentiation via AP-1 activation.

    PubMed

    Kushibiki, Toshihiro; Tu, Yupeng; Abu-Yousif, Adnan O; Hasan, Tayyaba

    2015-01-01

    In photodynamic therapy (PDT), cells are impregnated with a photosensitizing agent that is activated by light irradiation, thereby photochemically generating reactive oxygen species (ROS). The amounts of ROS produced depends on the PDT dose and the nature of the photosensitizer. Although high levels of ROS are cytotoxic, at physiological levels they play a key role as second messengers in cellular signaling pathways, pluripotency, and differentiation of stem cells. To investigate further the use of photochemically triggered manipulation of such pathways, we exposed mouse osteoblast precursor cells and rat primary mesenchymal stromal cells to low-dose PDT. Our results demonstrate that low-dose PDT can promote osteoblast differentiation via the activation of activator protein-1 (AP-1). Although PDT has been used primarily as an anti-cancer therapy, the use of light as a photochemical "molecular switch" to promote differentiation should expand the utility of this method in basic research and clinical applications. PMID:26279470

  17. Photodynamic activation as a molecular switch to promote osteoblast cell differentiation via AP-1 activation

    PubMed Central

    Kushibiki, Toshihiro; Tu, Yupeng; Abu-Yousif, Adnan O.; Hasan, Tayyaba

    2015-01-01

    In photodynamic therapy (PDT), cells are impregnated with a photosensitizing agent that is activated by light irradiation, thereby photochemically generating reactive oxygen species (ROS). The amounts of ROS produced depends on the PDT dose and the nature of the photosensitizer. Although high levels of ROS are cytotoxic, at physiological levels they play a key role as second messengers in cellular signaling pathways, pluripotency, and differentiation of stem cells. To investigate further the use of photochemically triggered manipulation of such pathways, we exposed mouse osteoblast precursor cells and rat primary mesenchymal stromal cells to low-dose PDT. Our results demonstrate that low-dose PDT can promote osteoblast differentiation via the activation of activator protein-1 (AP-1). Although PDT has been used primarily as an anti-cancer therapy, the use of light as a photochemical “molecular switch” to promote differentiation should expand the utility of this method in basic research and clinical applications. PMID:26279470

  18. Dynamic formation of single-atom catalytic active sites on ceria-supported gold nanoparticles

    DOE PAGESBeta

    Wang, Yanggang; Mei, Donghai; Glezakou, Vassiliki Alexandra; Li, Jun; Rousseau, Roger J.

    2015-03-04

    Ab initio Molecular Dynamics simulations and static Density Functional Theory calculations have been performed to investigate the reaction mechanism of CO oxidation on Au/CeO2 catalyst. It is found that under reaction condition CO adsorption significantly labializes the surface atoms of the Au cluster and leads to the formation of isolated Au+-CO species that resides on the support in the vicinity of the Au particle. In this context, we identified a dynamic single-atom catalytic mechanism at the interfacial area for CO oxidation on Au/CeO2 catalyst, which is a lower energy pathway than that of CO oxidation at the interface with themore » metal particle. This results from the ability of the single atom site to strongly couple with the redox properties of the support in a synergistic manner thereby lowering the barrier for redox reactions. We find that the single Au+ ion, which only exists under reaction conditions, breaks away from the Au cluster to catalyze CO oxidation and returns to the Au cluster after the catalytic cycle is completed. Generally, our study highlights the importance of the dynamic creation of active sites under reaction conditions and their essential role in a catalytic process.« less

  19. Structure of recombinant Leishmania donovani pteridine reductase reveals a disordered active site

    PubMed Central

    Barrack, Keri L.; Tulloch, Lindsay B.; Burke, Lynsey-Ann; Fyfe, Paul K.; Hunter, William N.

    2011-01-01

    Pteridine reductase (PTR1) is a potential target for drug development against parasitic Trypanosoma and Leishmania species, protozoa that are responsible for a range of serious diseases found in tropical and subtropical parts of the world. As part of a structure-based approach to inhibitor development, specifically targeting Leishmania species, well ordered crystals of L. donovani PTR1 were sought to support the characterization of complexes formed with inhibitors. An efficient system for recombinant protein production was prepared and the enzyme was purified and crystallized in an orthorhombic form with ammonium sulfate as the precipitant. Diffraction data were measured to 2.5 Å resolution and the structure was solved by molecular replacement. However, a sulfate occupies a phosphate-binding site used by NADPH and occludes cofactor binding. The nicotinamide moiety is a critical component of the active site and without it this part of the structure is disordered. The crystal form obtained under these conditions is therefore unsuitable for the characterization of inhibitor complexes. PMID:21206018

  20. Dynamic formation of single-atom catalytic active sites on ceria-supported gold nanoparticles

    SciTech Connect

    Wang, Yanggang; Mei, Donghai; Glezakou, Vassiliki Alexandra; Li, Jun; Rousseau, Roger J.

    2015-03-04

    Ab initio Molecular Dynamics simulations and static Density Functional Theory calculations have been performed to investigate the reaction mechanism of CO oxidation on Au/CeO2 catalyst. It is found that under reaction condition CO adsorption significantly labializes the surface atoms of the Au cluster and leads to the formation of isolated Au+-CO species that resides on the support in the vicinity of the Au particle. In this context, we identified a dynamic single-atom catalytic mechanism at the interfacial area for CO oxidation on Au/CeO2 catalyst, which is a lower energy pathway than that of CO oxidation at the interface with the metal particle. This results from the ability of the single atom site to strongly couple with the redox properties of the support in a synergistic manner thereby lowering the barrier for redox reactions. We find that the single Au+ ion, which only exists under reaction conditions, breaks away from the Au cluster to catalyze CO oxidation and returns to the Au cluster after the catalytic cycle is completed. Generally, our study highlights the importance of the dynamic creation of active sites under reaction conditions and their essential role in a catalytic process.

  1. Dynamic formation of single-atom catalytic active sites on ceria-supported gold nanoparticles

    SciTech Connect

    Wang, Yanggang; Mei, Donghai; Glezakou, Vassiliki Alexandra; Li, Jun; Rousseau, Roger J.

    2015-03-04

    Ab initio Molecular Dynamics simulations and static Density Functional Theory calculations have been performed to investigate the reaction mechanism of CO oxidation on Au/CeO2 catalyst. It is found that under reaction condition CO adsorption significantly labializes the surface atoms of the Au cluster and leads to the formation of isolated Au+-CO species that resides on the support in the vicinity of the Au particle. In this context, we identified a dynamic single-atom catalytic mechanism at the interfacial area for CO oxidation on Au/CeO2 catalyst, which is a lower energy pathway than that of CO oxidation at the interface with the metal particle. This results from the ability of the single atom site to strongly couple with the redox properties of the support in a synergistic manner thereby lowering the barrier for redox reactions. We find that the single Au+ ion, which only exists under reaction conditions, breaks away from the Au cluster to catalyze CO oxidation and returns to the Au cluster after the catalytic cycle is completed. Generally, our study highlights the importance of the dynamic creation of active sites under reaction conditions and their essential role in a catalytic process.

  2. COMPUTER-ASSISTED STUDIES OF MOLECULAR STRUCTURE-BIOLOGICAL ACTIVITY RELATIONSHIPS

    EPA Science Inventory

    Computer-assisted methods can be used to investigate the relationships between the molecular structures of compounds and their biological activity. A number of approaches have been reported in the literature, including correlations of activity with substituent constants, conforma...

  3. Identification of promiscuous ene-reductase activity by mining structural databases using active site constellations

    PubMed Central

    Steinkellner, Georg; Gruber, Christian C.; Pavkov-Keller, Tea; Binter, Alexandra; Steiner, Kerstin; Winkler, Christoph; Łyskowski, Andrzej; Schwamberger, Orsolya; Oberer, Monika; Schwab, Helmut; Faber, Kurt; Macheroux, Peter; Gruber, Karl

    2014-01-01

    The exploitation of catalytic promiscuity and the application of de novo design have recently opened the access to novel, non-natural enzymatic activities. Here we describe a structural bioinformatic method for predicting catalytic activities of enzymes based on three-dimensional constellations of functional groups in active sites (‘catalophores’). As a proof-of-concept we identify two enzymes with predicted promiscuous ene-reductase activity (reduction of activated C–C double bonds) and compare them with known ene-reductases, that is, members of the Old Yellow Enzyme family. Despite completely different amino acid sequences, overall structures and protein folds, high-resolution crystal structures reveal equivalent binding modes of typical Old Yellow Enzyme substrates and ligands. Biochemical and biocatalytic data show that the two enzymes indeed possess ene-reductase activity and reveal an inverted stereopreference compared with Old Yellow Enzymes for some substrates. This method could thus be a tool for the identification of viable starting points for the development and engineering of novel biocatalysts. PMID:24954722

  4. Molecular Interface of S100A8 with Cytochrome b558 and NADPH Oxidase Activation

    PubMed Central

    Berthier, Sylvie; Hograindleur, Marc-André; Paclet, Marie-Hélène; Polack, Benoît; Morel, Françoise

    2012-01-01

    S100A8 and S100A9 are two calcium binding Myeloid Related Proteins, and important mediators of inflammatory diseases. They were recently introduced as partners for phagocyte NADPH oxidase regulation. However, the precise mechanism of their interaction remains elusive. We had for aim (i) to evaluate the impact of S100 proteins on NADPH oxidase activity; (ii) to characterize molecular interaction of either S100A8, S100A9, or S100A8/S100A9 heterocomplex with cytochrome b558; and (iii) to determine the S100A8 consensus site involved in cytochrome b558/S100 interface. Recombinant full length or S100A9-A8 truncated chimera proteins and ExoS-S100 fusion proteins were expressed in E. coli and in P. aeruginosa respectively. Our results showed that S100A8 is the functional partner for NADPH oxidase activation contrary to S100A9, however, the loading with calcium and a combination with phosphorylated S100A9 are essential in vivo. Endogenous S100A9 and S100A8 colocalize in differentiated and PMA stimulated PLB985 cells, with Nox2/gp91phox and p22phox. Recombinant S100A8, loaded with calcium and fused with the first 129 or 54 N-terminal amino acid residues of the P. aeruginosa ExoS toxin, induced a similar oxidase activation in vitro, to the one observed with S100A8 in the presence of S100A9 in vivo. This suggests that S100A8 is the essential component of the S100A9/S100A8 heterocomplex for oxidase activation. In this context, recombinant full-length rS100A9-A8 and rS100A9-A8 truncated 90 chimera proteins as opposed to rS100A9-A8 truncated 86 and rS100A9-A8 truncated 57 chimeras, activate the NADPH oxidase function of purified cytochrome b558 suggesting that the C-terminal region of S100A8 is directly involved in the molecular interface with the hemoprotein. The data point to four strategic 87HEES90 amino acid residues of the S100A8 C-terminal sequence that are involved directly in the molecular interaction with cytochrome b558 and then in the phagocyte NADPH oxidase activation

  5. Selection and molecular characterization of cellulolytic-xylanolytic fungi from surface soil-biomass mixtures from Black Belt sites.

    PubMed

    Okeke, Benedict C; Hall, Rosine W; Nanjundaswamy, Ananda; Thomson, M Sue; Deravi, Yasaman; Sawyer, Leah; Prescott, Andrew

    2015-06-01

    Plant biomass is an abundant renewable natural resource that can be transformed into chemical feedstocks. Enzymes used in saccharification of lignocellulosic biomass are a major part of biofuel production costs. A cocktail of cellulolytic and xylanolytic enzymes are required for optimal saccharification of biomass. Accordingly, thirty-two fungal pure cultures were obtained from surface soil-biomass mixtures collected from Black Belt sites in Alabama by culturing on lignocellulosic biomass medium. The fungal strains were screened for the coproduction of cellulolytic and xylanolytic enzymes. Strains that displayed promising levels of cellulolytic and xylanolytic enzymes were characterized by molecular analysis of DNA sequences from the large subunit and internal transcribed spacer (ITS) of their ribosomal RNA gene. Nucleotide sequence analysis revealed that two most promising isolates FS22A and FS5A were most similar to Penicillium janthinellum and Trichoderma virens. Production dynamics of cellulolytic and xylanolytic enzymes from these two strains were explored in submerged fermentation. Volumetric productivity after 120 h incubation was 121.08 units/L/h and 348 units/L/h for the filter paper cellulase and xylanase of strain FS22A, and 90.83 units/L/h and 359 units/L/h, respectively for strain FS5A. Assays with 10 times dilution of enzymes revealed that the activities were much higher than that observed in the crude culture supernatant. Additionally, both FS22A and FS5A also produced amylase in lignocellulose medium. The enzyme profiles of these strains and their activities suggest potential applications in cost effective biomass conversion and biodegradation. PMID:25817459

  6. Laboratory Activities to Support Student Understanding of the Molecular Mechanisms of Mutation & Natural Selection

    ERIC Educational Resources Information Center

    Hubler, Tina; Adams, Patti; Scammell, Jonathan

    2015-01-01

    The molecular basis of evolution is an important and challenging concept for students to understand. In a previous article, we provided some of the scientific background necessary to teach this topic. This article features a series of laboratory activities demonstrating that molecular events can alter the genomes of organisms. These activities are…

  7. Benzimidazole-Based Quinazolines: In Vitro Evaluation, Quantitative Structure-Activity Relationship, and Molecular Modeling as Aurora Kinase Inhibitors.

    PubMed

    Sharma, Alka; Luxami, Vijay; Saxena, Sanjai; Paul, Kamaldeep

    2016-03-01

    A series of benzimidazole-based quinazoline derivatives with different substitutions of primary and secondary amines at the C2 position (1-12) were evaluated for their Aurora kinase inhibitory activities. All compounds except for 3 and 6 showed good activity against Aurora kinase inhibitors, with IC50 values in the range of 0.035-0.532 μM. The ligand efficiency (LE) of the compounds with Aurora A kinase was also determined. The structure-activity relationship and the quantitative structure-activity relationship revealed that the Aurora inhibitory activities of these derivatives primarily depend on the different substitutions of the amine present at the C2 position of the quinazoline core. Molecular docking studies in the active binding site also provided theoretical support for the experimental biological data acquired. The current study identifies a novel class of Aurora kinase inhibitors, which can further be used for the treatment of cancer. PMID:26773437

  8. A molecular characterization of the agonist binding site of a nematode cys-loop GABA receptor

    PubMed Central

    Kaji, Mark D; Kwaka, Ariel; Callanan, Micah K; Nusrat, Humza; Desaulniers, Jean-Paul; Forrester, Sean G

    2015-01-01

    Background and Purpose Cys-loop GABA receptors represent important targets for human chemotherapeutics and insecticides and are potential targets for novel anthelmintics (nematicides). However, compared with insect and mammalian receptors, little is known regarding the pharmacological characteristics of nematode Cys-loop GABA receptors. Here we have investigated the agonist binding site of the Cys-loop GABA receptor UNC-49 (Hco-UNC-49) from the parasitic nematode Haemonchus contortus. Experimental Approach We used two-electrode voltage-clamp electrophysiology to measure channel activation by classical GABA receptor agonists on Hco-UNC-49 expressed in Xenopus laevis oocytes, along with site-directed mutagenesis and in silico homology modelling. Key Results The sulphonated molecules P4S and taurine had no effect on Hco-UNC-49. Other classical Cys-loop GABAA receptor agonists tested on the Hco-UNC-49B/C heteromeric channel had a rank order efficacy of GABA > trans-4-aminocrotonic acid > isoguvacine > imidazole-4-acetic acid (IMA) > (R)-(−)-4-amino-3-hydroxybutyric acid [R(−)-GABOB] > (S)-(+)-4-amino-3-hydroxybutyric acid [S(+)-GABOB] > guanidinoacetic acid > isonipecotic acid > 5-aminovaleric acid (DAVA) (partial agonist) > β-alanine (partial agonist). In silico ligand docking revealed some variation in binding between agonists. Mutagenesis of a key serine residue in binding loop C to threonine had minimal effects on GABA and IMA but significantly increased the maximal response to DAVA and decreased twofold the EC50 for R(−)- and S(+)-GABOB. Conclusions and Implications The pharmacological profile of Hco-UNC-49 differed from that of vertebrate Cys-loop GABA receptors and insect resistance to dieldrin receptors, suggesting differences in the agonist binding pocket. These findings could be exploited to develop new drugs that specifically target GABA receptors of parasitic nematodes. PMID:25850584

  9. Structural mechanism of RuBisCO activation by carbamylation of the active site lysine

    PubMed Central

    Stec, Boguslaw

    2012-01-01

    Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a crucial enzyme in carbon fixation and the most abundant protein on earth. It has been studied extensively by biochemical and structural methods; however, the most essential activation step has not yet been described. Here, we describe the mechanistic details of Lys carbamylation that leads to RuBisCO activation by atmospheric CO2. We report two crystal structures of nitrosylated RuBisCO from the red algae Galdieria sulphuraria with O2 and CO2 bound at the active site. G. sulphuraria RuBisCO is inhibited by cysteine nitrosylation that results in trapping of these gaseous ligands. The structure with CO2 defines an elusive, preactivation complex that contains a metal cation Mg2+ surrounded by three H2O/OH molecules. Both structures suggest the mechanism for discriminating gaseous ligands by their quadrupole electric moments. We describe conformational changes that allow for intermittent binding of the metal ion required for activation. On the basis of these structures we propose the individual steps of the activation mechanism. Knowledge of all these elements is indispensable for engineering RuBisCO into a more efficient enzyme for crop enhancement or as a remedy to global warming. PMID:23112176

  10. Nature of Catalytic Active Sites Present on the Surface of Advanced Bulk Tantalum Mixed Oxide Photocatalysts

    SciTech Connect

    Phivilay, Somphonh; Puretzky, Alexander A; Domen, Kazunari Domen; Wachs, Israel

    2013-01-01

    The most active photocatalyst system for water splitting under UV irradiation (270 nm) is the promoted 0.2%NiO/NaTaO3:2%La photocatalyst with optimized photonic efficiency (P.E.) of 56%, but fundamental issues about the nature of the surface catalytic active sites and their involvement in the photocatalytic process still need to be clarified. This is the first study to apply cutting edge surface spectroscopic analyses to determine the surface nature of tantalum mixed oxide photocatalysts. Surface analysis with HR-XPS (1-3nm) and HS-LEIS (0.3nm) spectroscopy indicates that the NiO and La2O3 promoters are concentrated in the surface region of the bulk NaTaO3 phase. The La2O3 is concentrated on the NaTaO3 outermost surface layers while NiO is distributed throughout the NaTaO3 surface region (1-3nm). Raman and UV-vis spectroscopy revealed that the bulk molecular and electronic structures, respectively, of NaTaO3 were not modified by the addition of the La2O3 and NiO promoters, with La2O3 resulting in a slightly more ordered structure. Photoluminescence (PL) spectroscopy reveals that the addition of La2O3 and NiO produces a greater number of electron traps resulting in the suppression of the recombination of excited electrons/holes. In contrast to earlier reports, the La2O3 is only a textural promoter (increasing the BET surface area ~7x by stabilizing smaller NaTaO3 particles), but causes a ~3x decrease in the specific photocatalytic TORs ( mol H2/m2/h) rate because surface La2O3 blocks exposed catalytic active NaTaO3 sites. The NiO promoter was found to be a potent electronic promoter that enhances the NaTaO3 surface normalized TORs by a factor of ~10-50 and TOF by a factor of ~10. The level of NiO promotion is the same in the absence and presence of La2O3 demonstrating that there is no promotional synergistic interaction between the NiO and La2O3 promoters. This study demonstrates the important contributions of the photocatalyst surface properties to the fundamental

  11. Synthetic Molecular Machines for Active Self-Assembly: Prototype Algorithms, Designs, and Experimental Study

    NASA Astrophysics Data System (ADS)

    Dabby, Nadine L.

    behaviors. This class of behaviors includes any behavior where a passive physical system simply does not have enough physical energy to perform the specified tasks in the requisite amount of time. As we will demonstrate and prove, a sufficiently expressive implementation of an "active" molecular self-assembly approach can achieve these behaviors. Using an external source of fuel solves part of the problem, so the system is not "energetically incomplete." But the programmable system also needs to have sufficient expressive power to achieve the specified behaviors. Perhaps surprisingly, some of these systems do not even require Turing completeness to be sufficiently expressive. Building on a large variety of work by other scientists in the fields of DNA nanotechnology, chemistry and reconfigurable robotics, this thesis introduces several research contributions in the context of active self-assembly. We show that simple primitives such as insertion and deletion are able to generate complex and interesting results such as the growth of a linear polymer in logarithmic time and the ability of a linear polymer to treadmill. To this end we developed a formal model for active-self assembly that is directly implementable with DNA molecules. We show that this model is computationally equivalent to a machine capable of producing strings that are stronger than regular languages and, at most, as strong as context-free grammars. This is a great advance in the theory of active self-assembly as prior models were either entirely theoretical or only implementable in the context of macro-scale robotics. We developed a chain reaction method for the autonomous exponential growth of a linear DNA polymer. Our method is based on the insertion of molecules into the assembly, which generates two new insertion sites for every initial one employed. The building of a line in logarithmic time is a first step toward building a shape in logarithmic time. We demonstrate the first construction of a synthetic

  12. Recombinant human betacellulin. Molecular structure, biological activities, and receptor interaction.

    PubMed

    Watanabe, T; Shintani, A; Nakata, M; Shing, Y; Folkman, J; Igarashi, K; Sasada, R

    1994-04-01

    Soluble forms of human betacellulin (BTC) were purified to homogeneity from the conditioned medium of mouse A9 cells transfected with the BTC precursor cDNA. Three types of soluble BTC, designated BTC-1a, BTC-1b and BTC-2, were resolved by cation-exchange and size-exclusion column chromatography. Physicochemical analysis has revealed that BTC-1a represents the glycosylated, intact molecule composed of 80 amino acid residues (Asp32 to Tyr111 of the precursor molecule). BTC-1b appears to be a truncated molecule lacking 12 amino acid residues from the amino terminus of BTC-1a. BTC-2 was found to be a 50-amino acid molecule (Arg62 to Tyr111) that corresponds to the epidermal growth factor (EGF) structural unit. The biological activities of these BTC molecules were essentially identical as judged by their mitogenicity on Balb/c 3T3 fibroblasts. BTC and EGF were equipotent in stimulating Balb/c 3T3 cell proliferation and rat mesangial cell Ca2+ mobilization as well as in inhibiting the growth of human epidermoid carcinoma A431 cells. BTC and EGF antagonized each other with similar dose dependence for binding to A431 cells, indicating that these factors bind the same receptor molecules with equivalent avidity. The Kd value of EGF receptor (EGFR) and BTC is 0.5 nM as determined on Balb/c 3T3 cells. In addition, human mammary carcinoma MDA-MB-453 cells, which express multiple members of the EGFR family, were found to possess 2.7 x 10(3) BTC binding sites/cell, and the binding was readily quenched by EGF. These results suggest that the primary receptor for BTC is EGFR. PMID:8144591

  13. 78 FR 33908 - Commercial Wind Lease Issuance and Site Assessment Activities on the Atlantic Outer Continental...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-05

    ... identified Wind Energy Area (WEA) on the OCS offshore Rhode Island (RI) and Massachusetts (MA). The revised... from leasing, site characterization, and site assessment in and around the Call Area (76 FR 51391). The... Bureau of Ocean Energy Management Commercial Wind Lease Issuance and Site Assessment Activities on...

  14. 77 FR 39508 - Commercial Wind Lease Issuance and Site Assessment Activities on the Atlantic Outer Continental...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-03

    ... specific project proposals on those leases) in an identified Wind Energy Area (WEA) on the OCS offshore..., site characterization, and site assessment in and around the Call Area (76 FR 51391). The Call Area is... Bureau of Ocean Energy Management Commercial Wind Lease Issuance and Site Assessment Activities on...

  15. Active Layer and Moisture Measurements for Intensive Site 0 and 1, Barrow, Alaska

    DOE Data Explorer

    John Peterson

    2015-04-17

    These are measurements of Active Layer Thickness collected along several lines beginning in September, 2011 to the present. The data were collected at several time periods along the Site0 L2 Line, the Site1 AB Line, and an ERT Monitoring Line near Area A in Site1.

  16. Molecular pharmacology of hepsulfam, NSC 3296801: identification of alkylated nucleosides, alkylation site, and site of DNA cross-linking.

    PubMed

    Streeper, R T; Cotter, R J; Colvin, M E; Hilton, J; Colvin, O M

    1995-04-01

    We have determined that hepsulfam, in common with its structural homologue busulfan, alkylates both free guanosine and GMP in DNA at the 7 nitrogen. Mass spectral analysis of the products of the reaction of hepsulfam with guanosine has identified the mono- and bis-alkylated guanosine adducts. UV spectrophotometry and mass spectrometry were used to confirm that alkylation occurred at the 7 nitrogen by following the formation of the formamidopyrimidyl form of the hepsulfam-guanosine adduct at high pH. We have also isolated and identified 1-guanyl,7-hydroxyheptane, 1-guanyl,7-sulfamylheptane, and 1,7-bis(guanyl)heptane from in vitro reaction mixtures of hepsulfam and calf thymus DNA. We have isolated bis-(7-formamidopyrimidyldeoxyguanosinyl)-heptane from an enzymatic digest of DNA treated with hepsulfam. Finally, we have found that hepsulfam forms interstrand cross-links at 5'-GXC-3' sites in model oligonucleotides. PMID:7882358

  17. Molecular exploration of the α(1A)-adrenoceptor orthosteric site: binding site definition for epinephrine, HEAT and prazosin.

    PubMed

    Maïga, Arhamatoulaye; Dupont, Mélanie; Blanchet, Guillaume; Marcon, Elodie; Gilquin, Bernard; Servent, Denis; Gilles, Nicolas

    2014-12-20

    Despite the physiological and pharmacological importance of the α1A-adrenoreceptor, the mode of interactions of classical agonists and radioactive ligands with this receptor is not yet clearly defined. Here, we used mutagenesis studies and binding experiments to evaluate the importance of 11 receptor sites for the binding of (125)I-HEAT, (3)H-prazosin and epinephrine. Only one residue (F312) commonly interacts with the three molecules, and, surprisingly, D106 interacts only with epinephrine in a moderate way. Our docking model shows that prazosin and HEAT are almost superimposed into the orthosteric pocket with their tetralone and quinazoline rings close to the phenyl ring of the agonist. PMID:25447534

  18. Nuclear Site Security in the Event of Terrorist Activity

    SciTech Connect

    Thomson, M.L.; Sims, J.

    2008-07-01

    This paper, presented as a poster, identifies why ballistic protection should now be considered at nuclear sites to counter terrorist threats. A proven and flexible form of multi purpose protection is described in detail with identification of trial results that show its suitability for this role. (authors)

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

    SciTech Connect

    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.

  20. Hydride binding to the active site of [FeFe]-hydrogenase.

    PubMed

    Chernev, Petko; Lambertz, Camilla; Brünje, Annika; Leidel, Nils; Sigfridsson, Kajsa G V; Kositzki, Ramona; Hsieh, Chung-Hung; Yao, Shenglai; Schiwon, Rafael; Driess, Matthias; Limberg, Christian; Happe, Thomas; Haumann, Michael

    2014-11-17

    [FeFe]-hydrogenase from green algae (HydA1) is the most efficient hydrogen (H2) producing enzyme in nature and of prime interest for (bio)technology. Its active site is a unique six-iron center (H-cluster) composed of a cubane cluster, [4Fe4S]H, cysteine-linked to a diiron unit, [2Fe]H, which carries unusual carbon monoxide (CO) and cyanide ligands and a bridging azadithiolate group. We have probed the molecular and electronic configurations of the H-cluster in functional oxidized, reduced, and super-reduced or CO-inhibited HydA1 protein, in particular searching for intermediates with iron-hydride bonds. Site-selective X-ray absorption and emission spectroscopy were used to distinguish between low- and high-spin iron sites in the two subcomplexes of the H-cluster. The experimental methods and spectral simulations were calibrated using synthetic model complexes with ligand variations and bound hydride species. Distinct X-ray spectroscopic signatures of electronic excitation or decay transitions in [4Fe4S]H and [2Fe]H were obtained, which were quantitatively reproduced by density functional theory calculations, thereby leading to specific H-cluster model structures. We show that iron-hydride bonds are absent in the reduced state, whereas only in the super-reduced state, ligand rotation facilitates hydride binding presumably to the Fe-Fe bridging position at [2Fe]H. These results are in agreement with a catalytic cycle involving three main intermediates and at least two protonation and electron transfer steps prior to the H2 formation chemistry in [FeFe]-hydrogenases. PMID:25369169

  1. Blogs and Social Network Sites as Activity Systems: Exploring Adult Informal Learning Process through Activity Theory Framework

    ERIC Educational Resources Information Center

    Heo, Gyeong Mi; Lee, Romee

    2013-01-01

    This paper uses an Activity Theory framework to explore adult user activities and informal learning processes as reflected in their blogs and social network sites (SNS). Using the assumption that a web-based space is an activity system in which learning occurs, typical features of the components were investigated and each activity system then…

  2. Interplay of the Bacterial Ribosomal A-Site, S12 Protein Mutations and Paromomycin Binding: A Molecular Dynamics Study

    PubMed Central

    Panecka, Joanna; Mura, Cameron; Trylska, Joanna

    2014-01-01

    The conformational properties of the aminoacyl-tRNA binding site (A-site), and its surroundings in the Escherichia coli 30S ribosomal subunit, are of great relevance in designing antibacterial agents. The 30S subunit A-site is near ribosomal protein S12, which neighbors helices h27 and H69; this latter helix, of the 50S subunit, is a functionally important component of an intersubunit bridge. Experimental work has shown that specific point mutations in S12 (K42A, R53A) yield hyper-accurate ribosomes, which in turn confers resistance to the antibiotic ‘paromomycin’ (even when this aminoglycoside is bound to the A-site). Suspecting that these effects can be elucidated in terms of the local atomic interactions and detailed dynamics of this region of the bacterial ribosome, we have used molecular dynamics simulations to explore the motion of a fragment of the E. coli ribosome, including the A-site. We found that the ribosomal regions surrounding the A-site modify the conformational space of the flexible A-site adenines 1492/93. Specifically, we found that A-site mobility is affected by stacking interactions between adenines A1493 and A1913, and by contacts between A1492 and a flexible side-chain (K43) from the S12 protein. In addition, our simulations reveal possible indirect pathways by which the R53A and K42A mutations in S12 are coupled to the dynamical properties of the A-site. Our work extends what is known about the atomistic dynamics of the A-site, and suggests possible links between the biological effects of hyper-accurate mutations in the S12 protein and conformational properties of the ribosome; the implications for S12 dynamics help elucidate how the miscoding effects of paromomycin may be evaded in antibiotic-resistant mutants of the bacterial ribosome. PMID:25379961

  3. The drug binding sites and transport mechanism of the RND pumps from Mycobacterium tuberculosis: Insights from molecular dynamics simulations.

    PubMed

    Sandhu, Padmani; Akhter, Yusuf

    2016-02-15

    RND permease superfamily drug efflux pumps are involved in multidrug transport and are attractive to study them for therapeutic purpose. In previous work we have classified 14 members of MmpL proteins belong to RND superfamily from Mycobacterium tuberculosis (Mtb) within its families [Sandhu P. and Akhter Y., 2015. Int. J. Med. Microbiol., 305:413-423]. In this study, structures of these proteins are homology modelled. The drug binding sites and channels are identified using local micro-stereochemistry and charge densities. Potential transport mechanism based on differential structural behaviour in the absence and on the binding of drug molecules is explained using the molecular dynamics simulation results. Our studies show two potential drug binding sites positioned at opposite ends of the transport tunnel leading from cytoplasmic to the periplasmic space across MmpL5 trimer. The drug binding have effects on the structural conformation of the protein leading to molecular-scale peristaltic movements. The free binding energy calculations reveal that the subsequent binding events are interdependent and may have implications on transport mechanism. Two drug binding sites and a continuous channel in the RND pump have been reported. The proposed ligand binding mechanism shows peristaltic movements in the channel leading to the drug efflux. This study would be helpful in understanding the molecular basis of drugs resistance in Mtb. PMID:26792538

  4. Efficient soluble expression of active recombinant human cyclin A2 mediated by E. coli molecular chaperones.

    PubMed

    Grigoroudis, Asterios I; McInnes, Campbell; Premnath, Padmavathy Nandha; Kontopidis, George

    2015-09-01

    Bacterial expression of human proteins continues to present a critical challenge in protein crystallography and drug design. While human cyclin A constructs have been extensively characterized in complex with cyclin dependent kinase 2 (CDK2), efforts to express the monomeric human cyclin A2 in Escherichia coli in a stable form, without the kinase subunit, have been laden with technical difficulties, including solubility, yield and purity. Here, optimized conditions are described with the aim of generating for first time, sufficient quantities of human recombinant cyclin A2 in a soluble and active form for crystallization and ligand characterization purposes. The studies involve implementation of a His-tagged heterologous expression system under conditions of auto-induction and mediated by molecular chaperone-expressing plasmids. A high yield of human cyclin A2 was obtained in natively folded and soluble form, through co-expression with groups of molecular chaperones from E. coli in various combinations. A one-step affinity chromatography method was utilized to purify the fusion protein products to homogeneity, and the biological activity confirmed through ligand-binding affinity to inhibitory peptides, representing alternatives for the key determinants of the CDK2 substrate recruitment site on the cyclin regulatory subunit. As a whole, obtaining the active cyclin A without the CDK partner (referred to as monomeric in this work) in a straightforward and facile manner will obviate protein--production issues with the CDK2/cyclin A complex and enable drug discovery efforts for non-ATP competitive CDK inhibition through the cyclin groove. PMID:25956535

  5. Calculation of Vibrational Shifts of Nitrile Probes in the Active Site of Ketosteroid Isomerase upon Ligand Binding

    PubMed Central

    Layfield, Joshua P.

    2012-01-01

    The vibrational Stark effect provides insight into the roles of hydrogen bonding, electrostatics, and conformational motions in enzyme catalysis. In a recent application of this approach to the enzyme ketosteroid isomerase (KSI), thiocyanate probes were introduced in site-specific positions throughout the active site. This paper implements a quantum mechanical/molecular mechanical (QM/MM) approach for calculating the vibrational shifts of nitrile (CN) probes in proteins. This methodology is shown to reproduce the experimentally measured vibrational shifts upon binding of the intermediate analog equilinen to KSI for two different nitrile probe positions. Analysis of the molecular dynamics simulations provides atomistic insight into the roles that key residues play in determining the electrostatic environment and hydrogen-bonding interactions experienced by the nitrile probe. For the M116C-CN probe, equilinen binding reorients an active site water molecule that is directly hydrogen bonded to the nitrile probe, resulting in a more linear CNH angle and increasing the CN frequency upon binding. For the F86C-CN probe, equilinen binding orients the Asp103 residue, decreasing the hydrogen-bonding distance between the Asp103 backbone and the nitrile probe and slightly increasing the CN frequency. This QM/MM methodology is applicable to a wide range of biological systems and has the potential to assist in the elucidation of the fundamental principles underlying enzyme catalysis. PMID:23210919

  6. Effects of the cofactor binding sites on the activities of secondary alcohol dehydrogenase (SADH).

    PubMed

    Wang, Tao; Chen, Xiangjun; Han, Jun; Ma, Sichun; Wang, Jianmei; Li, Xufeng; Zhang, Hui; Liu, Zhibin; Yang, Yi

    2016-07-01

    SADHs from Thermoanaerobacter ethanolicus are enzymes that, together with various cofactors, catalyze the reversible reduction of carbonyl compounds to their corresponding alcohols. To explore how cofactors bind to SADH, TeSADH was cloned in this study, and Ser(199) and Arg(200) were replaced by Tyr and Asp, respectively. Both sites were expected to be inside or adjacent to the cofactor-binding domain according to computational a prediction. Analysis of TeSADH activities revealed that the enzymatic efficiency (kcat/Km) of the S199Y mutant was noticeably enhanced using by NADH, NADPH as cofactors, and similar with that of wild-type using by NADP(+), NAD(+). Conversely, the activity of the R200D mutant significantly decreased with all cofactors. Furthermore, in yeast, the S199Y mutant substantially elevated the ethanol concentration compared with the wild type. Molecular dynamics simulation results indicated the H-bonding network between TeSADH and the cofactors was stronger for the S199Y mutant and the binding energy was simultaneously increased. Moreover, the fluorescence results indicated the S199Y mutant exhibited an increased preference for NAD(P)H, binding with NAD(P)H more compactly compared with wild type. PMID:27016086

  7. Reverse evolution leads to genotypic incompatibility despite functional and active site convergence

    PubMed Central

    Kaltenbach, Miriam; Jackson, Colin J; Campbell, Eleanor C; Hollfelder, Florian; Tokuriki, Nobuhiko

    2015-01-01

    Understanding the extent to which enzyme evolution is reversible can shed light on the fundamental relationship between protein sequence, structure, and function. Here, we perform an experimental test of evolutionary reversibility using directed evolution from a phosphotriesterase to an arylesterase, and back, and examine the underlying molecular basis. We find that wild-type phosphotriesterase function could be restored (>104-fold activity increase), but via an alternative set of mutations. The enzyme active site converged towards its original state, indicating evolutionary constraints imposed by catalytic requirements. We reveal that extensive epistasis prevents reversions and necessitates fixation of new mutations, leading to a functionally identical sequence. Many amino acid exchanges between the new and original enzyme are not tolerated, implying sequence incompatibility. Therefore, the evolution was phenotypically reversible but genotypically irreversible. Our study illustrates that the enzyme's adaptive landscape is highly rugged, and different functional sequences may constitute separate fitness peaks. DOI: http://dx.doi.org/10.7554/eLife.06492.001 PMID:26274563

  8. Structural Insights into the Catalytic Active Site and Activity of Human Nit2/ω-Amidase

    PubMed Central

    Chien, Chin-Hsiang; Gao, Quan-Ze; Cooper, Arthur J. L.; Lyu, Jyun-Hong; Sheu, Sheh-Yi

    2012-01-01

    Human nitrilase-like protein 2 (hNit2) is a putative tumor suppressor, recently identified as ω-amidase. hNit2/ω-amidase plays a crucial metabolic role by catalyzing the hydrolysis of α-ketoglutaramate (the α-keto analog of glutamine) and α-ketosuccinamate (the α-keto analog of asparagine), yielding α-ketoglutarate and oxaloacetate, respectively. Transamination between glutamine and α-keto-γ-methiolbutyrate closes the methionine salvage pathway. Thus, hNit2/ω-amidase links sulfur metabolism to the tricarboxylic acid cycle. To elucidate the catalytic specificity of hNit2/ω-amidase, we performed molecular dynamics simulations on the wild type enzyme and its mutants to investigate enzyme-substrate interactions. Binding free energies were computed to characterize factors contributing to the substrate specificity. The predictions resulting from these computations were verified by kinetic analyses and mutational studies. The activity of hNit2/ω-amidase was determined with α-ketoglutaramate and succinamate as substrates. We constructed three catalytic triad mutants (E43A, K112A, and C153A) and a mutant with a loop 116–128 deletion to validate the role of key residues and the 116–128 loop region in substrate binding and turnover. The molecular dynamics simulations successfully verified the experimental trends in the binding specificity of hNit2/ω-amidase toward various substrates. Our findings have revealed novel structural insights into the binding of substrates to hNit2/ω-amidase. A catalytic triad and the loop residues 116–128 of hNit2 play an essential role in supporting the stability of the enzyme-substrate complex, resulting in the generation of the catalytic products. These observations are predicted to be of benefit in the design of new inhibitors or activators for research involving cancer and hyperammonemic diseases. PMID:22674578

  9. Methods for improved selectivity in photo-activation and detection of molecular diagnostic agents

    DOEpatents

    Wachter, Eric A.; Fisher, Walter G.; Dees, H. Craig

    2008-03-18

    A method for the imaging of a particular volume of plant or animal tissue, wherein the plant or animal tissue contains at least one photo-active molecular agent. The method comprises the steps of treating the particular volume of the plant or animal tissue with light sufficient to promote a simultaneous two-photon excitation of the photo-active molecular agent contained in the particular volume of the plant or animal tissue, photo-activating at least one of the at least one photo-active molecular agent in the particular volume of the plant or animal tissue, thereby producing at least one photo-activated molecular agent, wherein the at least one photo-activated molecular agent emits energy, detecting the energy emitted by the at least one photo-activated molecular agent, and producing a detected energy signal which is characteristic of the particular volume of plant or animal tissue. The present invention also provides a method for the imaging of a particular volume of material, wherein the material contains at least one photo-active molecular agent.

  10. Method for improved selectivity in photo-activation and detection of molecular diagnostic agents

    DOEpatents

    Wachter, E.A.; Fisher, W.G.; Dees, H.C.

    1998-11-10

    A method for the imaging of a particular volume of plant or animal tissue, wherein the plant or animal tissue contains at least one photo-active molecular agent. The method includes the steps of treating the particular volume of the plant or animal tissue with light sufficient to promote a simultaneous two-photon excitation of the photo-active molecular agent contained in the particular volume of the plant or animal tissue, photo-activating at least one of the at least one photo-active molecular agent in the particular volume of the plant or animal tissue, thereby producing at least one photo-activated molecular agent, wherein the at least one photo-activated molecular agent emits energy, detecting the energy emitted by the at least one photo-activated molecular agent, and producing a detected energy signal which is characteristic of the particular volume of plant or animal tissue. The present invention is also a method for the imaging of a particular volume of material, wherein the material contains at least one photo-active molecular agent. 13 figs.

  11. Method for improved selectivity in photo-activation and detection of molecular diagnostic agents

    DOEpatents

    Wachter, Eric A.; Fisher, Walter G.; Dees, H. Craig

    1998-01-01

    A method for the imaging of a particular volume of plant or animal tissue, wherein the plant or animal tissue contains at least one photo-active molecular agent. The method includes the steps of treating the particular volume of the plant or animal tissue with light sufficient to promote a simultaneous two-photon excitation of the photo-active molecular agent contained in the particular volume of the plant or animal tissue, photo-activating at least one of the at least one photo-active molecular agent in the particular volume of the plant or animal tissue, thereby producing at least one photo-activated molecular agent, wherein the at least one photo-activated molecular agent emits energy, detecting the energy emitted by the at least one photo-activated molecular agent, and producing a detected energy signal which is characteristic of the particular volume of plant or animal tissue. The present invention is also a method for the imaging of a particular volume of material, wherein the material contains at least one photo-active molecular agent.

  12. Activities, crystal structures and molecular dynamics of dihydro-1H-isoindole derivatives, inhibitors of HIV-1 integrase

    PubMed Central

    Métifiot, Mathieu; Maddali, Kasthuraiah; Johnson, Barry C.; Hare, Stephen; Smith, Steven J.; Zhao, XueZhi; Marchand, Christophe; Burke, Terrence R.; Hughes, Stephen H.; Cherepanov, Peter; Pommier, Yves

    2013-01-01

    Based on a series of lactam and phthalimide derivatives that inhibit HIV-1 integrase, we developed a new derivative, XZ-259, with biochemical and antiviral activities comparable to raltegravir. We determined the crystal structures of XZ-259 and four other derivatives in complex with the prototype foamy virus intasome. The compounds bind at the integrase-Mg2+-DNA interface of the integrase active site. In biochemical and antiviral assays, XZ-259 inhibits raltegravir-resistant HIV-1 integrases harboring the Y143R mutation. Molecular modeling is also presented suggesting that XZ-259 can bind in the HIV-1 intasome with its dimethyl sulfonamide group adopting two opposite orientations. Molecular dynamics analyses of the HIV-1 intasome highlight the importance of the viral DNA in drug potency. PMID:23075516

  13. Molecular Basis for Redox Activation of Epidermal Growth Factor Receptor Kinase.

    PubMed

    Truong, Thu H; Ung, Peter Man-Un; Palde, Prakash B; Paulsen, Candice E; Schlessinger, Avner; Carroll, Kate S

    2016-07-21

    Epidermal growth factor receptor (EGFR) is a target of signal-derived H2O2, and oxidation of active-site cysteine 797 to sulfenic acid enhances kinase activity. Although a major class of covalent drugs targets C797, nothing is known about its catalytic importance or how S-sulfenylation leads to activation. Here, we report the first detailed functional analysis of C797. In contrast to prior assumptions, mutation of C797 diminishes catalytic efficiency in vitro and cells. The experimentally determined pKa and reactivity of C797 toward H2O2 correspondingly distinguish this residue from the bulk of the cysteinome. Molecular dynamics simulation of reduced versus oxidized EGFR, reinforced by experimental testing, indicates that sulfenylation of C797 allows new electrostatic interactions to be formed with the catalytic loop. Finally, we show that chronic oxidative stress yields an EGFR subpopulation that is refractory to the FDA-approved drug afatinib. Collectively, our data highlight the significance of redox biology to understanding kinase regulation and drug pharmacology. PMID:27427230

  14. Active Site Structure and Peroxidase Activity of Oxidatively Modified Cytochrome c Species in Complexes with Cardiolipin.

    PubMed

    Capdevila, Daiana A; Oviedo Rouco, Santiago; Tomasina, Florencia; Tortora, Verónica; Demicheli, Verónica; Radi, Rafael; Murgida, Daniel H

    2015-12-29

    We report a resonance Raman and UV-vis characterization of the active site structure of oxidatively modified forms of cytochrome c (Cyt-c) free in solution and in complexes with cardiolipin (CL). The studied post-translational modifications of Cyt-c include methionine sulfoxidation and tyrosine nitration, which lead to altered heme axial ligation and increased peroxidase activity with respect to those of the wild-type protein. In spite of the structural and activity differences between the protein variants free in solution, binding to CL liposomes induces in all cases the formation of a spectroscopically identical bis-His axial coordination conformer that more efficiently promotes lipid peroxidation. The spectroscopic results indicate that the bis-His form is in equilibrium with small amounts of high-spin species, thus suggesting a labile distal His ligand as the basis for the CL-induced increase in enzymatic activity observed for all protein variants. For Cyt-c nitrated at Tyr74 and sulfoxidized at Met80, the measured apparent binding affinities for CL are ∼4 times larger than for wild-type Cyt-c. On the basis of these results, we propose that these post-translational modifications may amplify the pro-apoptotic signal of Cyt-c under oxidative stress conditions at CL concentrations lower than for the unmodified protein. PMID:26620444

  15. Molecular switch for CLC-K Cl- channel block/activation: optimal pharmacophoric requirements towards high-affinity ligands.

    PubMed

    Liantonio, Antonella; Picollo, Alessandra; Carbonara, Giuseppe; Fracchiolla, Giuseppe; Tortorella, Paolo; Loiodice, Fulvio; Laghezza, Antonio; Babini, Elena; Zifarelli, Giovanni; Pusch, Michael; Camerino, Diana Conte

    2008-01-29

    ClC-Ka and ClC-Kb Cl(-) channels are pivotal for renal salt reabsorption and water balance. There is growing interest in identifying ligands that allow pharmacological interventions aimed to modulate their activity. Starting from available ligands, we followed a rational chemical strategy, accompanied by computational modeling and electrophysiological techniques, to identify the molecular requisites for binding to a blocking or to an activating binding site on ClC-Ka. The major molecular determinant that distinguishes activators from blockers is the level of planarity of the aromatic portions of the molecules: only molecules with perfectly coplanar aromatic groups display potentiating activity. Combining several molecular features of various CLC-K ligands, we discovered that phenyl-benzofuran carboxylic acid derivatives yield the most potent ClC-Ka inhibitors so far described (affinity <10 microM). The increase in affinity compared with 3-phenyl-2-p-chlorophenoxy-propionic acid (3-phenyl-CPP) stems primarily from the conformational constraint provided by the phenyl-benzofuran ring. Several other key structural elements for high blocking potency were identified through a detailed structure-activity relationship study. Surprisingly, some benzofuran-based drugs inhibit ClC-Kb with a similar affinity of <10 microM, thus representing the first inhibitors for this CLC-K isoform identified so far. Based on our data, we established a pharmacophore model that will be useful for the development of drugs targeting CLC-K channels. PMID:18216243

  16. Beyond the Active Site: The Impact of the Outer Coordination Sphere on Electrocatalysts for Hydrogen Production and Oxidation

    SciTech Connect

    Ginovska-Pangovska, Bojana; Dutta, Arnab; Reback, Matthew L.; Linehan, John C.; Shaw, Wendy J.

    2014-08-19

    Hydrogenase enzymes provide inspiration for investigations of molecular catalysts utilizing structural and functional mimics of the active site. However, the resulting active site mimics cannot match the combination of high rates and low overpotentials of the enzyme, suggesting that the rest of the protein scaffold, i.e., the outer coordination sphere, is necessary for the efficiency of hydrogenase. Therefore, inclusion of outer coordination sphere elements onto molecular catalysts may enable us to achieve and ultimately surpass the overall enzymatic efficiency. In an effort to identify and include the missing enzymatic features, there has been recent effort to understand the effect of outer coordination sphere elements on molecular catalysts for hydrogen oxidation and production. Our focus has been to utilize amino acid or peptide based scaffolds on an active functional mimic for hydrogen oxidation and production, [Ni(PR2NR’2)2]2+. This bottom-up approach, i.e, building an outer coordination sphere around a functional molecular catalyst, has allowed us to evaluate individual contributions to catalysis, including enhancing proton movement, concentrating substrate and introducing structural features to control reactivity. Collectively, these studies have resulted in catalysts that can operate faster, can operate at lower overpotentials, have enhanced water solubility, and/or can provide more stability to oxygen or extreme conditions such as strongly acidic or basic conditions than their unmodified parent complexes. Common mechanisms have yet to be defined to predictably control these processes but our growing knowledge in this area is essential for the eventual mimicry of enzymes for developing efficient molecular catalysts for practical use. This account reviews previously published work supported by the US DOE Basic Energy Sciences (BES), Physical Bioscience program, the Office of Science Early Career Research Program through the USDOE, BES, the Center for

  17. Molecular modeling studies of 1,4-dihydro-4-oxoquinoline ribonucleosides with anti-HSV-1 activity

    NASA Astrophysics Data System (ADS)

    Yoneda, Julliane Diniz; Albuquerque, Magaly Girão; Leal, Kátia Zaccur; Seidl, Peter Rudolf; de Alencastro, Ricardo Bicca

    2011-12-01

    Eight human herpes viruses ( e.g., herpes simplex, varicella-zoster, Epstein-Barr, cytomegalovirus, Kaposi's sarcoma) are responsible for several diseases from sub-clinic manifestations to fatal infections, mostly in immunocompromised patients. The major limitations of the currently available antiviral drug therapy are drug resistance, host toxicity, and narrow spectrum of activity. However, some non-nucleoside 1,4-dihydro-4-oxoquinoline derivatives ( e.g., PNU-183792) [4] shows broad spectrum antiviral activity. We have developed molecular modeling studies, including molecular docking and molecular dynamics simulations, based on a model proposed by Liu and co-workers [14] in order to understand the mechanism of action of a 6-chloro substituted 1,4-dihydro-4-oxoquinoline ribonucleoside, synthesized by the synthetic group, which showed anti-HSV-1 activity [9]. The molecular docking simulations confirmed the Liu's model showing that the ligand needs to dislocate template residues from the active site in order to interact with the viral DNA polymerase enzyme, reinforcing that the interaction with the Val823 residue is pivotal for the inhibitory activity of non-nucleoside 1,4-dihydro-4-oxoquinoline derivatives, such as PNU-183792, with the HSV-1. The molecular dynamics simulations showed that the 6-chloro-benzyl group of PNU-183792 maintains its interaction with residues of the HSV-1 DNA polymerase hydrophobic pocket, considered important according to the Liu's model, and also showed that the methyl group bounded to the nitrogen atom from PNU-183792 is probably contributing to a push-pull effect with the carbonyl group.

  18. Molecular Modeling on Berberine Derivatives toward BuChE: An Integrated Study with Quantitative Structure-Activity Relationships Models, Molecular Docking, and Molecular Dynamics Simulations.

    PubMed

    Fang, Jiansong; Pang, Xiaocong; Wu, Ping; Yan, Rong; Gao, Li; Li, Chao; Lian, Wenwen; Wang, Qi; Liu, Ai-Lin; Du, Guan-Hua

    2016-05-01

    A dataset of 67 berberine derivatives for the inhibition of butyrylcholinesterase (BuChE) was studied based on the combination of quantitative structure-activity relationships models, molecular docking, and molecular dynamics methods. First, a series of berberine derivatives were reported, and their inhibitory activities toward butyrylcholinesterase (BuChE) were evaluated. By 2D- quantitative structure-activity relationships studies, the best model built by partial least-square had a conventional correlation coefficient of the training set (R(2) ) of 0.883, a cross-validation correlation coefficient (Qcv2) of 0.777, and a conventional correlation coefficient of the test set (Rpred2) of 0.775. The model was also confirmed by Y-randomization examination. In addition, the molecular docking and molecular dynamics simulation were performed to better elucidate the inhibitory mechanism of three typical berberine derivatives (berberine, C2, and C55) toward BuChE. The predicted binding free energy results were consistent with the experimental data and showed that the van der Waals energy term (ΔEvdw ) difference played the most important role in differentiating the activity among the three inhibitors (berberine, C2, and C55). The developed quantitative structure-activity relationships models provide details on the fine relationship linking structure and activity and offer clues for structural modifications, and the molecular simulation helps to understand the inhibitory mechanism of the three typical inhibitors. In conclusion, the results of this study provide useful clues for new drug design and discovery of BuChE inhibitors from berberine derivatives. PMID:26648584

  19. Identification of Ice Nucleation Active Sites on Feldspar Dust Particles

    PubMed Central

    2015-01-01

    Mineral dusts originating from Earth’s crust are known to be important atmospheric ice nuclei. In agreement with earlier studies, feldspar was found as the most active of the tested natural mineral dusts. Here we investigated in closer detail the reasons for its activity and the difference in the activity of the different feldspars. Conclusions are drawn from scanning electron microscopy, X-ray powder diffraction, infrared spectroscopy, and oil-immersion freezing experiments. K-feldspar showed by far the highest ice nucleation activity. Finally, we give a potential explanation of this effect, finding alkali-metal ions having different hydration shells and thus an influence on the ice nucleation activity of feldspar surfaces. PMID:25584435

  20. An improved molecular design of obtaining NLO active molecular glasses using triphenyl moieties as amorphous phase formation enhancers

    NASA Astrophysics Data System (ADS)

    Traskovskis, Kaspars; Mihailovs, Igors; Tokmakovs, Andrejs; Kokars, Valdis; Rutkis, Martins

    2012-06-01

    New molecular design of obtaining molecular glasses has been developed by linking triphenylmethyl moieties to chromophore core by flexible C-C bridge. Compounds capable of forming stable amorphous phase with good optical quality have been acquired with increased chemical and thermal sustainability compared to the previously reported design. NLO activity of compounds has been measured after corona discharge polling. Compared to previously synthesized trityloxy fragment containing compounds increase of d33 coefficient by up to 17 times was achieved for the same chromophore core containing compounds.

  1. Evolution & Phylogenetic Analysis: Classroom Activities for Investigating Molecular & Morphological Concepts

    ERIC Educational Resources Information Center

    Franklin, Wilfred A.

    2010-01-01

    In a flexible multisession laboratory, students investigate concepts of phylogenetic analysis at both the molecular and the morphological level. Students finish by conducting their own analysis on a collection of skeletons representing the major phyla of vertebrates, a collection of primate skulls, or a collection of hominid skulls.

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

    SciTech Connect

    Not Available

    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.

  3. Ultrafast ligand binding dynamics in the active site of native bacterial nitric oxide reductase.

    PubMed

    Kapetanaki, Sofia M; Field, Sarah J; Hughes, Ross J L; Watmough, Nicholas J; Liebl, Ursula; Vos, Marten H

    2008-01-01

    The active site of nitric oxide reductase from Paracoccus denitrificans contains heme and non-heme iron and is evolutionarily related to heme-copper oxidases. The CO and NO dynamics in the active site were investigated using ultrafast transient absorption spectroscopy. We find that, upon photodissociation from the active site heme, 20% of the CO rebinds in 170 ps, suggesting that not all the CO transiently binds to the non-heme iron. The remaining 80% does not rebind within 4 ns and likely migrates out of the active site without transient binding to the non-heme iron. Rebinding of NO to ferrous heme takes place in approximately 13 ps. Our results reveal that heme-ligand recombination in this enzyme is considerably faster than in heme-copper oxidases and are consistent with a more confined configuration of the active site. PMID:18420024

  4. Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination

    PubMed Central

    Rowley, Paul A.; Kachroo, Aashiq H.; Ma, Chien-Hui; Maciaszek, Anna D.; Guga, Piotr; Jayaram, Makkuni

    2015-01-01

    Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5′ to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions. PMID:25999343

  5. Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination.

    PubMed

    Rowley, Paul A; Kachroo, Aashiq H; Ma, Chien-Hui; Maciaszek, Anna D; Guga, Piotr; Jayaram, Makkuni

    2015-07-13

    Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5' to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions. PMID:25999343

  6. β-Glucuronidase activity and mitochondrial dysfunction: the sites where flavonoid glucuronides act as anti-inflammatory agents

    PubMed Central

    Kawai, Yoshichika

    2014-01-01

    Epidemiological and experimental studies suggest that the consumption of flavonoid-rich diets decreases the risk of various chronic diseases such as cardiovascular diseases. Although studies on the bioavailability of flavonoids have been well-characterized, the tissue and cellular localizations underlying their biological mechanisms are largely unknown. The development and application of novel monoclonal antibodies revealed that macrophages could be the major target of dietary flavonoids in vivo. Using macrophage-like cell lines in vitro, we examined the molecular basis of the interaction between the macrophages and flavonoids, especially the glucuronide metabolites. We have found that extracellular β-glucuronidase secreted from macrophages is essential for the bioactivation of the glucuronide conjugates into the aglycone, and that the enzymatic activity, which requires an acidic pH, is promoted by the increased secretion of lactate in response to the mitochondrial dysfunction. This review describes our recent findings indicating the molecular mechanisms responsible for the anti-inflammatory activity of dietary flavonoids within the inflammation sites. We propose that the extracellular activity of β-glucuronidase associated with the status of the mitochondrial function in the target cells might be important biomarkers for the specific sites where the glucuronides of dietary flavonoids can act as anti-atherosclerotic and anti-inflammatory agents in vivo. PMID:24895476

  7. Molecular Complexity via C–H Activation: A Dehydrogenative Diels-Alder Reaction

    PubMed Central

    Stang, Erik M.; White, M. Christina

    2011-01-01

    Traditionally, C–H oxidation reactions install oxidized functionality onto a preformed molecular skeleton, resulting in a local molecular change. The use of C–H activation chemistry to construct complex molecular scaffolds is a new area with tremendous potential in synthesis. We report a Pd(II)/bis-sulfoxide catalyzed dehydrogenative Diels-Alder reaction that converts simple terminal olefins into complex cycloadducts in a single operation. PMID:21842902

  8. Method for improved selectivity in photo-activation of molecular agents

    DOEpatents

    Fisher, Walter G.; Wachter, Eric A.; Dees, H. Craig

    2000-01-01

    An apparatus for the treatment of a particular volume of plant or animal tissue by treating the plant or animal tissue with at least one photo-active molecular agent, wherein the particular volume of the plant or animal tissue retains at least a portion of the at least one photo-active molecular agent, and then treating the particular volume of the plant or animal tissue with light sufficient to promote a simultaneous two-photon excitation of at least one of the at least one photo-active molecular agent retained in the particular volume of the plant or animal tissue, wherein the at least one photo-active molecular agent becomes active in the particular volume of the plant or animal tissue.

  9. How Intrinsic Molecular Dynamics Control Intramolecular Communication in Signal Transducers and Activators of Transcription Factor STAT5

    PubMed Central

    Langenfeld, Florent; Guarracino, Yann; Arock, Michel; Trouvé, Alain; Tchertanov, Luba

    2015-01-01

    Signal Transducer and Activator of Transcription STAT5 is a key mediator of cell proliferation, differentiation and survival. While STAT5 activity is tightly regulated in normal cells, its constitutive activation directly contributes to oncogenesis and is associated with a broad range of hematological and solid tumor cancers. Therefore the development of compounds able to modulate pathogenic activation of this protein is a very challenging endeavor. A crucial step of drug design is the understanding of the protein conformational features and the definition of putative binding site(s) for such modulators. Currently, there is no structural data available for human STAT5 and our study is the first footprint towards the description of structure and dynamics of this protein. We investigated structural and dynamical features of the two STAT5 isoforms, STAT5a and STAT5b, taken into account their phosphorylation status. The study was based on the exploration of molecular dynamics simulations by different analytical methods. Despite the overall folding similarity of STAT5 proteins, the MD conformations display specific structural and dynamical features for each protein, indicating first, sequence-encoded structural properties and second, phosphorylation-induced effects which contribute to local and long-distance structural rearrangements interpreted as allosteric event. Further examination of the dynamical coupling between distant sites provides evidence for alternative profiles of the communication pathways inside and between the STAT5 domains. These results add a new insight to the understanding of the crucial role of intrinsic molecular dynamics in mediating intramolecular signaling in STAT5. Two pockets, localized in close proximity to the phosphotyrosine-binding site and adjacent to the channel for communication pathways across STAT5, may constitute valid targets to develop inhibitors able to modulate the function-related communication properties of this signaling

  10. The GPS Motif Is a Molecular Switch for Bimodal Activities of Adhesion Class G Protein-Coupled Receptors

    PubMed Central

    Prömel, Simone; Frickenhaus, Marie; Hughes, Samantha; Mestek, Lamia; Staunton, David; Woollard, Alison; Vakonakis, Ioannis; Schöneberg, Torsten; Schnabel, Ralf; Russ, Andreas P.; Langenhan, Tobias

    2012-01-01

    Summary Adhesion class G protein-coupled receptors (aGPCR) form the second largest group of seven-transmembrane-spanning (7TM) receptors whose molecular layout and function differ from canonical 7TM receptors. Despite their essential roles in immunity, tumorigenesis, and development, the mechanisms of aGPCR activation and signal transduction have remained obscure to date. Here, we use a transgenic assay to define the protein domains required in vivo for the activity of the prototypical aGPCR LAT-1/Latrophilin in Caenorhabditis elegans. We show that the GPCR proteolytic site (GPS) motif, the molecular hallmark feature of the entire aGPCR class, is essential for LAT-1 signaling serving in two different activity modes of the receptor. Surprisingly, neither mode requires cleavage but presence of the GPS, which relays interactions with at least two different partners. Our work thus uncovers the versatile nature of aGPCR activity in molecular detail and places the GPS motif in a central position for diverse protein-protein interactions. PMID:22938866

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

    SciTech Connect

    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.

  12. The Role of an Active Site Mg2+ in HDV Ribozyme Self-Cleavage: Insights from QM/MM Calculations

    PubMed Central

    Mlýnský, Vojtěch; Šponer, Jiří

    2014-01-01

    The hepatitis delta virus (HDV) ribozyme is a catalytic RNA motif embedded in the human pathogenic HDV RNA. It catalyzes self-cleavage of its sugar-phosphate backbone with direct participation of the active site cytosine C75. Biochemical and structural data support a general acid role of C75. Here, we used hybrid quantum mechanical/molecular mechanical (QM/MM) calculations to probe the reaction mechanism and changes in Gibbs energy along the ribozyme's reaction pathway with an N3-protonated C75H+ in the active site, which acts as the general acid, and a partially hydrated Mg2+ ion with one deprotonated, inner-shell coordinated water molecule that acts as the general base. We followed eight reaction paths with distinct position and coordination of the catalytically important active site Mg2+ ion. For six of them, we observed feasible activation barriers ranging from 14.2 to 21.9 kcal/mol, indicating that the specific position of the Mg2+ ion in the active site is predicted to strongly affect the kinetics of self-cleavage. The deprotonation of the U-1(2′-OH) nucleophile and the nucleophilic attack of the resulting U-1(2′-O−) on the scissile phosphodiester are found to be separate steps, as deprotonation precedes the nucleophilic attack. This sequential mechanism of the HDV ribozyme differs from the concerted nucleophilic activation and attack suggested for the hairpin ribozyme. We estimated the pKa of the U-1(2′-OH) group to range from 8.8 to 11.2, suggesting that the pKa is lowered by several units from that of a free ribose, comparable to and most likely smaller than the pKa of the solvated active site Mg2+ ion. Our results thus support the notion that the structure of the HDV ribozyme, and particularly the positioning of the active site Mg2+ ion, facilitates deprotonation and activation of the 2′-OH nucleophile. PMID:25412464

  13. Understanding Molecular Genetics through a Drawing-Based Activity

    ERIC Educational Resources Information Center

    Rotbain, Yosi; Marbach-Ad, Gili; Stavy, Ruth

    2005-01-01

    The activity uses drawings typically found in textbooks, engaging students in physical and mental activities such as drawing, painting and figure completion--as well as finding missing words and answering guiding questions. The activity deals with three topics: the structure of DNA, DNA replication and protein synthesis. In this study we…

  14. Binding stability of peptides derived from 1ALA residue and 7GLY residues to sites near active center of fluctuating papain

    NASA Astrophysics Data System (ADS)

    Nishiyama, Katsuhiko

    2012-05-01

    We investigated the binding stability of peptides derived from 1ALA residue and 7GLY residues to sites near active center of fluctuating papain via molecular dynamics and docking simulations. Replacing GLY residue in 8GLY with ALA residue had a positive effect on binding stability to the sites in some cases although the replacing had a negative effect on it in other cases. Furthermore the replacing had a negative effect on the chance of binding to the sites. Residue in peptide should be replaced on the basis of systematic exploration of its position.

  15. 78 FR 7394 - Foreign-Trade Zone 121-Albany, NY; Authorization of Production Activity; Albany Molecular...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-01

    ... notice in the Federal Register inviting public comment (77 FR 63290, 10/16/2012). The FTZ Board has... Molecular Research, Inc.; Subzone 121A (Pharmaceutical Chemicals Production); Rensselaer, NY On September 26, 2012, Albany Molecular Research, Inc., submitted a notification of proposed production activity to...

  16. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand.

    PubMed

    Parashar, Abhinav; Venkatachalam, Avanthika; Gideon, Daniel Andrew; Manoj, Kelath Murali

    2014-12-12

    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. PMID:25449264

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

    SciTech Connect

    Khatri, Yogan; Gregory, Michael C.; Grinkova, Yelena V.; Denisov, Ilia G.; Sligar, Stephen G.

    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.

  18. Cholinesterase inhibitory activity versus aromatic core multiplicity: a facile green synthesis and molecular docking study of novel piperidone embedded thiazolopyrimidines.

    PubMed

    Basiri, Alireza; Murugaiyah, Vikneswaran; Osman, Hasnah; Kumar, Raju Suresh; Kia, Yalda; Hooda, Alysha; Parsons, Richard B

    2014-01-15

    Novel thiazolopyrimidine derivatives have been synthesized via microwave assisted, domino cascade methodology in ionic liquid and evaluated in vitro for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities. Among the newly synthesized compounds 6d, 6a, 6e and 6b displayed higher AChE inhibitory activity than standard drug, galanthamine, with IC50 values of 0.53, 1.47, 1.62 and 2.05μM, respectively. Interestingly, all the compounds except for 6m-r and 6x displayed higher BChE inhibitory potentials than galanthamine with IC50 values ranging from 1.09 to 18.56μM. Molecular docking simulations for 6d possessing the most potent AChE and BChE inhibitory activities, disclosed its binding interactions at the active site gorge of AChE and BChE enzymes. PMID:24369842

  19. Characterization of an Active Thermal Erosion Site, Caribou Creek, Alaska

    NASA Astrophysics Data System (ADS)

    Busey, R.; Bolton, W. R.; Cherry, J. E.; Hinzman, L. D.

    2013-12-01

    The goal of this project is to estimate volume loss of soil over time from this site, provide parameterizations on erodibility of ice rich permafrost and serve as a baseline for future landscape evolution simulations. Located in the zone of discontinuous permafrost, the interior region of Alaska (USA) is home to a large quantity of warm, unstable permafrost that is both high in ice content and has soil temperatures near the freezing point. Much of this permafrost maintains a frozen state despite the general warming air temperature trend in the region due to the presence of a thick insulating organic mat and a dense root network in the upper sub-surface of the soil column. At a rapidly evolving thermo-erosion site, located within the Caribou-Poker Creeks Research Watershed (part of the Bonanza Creek LTER) near Chatanika, Alaska (N65.140, W147.570), the protective organic layer and associated plants were disturbed by an adjacent traditional use trail and the shifting of a groundwater spring. These triggers have led to rapid geomorphological change on the landscape as the soil thaws and sediment is transported into the creek at the valley bottom. Since 2006 (approximately the time of initiation), the thermal erosion has grown to 170 meters length, 3 meters max depth, and 15 meters maximum width. This research combines several data sets: DGPS survey, imagery from an extremely low altitude pole-based remote sensing (3 to 5 meters above ground level), and imagery from an Unmanned Aerial System (UAS) at about 60m altitude.

  20. Molecular Network Analysis of Endometriosis Reveals a Novel Role for c-Jun Regulated Macrophage Activation

    PubMed Central

    Beste, Michael T.; Pfäffle-Doyle, Nicole; Prentice, Emily A.; Morris, Stephanie N.; Lauffenburger, Douglas A.; Isaacson, Keith B.; Griffith, Linda G.

    2014-01-01

    Clinical management of endometriosis is limited by the complex relationship between symptom severity, heterogeneous surgical presentations, and variability in clinical outcomes. As a complement to visual classification schemes, molecular profiles of disease activity may improve risk stratification to better inform treatment decisions and identify novel approaches to targeted treatment. Here, we employ a network analysis of information flow within and between inflammatory cells to discern consensus behaviors characterizing patient sub-populations. Unsupervised multivariate analysis of cytokine profiles quantified by multiplex immunoassays identified a subset of patients with a shared “consensus signature” of thirteen elevated cytokines that was associated with common clinical features, but was not observed among patient subpopulations defined by morphologic presentation alone. Enrichment analysis of consensus markers reinforced the primacy of peritoneal macrophage infiltration and activation, which was demonstrably elevated in ex vivo cultures. Although familiar targets of the NFκB family emerged among over-represented transcriptional binding sites for consensus markers, our analysis provides evidence for a previously unrecognized contribution from c-Jun, c-Fos, and AP-1 effectors of mitogen associated kinase signaling. Their crucial involvement in propagation of macrophage-driven inflammatory networks was confirmed via targeted inhibition of upstream kinases. Collectively, these analyses provide in vivo validation of a clinically relevant inflammatory network that may serve as an objective measure for guiding treatment decisions for endometriosis management, and in the future may provide a mechanistic endpoint for assessing efficacy of novel agents aimed at curtailing inflammatory mechanisms that drive disease progression. PMID:24500404

  1. Quantitative structure-activity relationship correlation between molecular structure and the Rayleigh enantiomeric enrichment factor.

    PubMed

    Jammer, S; Rizkov, D; Gelman, F; Lev, O

    2015-08-01

    It was recently demonstrated that under environmentally relevant conditions the Rayleigh equation is valid to describe the enantiomeric enrichment - conversion relationship, yielding a proportional constant called the enantiomeric enrichment factor, εER. In the present study we demonstrate a quantitative structure-activity relationship model (QSAR) that describes well the dependence of εER on molecular structure. The enantiomeric enrichment factor can be predicted by the linear Hansch model, which correlates biological activity with physicochemical properties. Enantioselective hydrolysis of sixteen derivatives of 2-(phenoxy)propionate (PPMs) have been analyzed during enzymatic degradation by lipases from Pseudomonas fluorescens (PFL), Pseudomonas cepacia (PCL), and Candida rugosa (CRL). In all cases the QSAR relationships were significant with R(2) values of 0.90-0.93, and showed high predictive abilities with internal and external validations providing QLOO(2) values of 0.85-0.87 and QExt(2) values of 0.8-0.91. Moreover, it is demonstrated that this model enables differentiation between enzymes with different binding site shapes. The enantioselectivity of PFL and PCL was dictated by electronic properties, whereas the enantioselectivity of CRL was determined by lipophilicity and steric factors. The predictive ability of the QSAR model demonstrated in the present study may serve as a helpful tool in environmental studies, assisting in source tracking of unstudied chiral compounds belonging to a well-studied homologous series. PMID:26153539

  2. Assembly of a Metalloporphyrin-Polyoxometalate Hybrid Material for Highly Efficient Activation of Molecular Oxygen.

    PubMed

    Zhu, Shu-Lan; Xu, Xuan; Ou, Sha; Zhao, Min; He, Wei-Long; Wu, Chuan-De

    2016-08-01

    Organic metalloporphyrins and inorganic polyoxometalates (POMs) are two kinds of efficient molecular catalysts to prompt a variety of chemical reactions. They have been used as active moieties for the synthesis of porous materials to realize highly efficient heterogeneous catalysis. Both of them are regarded as the organic/inorganic equivalent counterparts to complement the individual features. Therefore, the combination of metalloporphyrins and POMs in the same hybrid materials might generate interesting catalytic properties by emerging their unique individual functions. To avoid the random connections between metalloporphyrins, POMs, and lanthanide connecting nodes, we have developed a "step-by-step" aggregation strategy, including the reaction of POMs with metal ions to bind metal nodes on the surfaces of POMs at the first step and the reaction of the resulting POM derivatives with metalloporphyrin linkers to result in hybrid materials at the second step. Catalytic experiments demonstrate that the resulting hybrid material exhibits interesting catalytic properties in the heterogeneous epoxidation of olefins, in which the conversion, epoxide selectivity, turnover number, and turnover frequency for the epoxidation of styrene to (1,2-epoxyethyl)benzene are >99%, 94%, 220000, and 22000 h(-1), respectively. These results demonstrate that the collaborative work of multiple active sites in hybrid materials can achieve superior high efficiency in heterogeneous catalysis. PMID:27408952

  3. Effect of Active-site Mutation at Asn67 on the Proton Transfer Mechanism of Human Carbonic Anhydrase II

    PubMed Central

    Maupin, C. Mark; Zheng, Jiayin; Tu, Chingkuang; McKenna, Robert; Silverman, David N.; Voth, Gregory A.

    2009-01-01

    The rate-limiting proton transfer (PT) event in the site-specific mutant N67L of human carbonic anhydrase II (HCA II) has been examined by kinetic, x-ray, and simulation approaches. The x-ray crystallography, which were previously reported, and molecular dynamics (MD) simulations indicate that the proton shuttling residue, His64, predominantly resides in the outward orientation with a significant disruption of the ordered water in the active site for the dehydration pathway. While disorder is seen in the active-site water, water cluster analysis indicates that the N67L mutant may form water clusters similar to those seen in the wild-type (WT). For the hydration pathway of the enzyme, the active site water cluster analysis reveals an inability of the N67L mutant to stabilize water clusters when His64 is in the inward orientation, thereby favoring PT when His64 is in the outward orientation. The preference of the N67L mutant to carry out the PT when His64 is in the outward orientation for both the hydration and dehydration pathway is reasoned to be the main cause of the observed reduction in the overall rate. To probe the mechanism of PT, solvent H/D kinetic isotope effects (KIEs) were experimentally studied with catalysis measured by the exchange of 18O between CO2 and water. The values obtained from the KIEs were determined as a function of the deuterium content of solvent, using the proton inventory method. No differences were detected in the overarching mechanism of PT between WT and N67L HCA II, despite changes in the active-site water structure and/or the orientation of His64. PMID:19634894

  4. Probing Binding Sites and Mechanisms of Action of an IKs Activator by Computations and Experiments

    PubMed Central

    Xu, Yu; Wang, Yuhong; Zhang, Mei; Jiang, Min; Rosenhouse-Dantsker, Avia; Wassenaar, Tsjerk; Tseng, Gea-Ny

    2015-01-01

    The slow delayed rectifier (IKs) channel is composed of the KCNQ1 channel and KCNE1 auxiliary subunit, and functions to repolarize action potentials in the human heart. IKs activators may provide therapeutic efficacy for treating long QT syndromes. Here, we show that a new KCNQ1 activator, ML277, can enhance IKs amplitude in adult guinea pig and canine ventricular myocytes. We probe its binding site and mechanism of action by computational analysis based on our recently reported KCNQ1 and KCNQ1/KCNE1 3D models, followed by experimental validation. Results from a pocket analysis and docking exercise suggest that ML277 binds to a side pocket in KCNQ1 and the KCNE1-free side pocket of KCNQ1/KCNE1. Molecular-dynamics (MD) simulations based on the most favorable channel/ML277 docking configurations reveal a well-defined ML277 binding space surrounded by the S2-S3 loop and S4-S5 helix on the intracellular side, and by S4–S6 transmembrane helices on the lateral sides. A detailed analysis of MD trajectories suggests two mechanisms of ML277 action. First, ML277 restricts the conformational dynamics of the KCNQ1 pore, optimizing K+ ion coordination in the selectivity filter and increasing current amplitudes. Second, ML277 binding induces global motions in the channel, including regions critical for KCNQ1 gating transitions. We conclude that ML277 activates IKs by binding to an intersubunit space and allosterically influencing pore conductance and gating transitions. KCNE1 association protects KCNQ1 from an arrhythmogenic (constitutive current-inducing) effect of ML277, but does not preclude its current-enhancing effect. PMID:25564853

  5. Marine Biology Field Trip Sites. Ocean Related Curriculum Activities.

    ERIC Educational Resources Information Center

    Pauls, John

    The ocean affects all of our lives. Therefore, awareness of and information about the interconnections between humans and oceans are prerequisites to making sound decisions for the future. Project ORCA (Ocean Related Curriculum Activities) has developed interdisciplinary curriculum materials designed to meet the needs of students and teachers…

  6. The first self-assembled trimetallic lanthanide helicate: different coordination sites in symmetrical molecular architectures.

    PubMed

    Bocquet, Bernard; Bernardinelli, Gérald; Ouali, Nadjet; Floquet, Sebastien; Renaud, Fabien; Hopfgartner, Gérard; Piguet, Claude

    2002-05-01

    A tris-tridentate segmental ligand has been designed for the self-assembly of homotrimetallic triple-stranded lanthanide helicates possessing different coordination sites along the threefold axis. PMID:12123053

  7. Modulation of environmental dynamics at the active site and activity of an enzyme under nanoscopic confinement: Subtilisin Carlsberg in anionic AOT reverse micelle.

    PubMed

    Rakshit, Surajit; Saha, Ranajay; Pal, Samir Kumar

    2013-10-01

    Hydration dynamics plays a crucial role in determining the structure, function, dynamics, and stability of an enzyme. These dynamics involve the trapped-water motions within small distance along with the total protein dynamics. However, the exact molecular basis for the induction of enzyme function by water dynamics is still remain unclear. Here, we have studied both enzymatic activity and environmental dynamics at the active site of an enzyme, Subtilisin Carlsberg (SC), under confined environment of the reverse micelle (RM) retaining the structural integrity of the protein. Kinetic measurements show that enzymatic activity increases with increasing the water content of the RM. The picosecond-resolved fluorescence Stokes shift studies indicate faster hydration dynamics at the active site of the enzyme with increasing the water content in the RM (w0 values). Temperature-dependent hydration dynamics studies demonstrate the increased flexibility of the protein at higher temperature under confinement. From temperature-dependent solvation dynamics study, we have also calculated the activation energy that has to be overcome for full orientational freedom to the water molecules from bound to free-state. The results presented here establish a correlation between the enzymatic activity and dynamics of hydration of the encapsulated protein SC in cell-like confined environment within the structural integrity of the enzyme. PMID:24004033

  8. Molecular genotyping of Echinococcus granulosus using formalin-fixed paraffin-embedded preparations from human isolates in unusual tissue sites.

    PubMed

    Hizem, A; M'rad, S; Oudni-M'rad, M; Mestiri, S; Hammedi, F; Mezhoud, H; Zakhama, A; Mokni, M; Babba, H

    2016-07-01

    Cystic echinococcosis (CE) caused by Echinococcus granulosus remains a serious problem worldwide for issues relating to public health and the economy. The most predominantly affected sites are the liver and the lungs, but other organs such as the heart, the spleen and the peritoneum can also be infected. Access to cysts from uncommon sites has limited genomic and molecular investigations. In the present study, genotypes of E. granulosus sensu lato were identified from formalin-fixed paraffin-embedded tissues (FF-PETs) implicated in human CE. Tissue samples were obtained from 57 patients with histologically confirmed CE. DNA samples were analysed using Egss 1 polymerase chain reaction (PCR) specific to the mitochondrial 12S rRNA gene of E. granulosus sensu stricto. All cysts were typed as E. granulosus sensu stricto with up to 35% of the liver and 16.6% of lungs being the most frequently infected, and up to 48.4% of samples being from rare sites. No correlation was found between cyst site and either the gender or the age of patients. This study demonstrates the possibility of exploiting atypical cysts using FF-PET samples and highlights the predominance of E. granulosus sensu stricto species in the Tunisian population, even in unusual infection sites. PMID:26190231

  9. A model of compensatory molecular evolution involving multiple sites in RNA molecules.

    PubMed

    Kusumi, Junko; Ichinose, Motoshi; Takefu, Masasuke; Piskol, Robert; Stephan, Wolfgang; Iizuka, Masaru

    2016-01-01

    Consider two sites under compensatory fitness interaction, such as a Watson-Crick base pair in an RNA helix or two interacting residues in a protein. A mutation at any one of these two sites may reduce the fitness of an individual. However, fitness may be restored by the occurrence of a second mutation at the other site. Kimura modeled this process using a two-locus haploid fitness scheme with two alleles at each locus. He predicted that compensatory evolution following this model is very rare unless selection against the deleterious single mutations is weak and linkage between the interacting sites is tight. Here we investigate the question whether the rate of compensatory evolution increases if we take the context of the two directly interacting sites into account. By "context", we mean the effect of neighboring sites in an RNA helix. Interaction between the focal pair of sites under consideration and the context may lead to so-called indirect compensation. Thus, extending Kimura's classical model of compensatory evolution, we study the effects of both direct and indirect compensation on the rate of compensatory evolution. It is shown that the effects of indirect compensation are very strong. We find that recombination does not slow down the rate of compensatory evolution as predicted by the classical model. Instead, compensatory substitutions may be relatively frequent, even if linkage between the focal interacting sites is loose, selection against deleterious mutations is strong, and mutation rate is low. We compare our theoretical results with data on RNA secondary structures from vertebrate introns. PMID:26506471

  10. Molecular Cloning, Characterization and Positively Selected Sites of the Glutathione S-Transferase Family from Locusta migratoria

    PubMed Central

    Zhang, Min; Qin, Guohua; Li, Daqi; Zhu, Kun Yan; Ma, Enbo; Zhang, Jianzhen

    2014-01-01

    Glutathione S-transferases (GSTs) are multifunctional enzymes that are involved in the metabolism of endogenous and exogenous compounds and are related to insecticide resistance. The purpose of this study was to provide new information on the molecular characteristics and the positive selection of locust GSTs. Based on the transcriptome database, we sequenced 28 cytosolic GSTs and 4 microsomal GSTs from the migratory locust (Locusta migratoria). We assigned the 28 cytosolic GSTs into 6 classes—sigma, epsilon, delta, theta, omega and zeta, and the 4 microsomal GSTs into 2 subclasses—insect and MGST3. The tissue- and stage-expression patterns of the GSTs differed at the mRNA level. Further, the substrate specificities and kinetic constants of the cytosolic GSTs differed markedly at the protein level. The results of likelihood ratio tests provided strong evidence for positive selection in the delta class. The result of Bayes Empirical Bayes analysis identified 4 amino acid sites in the delta class as positive selection sites. These sites were located on the protein surface. Our findings will facilitate the elucidation of the molecular characteristics and evolutionary aspects of insect GST superfamily. PMID:25486043

  11. Identification of the antiepileptic racetam binding site in the synaptic vesicle protein 2A by molecular dynamics and docking simulations

    PubMed Central

    Correa-Basurto, José; Cuevas-Hernández, Roberto I.; Phillips-Farfán, Bryan V.; Martínez-Archundia, Marlet; Romo-Mancillas, Antonio; Ramírez-Salinas, Gema L.; Pérez-González, Óscar A.; Trujillo-Ferrara, José; Mendoza-Torreblanca, Julieta G.

    2015-01-01

    Synaptic vesicle protein 2A (SV2A) is an integral membrane protein necessary for the proper function of the central nervous system and is associated to the physiopathology of epilepsy. SV2A is the molecular target of the anti-epileptic drug levetiracetam and its racetam analogs. The racetam binding site in SV2A and the non-covalent interactions between racetams and SV2A are currently unknown; therefore, an in silico study was performed to explore these issues. Since SV2A has not been structurally characterized with X-ray crystallography or nuclear magnetic resonance, a three-dimensional (3D) model was built. The model was refined by performing a molecular dynamics simulation (MDS) and the interactions of SV2A with the racetams were determined by docking studies. A reliable 3D model of SV2A was obtained; it reached structural equilibrium during the last 15 ns of the MDS (50 ns) with remaining structural motions in the N-terminus and long cytoplasmic loop. The docking studies revealed that hydrophobic interactions and hydrogen bonds participate importantly in ligand recognition within the binding site. Residues T456, S665, W666, D670 and L689 were important for racetam binding within the trans-membrane hydrophilic core of SV2A. Identifying the racetam binding site within SV2A should facilitate the synthesis of suitable radio-ligands to study treatment response and possibly epilepsy progression. PMID:25914622

  12. Synthesis, biochemical evaluation and molecular modeling studies of novel rhodium complexes with nanomolar activity against Platelet Activating Factor.

    PubMed

    Tsoupras, Alexandros B; Papakyriakou, Athanasios; Demopoulos, Constantinos A; Philippopoulos, Athanassios I

    2013-03-01

    Two square planar Rh(I) organometallic complexes namely [Rh(L(1))(cod)]Cl (cod = cycloocta-1,5-diene, L(1)=2,2'-pyridylquinoxaline (1-Cl), [Rh(L1)(cod)](NO3) (1-NO(3)) and a series of novel octahedral rhodium(III) complexes of the general formulae mer-[Rh(L(1))Cl(3)(MeOH)] (2) and cis-[Rh(L(2))(2)Cl(2)]Cl (L(2)=4 carboxy 2 (2' pyridyl)quinoline (3), L(3)=2,2' bipyridine 4,4' dicarboxylic acid (4) were synthesized and characterized spectroscopically. All the synthesized compounds including the previously prepared cis-[Rh(L(1))(2)Cl(2)]Cl complex (5) were biologically evaluated as potential inhibitors of the Platelet Activation Factor (PAF) and thrombin induced aggregation. In particular compounds 1-Cl and 1-NO(3) were found to be strong inhibitors of PAF with IC(50) values in the range of 16 nM and 15 nM rendering them good candidates for further investigation. Their potency is comparable to that of the widely used PAF receptor antagonists WEB2170, BN52021, and Rupatadine (IC(50) of 20, 30 and 260 nM respectively). Molecular docking calculations suggest that 1-Cl, 1-NO3 and 2 can be accommodated within the ligand-binding site of PAF receptor and block the activity of PAF. On the other hand, the octahedral rhodium(III) complexes 3-5 that cannot fit the ligand-binding domain, could potentially exhibit their activity at the extracellular domain of the receptor. PMID:23318288

  13. Active site dynamics in the zinc-dependent medium chain alcohol dehydrogenase superfamily

    PubMed Central

    Baker, Patrick J.; Britton, K. Linda; Fisher, Martin; Esclapez, Julia; Pire, Carmen; Bonete, Maria Jose; Ferrer, Juan; Rice, David W.

    2009-01-01

    Despite being the subject of intensive investigations, many aspects of the mechanism of the zinc-dependent medium chain alcohol dehydrogenase (MDR) superfamily remain contentious. We have determined the high-resolution structures of a series of binary and ternary complexes of glucose dehydrogenase, an MDR enzyme from Haloferax mediterranei. In stark contrast to the textbook MDR mechanism in which the zinc ion is proposed to remain stationary and attached to a common set of protein ligands, analysis of these structures reveals that in each complex, there are dramatic differences in the nature of the zinc ligation. These changes arise as a direct consequence of linked movements of the zinc ion, a zinc-bound bound water molecule, and the substrate during progression through the reaction. These results provide evidence for the molecular basis of proton traffic during catalysis, a structural explanation for pentacoordinate zinc ion intermediates, a unifying view for the observed patterns of metal ligation in the MDR family, and highlight the importance of dynamic fluctuations at the metal center in changing the electrostatic potential in the active site, thereby influencing the proton traffic and hydride transfer events. PMID:19131516

  14. The Role of OOH Binding Site and Pt Surface Structure on ORR Activities

    PubMed Central

    Jia, Qingying; Caldwell, Keegan; Ziegelbauer, Joseph M.; Kongkanand, Anusorn; Wagner, Frederick T.; Mukerjee, Sanjeev; Ramaker, David E.

    2015-01-01

    We present experimentally observed molecular adsorbate coverages (e.g., O(H), OOH and HOOH) on real operating dealloyed bimetallic PtMx (M = Ni or Co) catalysts under oxygen reduction reaction (ORR) conditions obtained using X-ray absorption near edge spectroscopy (XANES). The results reveal a complex Sabatier catalysis behavior and indicate the active ORR mechanism changes with Pt–O bond weakening from the O2 dissociative mechanism, to the peroxyl mechanism, and finally to the hydrogen peroxide mechanism. An important rearrangement of the OOH binding site, an intermediate in the ORR, enables facile H addition to OOH and faster O–O bond breaking on 111 faces at optimal Pt–O bonding strength, such as that occurring in dealloyed PtM core-shell nanoparticles. This rearrangement is identified by previous DFT calculations and confirmed from in situ measured OOH adsorption coverages during the ORR. The importance of surface structural effects and 111 ordered faces is confirmed by the higher specific ORR rates on solid core vs porous multi-core nanoparticles. PMID:26190857

  15. In-silico analysis of binding site features and substrate selectivity in plant flavonoid-3-O glycosyltransferases (F3GT) through molecular modeling, docking and dynamics simulation studies.

    PubMed

    Sharma, Ranu; Panigrahi, Priyabrata; Suresh, C G

    2014-01-01

    Flavonoids are a class of plant secondary metabolites that act as storage molecules, chemical messengers, as well as participate in homeostasis and defense processes. They possess pharmaceutical properties important for cancer treatment such as antioxidant and anti-tumor activities. The drug-related properties of flavonoids can be improved by glycosylation. The enzymes glycosyltransferases (GTs) glycosylate acceptor molecules in a regiospecific manner with the help of nucleotide sugar donor molecules. Several plant GTs have been characterized and their amino acid sequences determined. However, three-dimensional structures of only a few are reported. Here, phylogenetic analysis using amino acid sequences have identified a group of GTs with the same regiospecific activity. The structures of these closely related GTs were modeled using homologous GT structures. Their substrate binding sites were elaborated by docking flavonoid acceptor and UDP-sugar donor molecules in the modeled structures. Eight regions near the acceptor binding site in the N- and C- terminal domain of GTs have been identified that bind and specifically glycosylate the 3-OH group of acceptor flavonoids. Similarly, a conserved motif in the C-terminal domain is known to bind a sugar donor substrate. In certain GTs, the substitution of a specific glutamine by histidine in this domain changes the preference of sugar from glucose to galactose as a result of changed pattern of interactions. The molecular modeling, docking, and molecular dynamics simulation studies have revealed the chemical and topological features of the binding site and thus provided insights into the basis of acceptor and donor recognition by GTs. PMID:24667893

  16. In-Silico Analysis of Binding Site Features and Substrate Selectivity in Plant Flavonoid-3-O Glycosyltransferases (F3GT) through Molecular Modeling, Docking and Dynamics Simulation Studies

    PubMed Central

    Sharma, Ranu; Panigrahi, Priyabrata; Suresh, C.G.

    2014-01-01

    Flavonoids are a class of plant secondary metabolites that act as storage molecules, chemical messengers, as well as participate in homeostasis and defense processes. They possess pharmaceutical properties important for cancer treatment such as antioxidant and anti-tumor activities. The drug-related properties of flavonoids can be improved by glycosylation. The enzymes glycosyltransferases (GTs) glycosylate acceptor molecules in a regiospecific manner with the help of nucleotide sugar donor molecules. Several plant GTs have been characterized and their amino acid sequences determined. However, three-dimensional structures of only a few are reported. Here, phylogenetic analysis using amino acid sequences have identified a group of GTs with the same regiospecific activity. The structures of these closely related GTs were modeled using homologous GT structures. Their substrate binding sites were elaborated by docking flavonoid acceptor and UDP-sugar donor molecules in the modeled structures. Eight regions near the acceptor binding site in the N- and C- terminal domain of GTs have been identified that bind and specifically glycosylate the 3-OH group of acceptor flavonoids. Similarly, a conserved motif in the C-terminal domain is known to bind a sugar donor substrate. In certain GTs, the substitution of a specific glutamine by histidine in this domain changes the preference of sugar from glucose to galactose as a result of changed pattern of interactions. The molecular modeling, docking, and molecular dynamics simulation studies have revealed the chemical and topological features of the binding site and thus provided insights into the basis of acceptor and donor recognition by GTs. PMID:24667893

  17. DESIGN, SYNTHESIS, MOLECULAR DOCKING AND ANTI-BREAST CANCER ACTIVITY OF NOVEL QUINAZOLINONES TARGETING ESTROGEN RECEPTOR α.

    PubMed

    Ahmed, Marwa F; Youns, Mahmoud; Belal, Amany

    2016-01-01

    A new series of 6,8-dibromo-2-(4-chlorophenyl)-4-oxo-4H-quinazoline derivatives II-VI were syn- thesized, their chemical structures were confirmed by spectroscopic means and elemental analyses. All these compounds were tested in vitro against human breast cancer cell line (MCF-7) using resazurin reduction assay method and doxorubicin as a reference drug. Most of the tested compounds showed better activity than dox- orubicin. Compound IVh was the best active one, its IC₅₀ is 8.52 µg/mL. Molecular docking studies for the best active compounds IVb, IVc, IVf, IVh and Va were performed on the active site of estrogen receptor α (ERα) subtype to explore the estrogen receptor binding ability of these compounds. All the docked compounds showed good fitting score energy with the active site of ERα subtype and compound IVh showed the best docking score energy(-25.3 kcal/mol). Estrogen binding evaluation assay was performed for the docked compounds to ensure that their activity against MCF7 go through inhibition of ERα, they showed ERα inhibition at 41-85% and compound IVh was the most active one (85%). PMID:27008806

  18. Molecular vibration-activity relationship in the agonism of adenosine receptors.

    PubMed

    Chee, Hyun Keun; Oh, S June

    2013-12-01

    The molecular vibration-activity relationship in the receptor-ligand interaction of adenosine receptors was investigated by structure similarity, molecular vibration, and hierarchical clustering in a dataset of 46 ligands of adenosine receptors. The resulting dendrogram was compared with those of another kind of fingerprint or descriptor. The dendrogram result produced by corralled intensity of molecular vibrational frequency outperformed four other analyses in the current study of adenosine receptor agonism and antagonism. The tree that was produced by clustering analysis of molecular vibration patterns showed its potential for the functional classification of adenosine receptor ligands. PMID:24465242

  19. Molecular simulation of receptors of physiologically active compounds for purposes of medical chemistry

    NASA Astrophysics Data System (ADS)

    Baskin, Igor I.; Palyulin, Vladimir A.; Zefirov, Nikolai S.

    2009-06-01

    The general strategy of the molecular simulation of biological receptors and their interaction with ligands is considered. The procedures for construction of 3D protein models, molecular docking, evaluation of model quality, determination of the free energy of protein binding with ligands are discussed. The methods of molecular design of new medicaments based on molecular models of biological targets: virtual screening and de novo design, are presented. Examples of the above-listed approaches for the simulation of a number of pharmacologically significant receptors, analysis of receptor-ligand interactions and design of new biologically active organic compounds are given.

  20. Molecular Vibration-Activity Relationship in the Agonism of Adenosine Receptors

    PubMed Central

    Chee, Hyun Keun

    2013-01-01

    The molecular vibration-activity relationship in the receptor-ligand interaction of adenosine receptors was investigated by structure similarity, molecular vibration, and hierarchical clustering in a dataset of 46 ligands of adenosine receptors. The resulting dendrogram was compared with those of another kind of fingerprint or descriptor. The dendrogram result produced by corralled intensity of molecular vibrational frequency outperformed four other analyses in the current study of adenosine receptor agonism and antagonism. The tree that was produced by clustering analysis of molecular vibration patterns showed its potential for the functional classification of adenosine receptor ligands. PMID:24465242

  1. MOLECULAR INTERACTION POTENTIALS FOR THE DEVELOPMENT OF STRUCTURE-ACTIVITY RELATIONSHIPS

    EPA Science Inventory

    Abstract
    One reasonable approach to the analysis of the relationships between molecular structure and toxic activity is through the investigation of the forces and intermolecular interactions responsible for chemical toxicity. The interaction between the xenobiotic and the bio...

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

    PubMed

    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

  3. Reduction of Urease Activity by Interaction with the Flap Covering the Active Site

    PubMed Central

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

    2015-01-01

    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

  4. Encroachment of Human Activity on Sea Turtle Nesting Sites

    NASA Astrophysics Data System (ADS)

    Ziskin, D.; Aubrecht, C.; Elvidge, C.; Tuttle, B.; Baugh, K.; Ghosh, T.

    2008-12-01

    The encroachment of anthropogenic lighting on sea turtle nesting sites poses a serious threat to the survival of these animals [Nicholas, 2001]. This danger is quantified by combining two established data sets. The first is the Nighttime Lights data produced by the NOAA National Geophysical Data Center [Elvidge et al., 1997]. The second is the Marine Turtle Database produced by the World Conservation Monitoring Centre (WCMC). The technique used to quantify the threat of encroachment is an adaptation of the method described in Aubrecht et al. [2008], which analyzes the stress on coral reef systems by proximity to nighttime lights near the shore. Nighttime lights near beaches have both a direct impact on turtle reproductive success since they disorient hatchlings when they mistake land-based lights for the sky-lit surf [Lorne and Salmon, 2007] and the lights are also a proxy for other anthropogenic threats. The identification of turtle nesting sites with high rates of encroachment will hopefully steer conservation efforts to mitigate their effects [Witherington, 1999]. Aubrecht, C, CD Elvidge, T Longcore, C Rich, J Safran, A Strong, M Eakin, KE Baugh, BT Tuttle, AT Howard, EH Erwin, 2008, A global inventory of coral reef stressors based on satellite observed nighttime lights, Geocarto International, London, England: Taylor and Francis. In press. Elvidge, CD, KE Baugh, EA Kihn, HW Kroehl, ER Davis, 1997, Mapping City Lights with Nighttime Data from the DMSP Operational Linescan System, Photogrammatic Engineering and Remote Sensing, 63:6, pp. 727-734. Lorne, JK, M Salmon, 2007, Effects of exposure to artificial lighting on orientation of hatchling sea turtles on the beach and in the ocean, Endangered Species Research, Vol. 3: 23-30. Nicholas, M, 2001, Light Pollution and Marine Turtle Hatchlings: The Straw that Breaks the Camel's Back?, George Wright Forum, 18:4, p77-82. Witherington, BE, 1999, Reducing Threats To Nesting Habitat, Research and Management Techniques for

  5. Small Molecule Active Site Directed Tools for Studying Human Caspases.

    PubMed

    Poreba, Marcin; Szalek, Aleksandra; Kasperkiewicz, Paulina; Rut, Wioletta; Salvesen, Guy S; Drag, Marcin

    2015-11-25

    Caspases are proteases of clan CD and were described for the first time more than two decades ago. They play critical roles in the control of regulated cell death pathways including apoptosis and inflammation. Due to their involvement in the development of various diseases like cancer, neurodegenerative diseases, or autoimmune disorders, caspases have been intensively investigated as potential drug targets, both in academic and industrial laboratories. This review presents a thorough, deep, and systematic assessment of all technologies developed over the years for the investigation of caspase activity and specificity using substrates and inhibitors, as well as activity based probes, which in recent years have attracted considerable interest due to their usefulness in the investigation of biological functions of this family of enzymes. PMID:26551511

  6. Activation of brown adipose tissue mitochondrial GDP binding sites

    SciTech Connect

    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.

  7. Using Active Learning in a Studio Classroom to Teach Molecular Biology

    ERIC Educational Resources Information Center

    Nogaj, Luiza A.

    2013-01-01

    This article describes the conversion of a lecture-based molecular biology course into an active learning environment in a studio classroom. Specific assignments and activities are provided as examples. The goal of these activities is to involve students in collaborative learning, teach them how to participate in the learning process, and give…

  8. Comparison of antimicrobial activities of newly obtained low molecular weight scorpion chitosan and medium molecular weight commercial chitosan.

    PubMed

    Kaya, Murat; Asan-Ozusaglam, Meltem; Erdogan, Sevil

    2016-06-01

    In this study the antimicrobial activity of low molecular weight (3.22 kDa) chitosan, obtained for the first time from a species belonging to the Scorpiones, was screened against nine pathogenic microorganisms (seven bacteria and two yeasts) and compared with that of medium molecular weight commercial chitosan (MMWCC). It was observed that the antimicrobial activity of the low molecular weight scorpion chitosan (LMWSC) was specific to bacterial species in general rather than gram-negative or gram-positive bacterial groups. It was also determined that LMWSC had a stronger inhibitory effect than the MMWCC, particularly on the bacterium Listeria monocytogenes and the yeast Candida albicans, which are important pathogens for public health. In addition, it was recorded that the MMWCC had a greater inhibitory effect on Bacillus subtilis than LMWSC. According to the results obtained by the disc diffusion method, the antibacterial activity of both LMWSC and MMWCC against B. subtilis and Salmonella enteritidis was higher than the widely used antibiotic Gentamicin (CN, 10 μg/disc). PMID:26702952

  9. High and low molecular weight hyaluronic acid differentially influence macrophage activation

    PubMed Central

    Rayahin, Jamie E.; Buhrman, Jason S.; Zhang, Yu; Koh, Timothy J.; Gemeinhart, Richard A.

    2015-01-01

    Macrophages exhibit phenotypic diversity permitting wide-ranging roles in maintaining physiologic homeostasis. Hyaluronic acid, a major glycosaminoglycan of the extracellular matrix, has been shown to have differential signaling based on its molecular weight. With this in mind, the main objective of this study was to elucidate the role of hyaluronic acid molecular weight on macrophage activation and reprogramming. Changes in macrophage activation were assessed by activation state selective marker measurement, specifically quantitative real time polymerase chain reaction, and cytokine enzyme-linked immunoassays, after macrophage treatment with differing molecular weights of hyaluronic acid under four conditions: the resting state, concurrent with classical activation, and following inflammation involving either classically or alternatively activated macrophages. Regardless of initial polarization state, low molecular weight hyaluronic acid induced a classically activated-like state, confirmed by up-regulation of pro-inflammatory genes, including nos2, tnf, il12b, and cd80, and enhanced secretion of nitric oxide and TNF-α. High molecular weight hyaluronic acid promoted an alternatively activated-like state, confirmed by up regulation of pro-resolving gene transcription, including arg1, il10, and mrc1, and enhanced arginase activity. Overall, our observations suggest that macrophages undergo phenotypic changes dependent on molecular weight of hyaluronan that correspond to either (1) pro-inflammatory response for low molecular weight HA or (2) pro-resolving response for high molecular weight HA. These observations bring significant further understanding of the influence of extracellular matrix polymers, hyaluronic acid in particular, on regulating the inflammatory response of macrophages. This knowledge can be used to guide the design of HA-containing biomaterials to better utilize the natural response to HAs. PMID:26280020

  10. Leukocyte arrest: Biomechanics and molecular mechanisms of β2 integrin activation

    PubMed Central

    Fan, Zhichao; Ley, Klaus

    2016-01-01

    Integrins are a group of heterodimeric transmembrane receptors that play essential roles in cell–cell and cell–matrix interaction. Integrins are important in many physiological processes and diseases. Integrins acquire affinity to their ligand by undergoing molecular conformational changes called activation. Here we review the molecular biomechanics during conformational changes of integrins, integrin functions in leukocyte biorheology (adhesive functions during rolling and arrest) and molecules involved in integrin activation. PMID:26684674

  11. Ligand uptake in Mycobacterium tuberculosis truncated hemoglobins is controlled by both internal tunnels and active site water molecules

    PubMed Central

    Davidge, Kelly S; Singh, Sandip; Bowman, Lesley AH; Tinajero-Trejo, Mariana; Carballal, Sebastián; Radi, Rafael; Poole, Robert K; Dikshit, Kanak; Estrin, Dario A; Marti, Marcelo A; Boechi, Leonardo

    2015-01-01

    Mycobacterium tuberculosis, the causative agent of human tuberculosis, has two proteins belonging to the truncated hemoglobin (trHb) family. Mt-trHbN presents well-defined internal hydrophobic tunnels that allow O 2 and •NO to migrate easily from the solvent to the active site, whereas Mt-trHbO possesses tunnels that are partially blocked by a few bulky residues, particularly a tryptophan at position G8. Differential ligand migration rates allow Mt-trHbN to detoxify •NO, a crucial step for pathogen survival once under attack by the immune system, much more efficiently than Mt-trHbO. In order to investigate the differences between these proteins, we performed experimental kinetic measurements, •NO decomposition, as well as molecular dynamics simulations of the wild type Mt-trHbN and two mutants, VG8F and VG8W. These mutations introduce modifications in both tunnel topologies and affect the incoming ligand capacity to displace retained water molecules at the active site. We found that a single mutation allows Mt-trHbN to acquire ligand migration rates comparable to those observed for Mt-trHbO, confirming that ligand migration is regulated by the internal tunnel architecture as well as by water molecules stabilized in the active site. PMID:26478812

  12. Ligand uptake in Mycobacterium tuberculosis truncated hemoglobins is controlled by both internal tunnels and active site water molecules.

    PubMed

    Boron, Ignacio; Bustamante, Juan Pablo; Davidge, Kelly S; Singh, Sandip; Bowman, Lesley Ah; Tinajero-Trejo, Mariana; Carballal, Sebastián; Radi, Rafael; Poole, Robert K; Dikshit, Kanak; Estrin, Dario A; Marti, Marcelo A; Boechi, Leonardo

    2015-01-01

    Mycobacterium tuberculosis, the causative agent of human tuberculosis, has two proteins belonging to the truncated hemoglobin (trHb) family. Mt-trHbN presents well-defined internal hydrophobic tunnels that allow O 2 and (•)NO to migrate easily from the solvent to the active site, whereas Mt-trHbO possesses tunnels that are partially blocked by a few bulky residues, particularly a tryptophan at position G8. Differential ligand migration rates allow Mt-trHbN to detoxify (•)NO, a crucial step for pathogen survival once under attack by the immune system, much more efficiently than Mt-trHbO. In order to investigate the differences between these proteins, we performed experimental kinetic measurements, (•)NO decomposition, as well as molecular dynamics simulations of the wild type Mt-trHbN and two mutants, VG8F and VG8W. These mutations introduce modifications in both tunnel topologies and affect the incoming ligand capacity to displace retained water molecules at the active site. We found that a single mutation allows Mt-trHbN to acquire ligand migration rates comparable to those observed for Mt-trHbO, confirming that ligand migration is regulated by the internal tunnel architecture as well as by water molecules stabilized in the active site. PMID:26478812

  13. Molecular Recognition of Agonist and Antagonist for Peroxisome Proliferator-Activated Receptor-α Studied by Molecular Dynamics Simulations

    PubMed Central

    Liu, Mengyuan; Wang, Lushan; Zhao, Xian; Sun, Xun

    2014-01-01

    Peroxisome proliferator activated receptor-α (PPAR-α) is a ligand-activated transcription factor which plays important roles in lipid and glucose metabolism. The aim of this work is to find residues which selectively recognize PPAR-α agonists and antagonists. To achieve this aim, PPAR-α/13M and PPAR-α/471 complexes were subjected to perform molecular dynamics simulations. This research suggests that several key residues only participate in agonist recognition, while some other key residues only contribute to antagonist recognition. It is hoped that such work is useful for medicinal chemists to design novel PPAR-α agonists and antagonists. PMID:24837836

  14. Molecular epidemiology of Staphylococcus aureus isolates at different sites in the milk producing dairy farms.

    PubMed

    Souza, Viviane; Nader Filho, Antonio; de Castro Melo, Poliana; Ferraudo, Guilherme Moraes; Antônio Sérgio, Ferraudo; de Oliveira Conde, Sandra; Fogaça Junior, Flavio Augusto

    2012-10-01

    The epidemiological relationships between isolated Staphylococcus aureus strains in milk samples of dairy cows, reagent to California Mastitis Test, individual and group milk was demonstrated in different sites of the production fluxogram, in 12 milk-producing farms in the Gameleira region, municipality of Sacramento MG Brazil, so that localization and transmission modes may be identified. Two hundred and forty-four strains out of 446 samples collected at several sites were isolated and bio-chemically characterized as coagulase-positive staphylococcus. Specific chromosome DNA fragment of the species Staphylococcus aureus was amplified to 106 strains and 103 underwent (PFGE). Samples' collection sites with the highest isolation frequency of Staphylococcus aureus strains comprised papillary ostia (31.1%), CMT-reagent cow milk (21.7%), mechanical milking machines' insufflators (21,7%), milk in milk pails (6.6%) and the milk in community bulk tanks (5.6%). Genetic heterogeneity existed among the isolated 103 Staphylococcus aureus strains, since 32 different pulse-types were identified. Pulse-type 1 had the highest similarity among the isolated strains within the different sites of the milk-production fluxogram. Highest occurrence of pulsetype 1 isolates of Staphylococcus aureus strains was reported in samples collected from the papillary ostia (10.6%), followed by milk samples from CMT-reagent dairy cows (5.8%) and mechanical milking machine insufflators (3.8%). The above shows the relevance of these sites in the agents' transmission mechanism within the context of the farms investigated. PMID:24031997

  15. Molecular epidemiology of Staphylococcus aureus isolates at different sites in the milk producing dairy farms

    PubMed Central

    Souza, Viviane; Nader Filho, Antonio; de Castro Melo, Poliana; Ferraudo, Guilherme Moraes; Antônio Sérgio, Ferraudo; de Oliveira Conde, Sandra; Fogaça Junior, Flavio Augusto

    2012-01-01

    The epidemiological relationships between isolated Staphylococcus aureus strains in milk samples of dairy cows, reagent to California Mastitis Test, individual and group milk was demonstrated in different sites of the production fluxogram, in 12 milk-producing farms in the Gameleira region, municipality of Sacramento MG Brazil, so that localization and transmission modes may be identified. Two hundred and forty-four strains out of 446 samples collected at several sites were isolated and bio-chemically characterized as coagulase-positive staphylococcus. Specific chromosome DNA fragment of the species Staphylococcus aureus was amplified to 106 strains and 103 underwent (PFGE). Samples’ collection sites with the highest isolation frequency of Staphylococcus aureus strains comprised papillary ostia (31.1%), CMT-reagent cow milk (21.7%), mechanical milking machines’ insufflators (21,7%), milk in milk pails (6.6%) and the milk in community bulk tanks (5.6%). Genetic heterogeneity existed among the isolated 103 Staphylococcus aureus strains, since 32 different pulse-types were identified. Pulse-type 1 had the highest similarity among the isolated strains within the different sites of the milk-production fluxogram. Highest occurrence of pulsetype 1 isolates of Staphylococcus aureus strains was reported in samples collected from the papillary ostia (10.6%), followed by milk samples from CMT-reagent dairy cows (5.8%) and mechanical milking machine insufflators (3.8%). The above shows the relevance of these sites in the agents’ transmission mechanism within the context of the farms investigated. PMID:24031997

  16. Study of the Differential Activity of Thrombin Inhibitors Using Docking, QSAR, Molecular Dynamics, and MM-GBSA

    PubMed Central

    Mena-Ulecia, Karel; Tiznado, William; Caballero, Julio

    2015-01-01

    Non-peptidic thrombin inhibitors (TIs; 177 compounds) with diverse groups at motifs P1 (such as oxyguanidine, amidinohydrazone, amidine, amidinopiperidine), P2 (such as cyanofluorophenylacetamide, 2-(2-chloro-6-fluorophenyl)acetamide), and P3 (such as phenylethyl, arylsulfonate groups) were studied using molecular modeling to analyze their interactions with S1, S2, and S3 subsites of the thrombin binding site. Firstly, a protocol combining docking and three dimensional quantitative structure–activity relationship was performed. We described the orientations and preferred active conformations of the studied inhibitors, and derived a predictive CoMSIA model including steric, donor hydrogen bond, and acceptor hydrogen bond fields. Secondly, the dynamic behaviors of some selected TIs (compounds 26, 133, 147, 149, 162, and 177 in this manuscript) that contain different molecular features and different activities were analyzed by creating the solvated models and using molecular dynamics (MD) simulations. We used the conformational structures derived from MD to accomplish binding free energetic calculations using MM-GBSA. With this analysis, we theorized about the effect of van der Waals contacts, electrostatic interactions and solvation in the potency of TIs. In general, the contents reported in this article help to understand the physical and chemical characteristics of thrombin-inhibitor complexes. PMID:26599107

  17. Study of the Differential Activity of Thrombin Inhibitors Using Docking, QSAR, Molecular Dynamics, and MM-GBSA.

    PubMed

    Mena-Ulecia, Karel; Tiznado, William; Caballero, Julio

    2015-01-01

    Non-peptidic thrombin inhibitors (TIs; 177 compounds) with diverse groups at motifs P1 (such as oxyguanidine, amidinohydrazone, amidine, amidinopiperidine), P2 (such as cyanofluorophenylacetamide, 2-(2-chloro-6-fluorophenyl)acetamide), and P3 (such as phenylethyl, arylsulfonate groups) were studied using molecular modeling to analyze their interactions with S1, S2, and S3 subsites of the thrombin binding site. Firstly, a protocol combining docking and three dimensional quantitative structure-activity relationship was performed. We described the orientations and preferred active conformations of the studied inhibitors, and derived a predictive CoMSIA model including steric, donor hydrogen bond, and acceptor hydrogen bond fields. Secondly, the dynamic behaviors of some selected TIs (compounds 26, 133, 147, 149, 162, and 177 in this manuscript) that contain different molecular features and different activities were analyzed by creating the solvated models and using molecular dynamics (MD) simulations. We used the conformational structures derived from MD to accomplish binding free energetic calculations using MM-GBSA. With this analysis, we theorized about the effect of van der Waals contacts, electrostatic interactions and solvation in the potency of TIs. In general, the contents reported in this article help to understand the physical and chemical characteristics of thrombin-inhibitor complexes. PMID:26599107

  18. Beyond the active site: the impact of the outer coordination sphere on electrocatalysts for hydrogen production and oxidation.

    PubMed

    Ginovska-Pangovska, Bojana; Dutta, Arnab; Reback, Matthew L; Linehan, John C; Shaw, Wendy J

    2014-08-19

    Redox active metalloenzymes play a major role in energy transformation reactions in biological systems. Examples include formate dehydrogenases, nitrogenases, CO dehydrogenase, and hydrogenases. Many of these reactions are also of interest to humans as potential energy storage or utilization reactions for photoelectrochemical, electrolytic, and fuel cell applications. These metalloenzymes consist of redox active metal centers where substrates are activated and undergo transformation to products accompanied by electron and proton transfer to or from the substrate. These active sites are typically buried deep within a protein matrix of the enzyme with channels for proton transport, electron transport, and substrate/product transport between the active site and the surface of the protein. In addition, there are amino acid residues that lie in close proximity to the active site that are thought to play important roles in regulating and enhancing enzyme activity. Directly studying the outer coordination sphere of enzymes can be challenging due to their complexity, and the use of modified molecular catalysts may allow us to provide some insight. There are two fundamentally different approaches to understand these important interactions. The "bottom-up" approach involves building an amino acid or peptide containing outer coordination sphere around a functional molecular catalyst, and the "top-down" approach involves attaching molecular catalyst to a structured protein. Both of these approaches have been undertaken for hydrogenase mimics and are the emphasis of this Account. Our focus has been to utilize amino acid or peptide based scaffolds on an active functional enzyme mimic for H2 oxidation and production, [Ni(P(R)2N(R('))2)2](2+). This "bottom-up" approach has allowed us to evaluate individual functional group and structural contributions to electrocatalysts for H2 oxidation and production. For instance, using amine, ether, and carboxylic acid functionalities in the

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

    PubMed

    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

  20. Hydrogen Bond Migration between Molecular Sites Observed with Ultrafast 2D IR Chemical Exchange Spectroscopy

    PubMed Central

    Rosenfeld, Daniel E.; Kwak, Kyungwon; Gengeliczki, Zsolt

    2010-01-01

    Hydrogen bonded complexes between phenol and phenylacetylene are studied using ultrafast two-dimensional infrared (2D IR) chemical exchange spectroscopy. Phenylacetylene has two possible π hydrogen bonding acceptor sites (phenyl or acetylene) that compete for hydrogen bond donors in solution at room temperature. The OD stretch frequency of deuterated phenol is sensitive to which acceptor site it is bound. The appearance of off-diagonal peaks between the two vibrational frequencies in the 2D IR spectrum reports on the exchange process between the two competitive hydrogen bonding sites of phenol-phenylacetylene complexes in the neat phenylacetylene solvent. The chemical exchange process occurs in ∼5 ps, and is assigned to direct hydrogen bond migration along the phenylacetylene molecule. Other non-migration mechanisms are ruled out by performing 2D IR experiments on phenol dissolved in the phenylacetylene/carbon tetrachloride mixed solvent. The observation of direct hydrogen bond migration can have implications for macromolecular systems. PMID:20121275

  1. An Accessory Agonist Binding Site Promotes Activation of α4β2* Nicotinic Acetylcholine Receptors*

    PubMed Central

    Wang, Jingyi; Kuryatov, Alexander; Sriram, Aarati; Jin, Zhuang; Kamenecka, Theodore M.; Kenny, Paul J.; Lindstrom, Jon